Counteracting Immunosenescence: Effects of Biomodulina T® on B-1a Lymphocytes in Older Cuban Adults

Counteracting Immunosenescence: Effects of Biomodulina T® on B-1a Lymphocytes in Older Cuban Adults | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Counteracting Immunosenescence: Effects of Biomodulina T® on B-1a Lymphocytes in Older Cuban Adults Vianed Marsan Suarez, Imilla Casado Hernandez, Elizabeth Hernández Ramos, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8649614/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Introduction: B1a lymphocytes are components of the innate immune response that maintain their population through self-renewal, a process that declines with age. Objective To evaluate the effect of Biomodulina T® (BT) on B1a lymphocyte subpopulations in older Cuban adults. Methods In this non-randomized clinical trial, 30 institutionalized adults aged ≥ 60 years received intramuscular BT (3 mg) twice weekly for six weeks. B1a lymphocyte subpopulations (CD19⁺CD5⁺, CD20⁺CD5⁺, and CD19⁺CD20⁺) were quantified by flow cytometry at baseline, six weeks, and six months post-treatment. Results The median percentages and absolute counts of B1a CD5⁺CD20⁺ lymphocytes increased after six weeks of treatment with BT (0.96 vs. 2.11, SD 6.02 vs. 2.38) and (17.02 vs. 40.58, SD 80.09 vs. 39.70), with statistical significance (p = 0.026 and p = 0.014, respectively). In contrast, the percentage and absolute count of lymphocytes coexpressing CD19⁺ and CD20⁺ antigens decreased after six weeks of BT treatment (4.87 vs. 1.94, SD 4.69 vs. 4.57) and (88.48 vs. 31.03, SD 62.14 vs. 65.54), with statistical significance (p = 0.014 and p = 0.018, respectively). Six months after completing the treatment, both the percentage and absolute count of these cells continued to increase (4.87 vs. 8.47, SD 4.69 vs. 26.69) and (88.48 vs. 173.5, SD 62.14 vs. 323.8), reaching statistical significance (p = 0.012 and p = 0.011, respectively). Conclusions BT exerts an immunomodulatory effect on B1a lymphocytes in older adults, with partial persistence six months after treatment, suggesting potential benefits in preventing age-related immune decline. B-1a lymphocytes Flow cytometry Immunosenescence Immunomodulation Thymic peptides Figures Figure 1 1. Introduction In humans, B lymphocytes develop in the bone marrow and, after selection and maturation, migrate to peripheral lymphoid organs such as the spleen, lymph nodes, and mucosa-associated lymphoid tissue. Their primary functions include antibody production and antigen presentation, but they can also mediate cell-based immunity by activating antigen-specific CD4⁺ and CD8⁺ T cells, as well as exert regulatory and cytotoxic effects. B lymphocytes are generally classified into three main subsets: B-1 cells, B-2 cells, and regulatory B cells (Breg). Among these, B-1a lymphocytes are of particular interest in aging, as they contribute to innate immunity and display unique phenotypic and functional characteristics that may be altered during immunosenescence. [ 1 ]. B-2 lymphocytes are the most abundant subset in humans. Their development and maturation are characterized by the generation of a specific surface immunoglobulin receptor (IgM or IgD) and the production of immunoglobulin isotypes IgM, IgG, IgA, and IgE [ 1 ]. Regulatory B cells (Breg) represent less than 1% of peripheral blood mononuclear cells. They secrete interleukin (IL)-10, IL-35, and transforming growth factor beta (TGF-β), inhibit antigen presentation by monocytes and macrophages, and exert immunomodulatory functions by suppressing inflammation [ 1 ]. B-1a lymphocytes were first described by Lee Herzenberg in mice [ 2 , 3 ]. They originate from Lin-CD34 + CD38 low hematopoietic stem cells [ 4 ]. In humans, they are found mainly in the peritoneal and pleural cavities, where they exhibit features of activated lymphoid cells, including larger size and greater cytoplasmic complexity [ 2 ]. During fetal and neonatal life, B-1a cells predominate in umbilical cord blood, representing 60–80% of total B cells, but their proportion decreases progressively with age [ 2 , 3 ]. B-1a lymphocytes are distinguished from B-1b cells by expression of the CD5 antigen, which is absent in the latter [ 1 ]. They contribute to innate immunity, constitutively express antigen-presentation and co-stimulatory molecules such as MHC-II, CD80, and CD86, and can perform phagocytosis similarly to macrophages via clearance receptors, complement receptors, Fc receptors, and integrins [ 1 , 5 , 6 ]. They provide a first line of defense against infectious microorganisms and are considered the sole immune protection against encapsulated bacteria. Zimecki et al. reported that B-1 cells may surpass conventional B-2 lymphocytes in antigen presentation, eliciting greater proliferation of antigen-specific T cells [ 7 ]. B-1a lymphocytes produce natural antibodies in a T-independent manner and contribute to barrier immunity by promoting isotype switching to IgA, thereby aiding in the control of microorganisms at mucosa-associated lymphoid tissues. They also cross-react with epitopes on apoptotic and senescent cells, contributing to their clearance and maintaining immune homeostasis [ 8 , 9 ]. Griffin et al. identified a novel B-1a phenotype in peripheral and umbilical cord blood, characterized by coexpression of CD20⁺CD27⁺CD43⁺ antigens and the absence of CD70; notably, 70% of these cells also expressed CD5 [ 10 ]. B-1a lymphocytes maintain their population through self-renewal of pre-existing cells, a capacity that declines with age, as part of the phenomenon known as immunosenescence. This age-related change is associated with increased susceptibility to infections and chronic non-communicable diseases [ 11 ]. Immunosenescence as a dynamic process can be modified by nutritional and pharmacological interventions. Components of both innate and adaptive immunity may be renewed or activated through cytokines and other immunomodulatory agents [ 12 ]. Biomodulina T ® (BT) is a Cuban biological immunomodulator, registered in 1994 product from the Biopreparations Center (BioCen) registered in 1994 for the treatment of recurrent infections in older adults. It is a biological immunomodulator of natural origin, not derived from blood and composed of specific fractions of natural bovine thymus polypeptides [ 13 ]. Previous studies have reported its benefits in reducing the frequency and severity of infections in both children and adults [ 14 , 15 ], including patients with thymic hypoplasia, chronic obstructive pulmonary disease, and immune system deterioration associated with cancer [ 16 – 20 ]. Recently, BT demonstrated safety and efficacy in older adults during the COVID-19 epidemic in Cuba [ 21 , 22 ]. This study aimed to evaluate the effect of BT on B-1a lymphocyte subpopulations in older Cuban adults. To address this objective, we carried out a clinical investigation in a cohort of institutionalized older adults. 2. Methods 2.1 Design and subjects This study was derived from an uncontrolled, non-randomized, experimental clinical trial entitled “Evaluation of the efficacy and safety of a new dosage regimen of Biomodulina T® for the prevention of infections, including COVID-19, in older adults in Cuba”, sponsored by the Biopreparations Center (BioCen) and coordinated by the National Clinical Trials Coordinating Center (CENCEC). The trial is publicly registered at http://registroclinico.sld.cu/ensayos/RPCEC00000319-Sp . For the present analysis, conducted in March 2020, the source population comprised 68 residents of the “Alfredo Gómez Gendra” Nursing Home in Havana, Cuba. Based on predefined inclusion and exclusion criteria, 30 older adults aged 60–99 years were selected. 2.2 Inclusion criteria Older adults aged 60 years or above, of any sex and skin color, who provided written informed consent to participate in the study. 2.3 Exclusion criteria Older adults were excluded if they had received BT in the previous two months; had known hypersensitivity to any component of the formulation; presented with acute allergic conditions or a history of severe allergic reactions; or had uncontrolled intercurrent illnesses, including acute infections with fever, symptomatic heart failure, unstable angina, or were receiving immunosuppressive treatment. 2.4 Treatment Participants received intramuscular BT at a dose of 3 mg (one vial) twice weekly for six weeks. Each participant served as their control, with baseline measurements obtained before BT administration to evaluate the potential immunomodulatory effect on the selected lymphocyte populations. 2.5 Obtaining and procedure of the biological sample From each participant, 3 mL of peripheral blood was collected in EDTA-containing Vacutainer® tubes at three time points: before BT treatment, one week after completing the six-week regimen (week 7), and six months later. B-1a lymphocyte subpopulations (CD19⁺CD5⁺, CD20⁺CD5⁺, and CD19⁺CD20⁺) were quantified by flow cytometry at the Department of Immunochemistry and Immunology, Institute of Hematology and Immunology, Havana, Cuba. For staining, 50 µL of whole blood were placed in 15 mL tubes, and 5 µL of fluorochrome-conjugated monoclonal antibodies were added: anti-CD20-FITC, anti-CD5-PE, anti-CD19-APC (DaKo Multi Mix™), and anti-CD45-PerCP (Miltenyi Biotec). Samples were incubated at room temperature (RT) for 20 minutes, protected from light. Red blood cells were lysed with lysing solution for 10 minutes at RT, then washed twice with 0.9% sodium chloride and centrifuged for 10 minutes at 1500 rpm Data acquisition was performed on a Gallios™ Beckman Coulter flow cytometer, collecting 100,000 events per tube. Analysis was carried out using Kaluza Analysis® software version 1.2. The lymphocyte subpopulations were compared against previously established reference values: ≤12% for CD19⁺CD5⁺ and CD20⁺CD5⁺ B lymphocytes, and 5–15% for CD19⁺CD20⁺ cells [ 23 ]. Gating strategy included initial singlet selection using FSC-H versus FSC-A to exclude aggregates (Fig. 1 A), followed by lymphocyte identification on SSC-A versus CD45-PerCP-A plots (Fig. 1 B). Subsequent analyses of CD45⁺ lymphocytes included: (C) CD19-APC-A/CD5-PE-A for B-1a CD19⁺CD5⁺ cells, (D) CD20-FITC-A/CD5-PE-A for B-1a CD20⁺CD5⁺ cells, and (E) CD19-APC-A/CD20-FITC-A for CD19⁺CD20⁺ cells. 2.6 Statistical processing A database was created in Microsoft Excel to store the counts and percentages of CD5⁺CD19⁺, CD5⁺CD20⁺, and CD19⁺CD20⁺ B-1a lymphocytes. The Shapiro–Wilk test was used to assess the normality of data distribution [ 24 ]. Descriptive statistics included the median and standard deviation (SD) at three points: baseline (before treatment), six weeks after treatment initiation, and six months after completing BT therapy. Binary logistic regression was applied to estimate the odds of observing an increase (yes/no) in the percentage or absolute count of CD5⁺CD19⁺ lymphocytes after treatment, compared to baseline values. Odds ratios (OR) and 95% confidence intervals (CI) were calculated using the compare Groups package in the R statistical environment [ 25 ]. A significance threshold of p ≤ 0.05 was adopted. All statistical analyses were performed using GraphPad Prism software, version 9.5.0. 2.7 Bioethical aspects The study was conducted in accordance with the principles of the Declaration of Helsinki of the World Medical Association, last updated at the 64th General Assembly, Fortaleza, Brazil, 2013 [ 26 ]. The protocol was reviewed and approved by the Research Ethics Committee of the Center for Research on Longevity, Aging, and Health (CITED). All participants’ data were kept confidential, and no conflicts of interest were declared among the investigators. 3. Results The most common comorbidities among participants were cardiovascular disease (n = 19, 63.3%), dementia (46.7%), cerebrovascular disease (30%), diabetes mellitus (26.7%), chronic obstructive pulmonary disease (20%), bronchial asthma (10%), and neoplasms (6.7%). Analysis of the percentage of B1a CD5⁺CD19⁺ lymphocytes showed a slight decrease in the median value (5.48 vs 4.83; SD 7.77 vs 4.24) after six weeks of BT treatment, whereas the absolute count increased (57.87 vs 79.78; SD 193.7 vs 70.91), without reaching statistical significance. At six months post-treatment, both the percentage and absolute count were higher than baseline (5.48 vs 8.38; SD 7.77 vs 7.58, and 57.87 vs 139.9; SD 193.7 vs 128.5, respectively), although these differences were not statistically significant (Table). Analysis of B1a CD5⁺CD20⁺ lymphocytes showed an increase in both percentage and absolute count after six weeks of BT treatment (0.96 vs 2.11; SD 6.02 vs 2.38, and 17.02 vs 40.58; SD 80.09 vs 39.70), with statistical significance (p = 0.026 and p = 0.014, respectively). These values remained elevated at six months post-treatment (0.96 vs 2.12; SD 6.02 vs 4.28, and 17.02 vs 38.91; SD 80.09 vs 62.86), although without statistical significance. In contrast, the percentage and absolute count of lymphocytes co-expressing CD19⁺ and CD20⁺ antigens decreased after six weeks of BT treatment (4.87 vs 1.94; SD 4.69 vs 4.57, and 88.48 vs 31.03; SD 62.14 vs 65.54), with statistical significance (p = 0.014 and p = 0.018, respectively). Six months after treatment, both parameters were higher than baseline (4.87 vs 8.47; SD 4.69 vs 26.69, and 88.48 vs 173.5; SD 62.14 vs 323.8), with statistical significance (p = 0.012 and p = 0.011, respectively) (Table 1 ). Table 1 Distribution of B1a lymphocytes in older adults before and after treatment with Biomodulina T® Immunophenotype % /cells/µL (RRV) Timepoint Median before (SD) Median after (SD) Absolute Δ OR (95% CI) p-value CD5⁺CD19⁺ % (≤ 12%) 6 weeks 5.48 (7.77) 4.83 (4.24) − 0.65 0.96 [0.88–1.04] 0.976 6 months 8.38 (7.58) + 2.9 1.05 [0.97–1.12] 0.09 CD5⁺CD19⁺ (cells/µL) (26.2–40.1) 6 weeks 57.87 (193.7) 79.78 (70.91) + 21.91 1.00 [0.99–1.00] 0.701 6 months 139.9 (128.5) + 82.03 1.00 [1.00–1.00] 0.121 CD5⁺CD20⁺ % (≤ 12%) 6 weeks 0.96 (6.02) 2.11 (2.38) + 1.15 0.97 [0.87–1.09] 0.026* 6 months 2.12 (4.28) + 1.16 1.02 [0.92–1.12] 0.131 CD5⁺CD20⁺ (cells/µL) (26.2–40.1) 6 weeks 17.02 (80.09) 40.58 (39.70) + 23.56 1.00 [0.99–1.01] 0.014* 6 months 38.91 (62.86) + 21.89 1.00 [0.99;1.01] 0.299 CD19⁺CD20⁺ % (5.4–49.5) 6 weeks 4.87 (4.69) 1.94 (4.57) − 2.93 0.91 [0.81–1.02] 0.014* 6 months 8.47 (26.69) + 3.6 1.10 [1.02–1.18] 0.012* CD19⁺CD20⁺ cells/µL (114–1491) 6 weeks 88.48 (62.14) 31.03 (65.54) − 57.45 0.99 [0.98–1.00] 0.018* 6 months 173.5 (323.8) + 85.02 1.01 [1.00–1.01] 0.011* Abbreviations : RRV: range of normal reference values; SD: standard deviation; OR: odds ratios; CI: confidence interval; * statistically significant differences between the groups before and after treatment. 4. Discussion Older adults are more susceptible to recurrent infections, neurodegenerative disorders, cardiovascular diseases, and neoplasms of mature B cells, such as small lymphocytic lymphoma, diffuse large B-cell lymphoma, and mantle cell lymphoma [ 27 ]. With advancing age, both the composition and function of the immune system undergo significant changes. One of the most evident effects on the B-cell compartment is the marked reduction in circulating B lymphocytes [ 28 , 29 ]. Several studies have reported a decline in B1a CD5⁺CD19⁺ and CD5⁺CD20⁺ lymphocytes in older adults, particularly in those over 80 years of age [ 30 , 31 ]. A deficiency in CD5⁺CD19⁺ B lymphocytes has also been reported in the peripheral blood of patients with inflammatory bowel diseases, including ulcerative colitis and Crohn’s disease. In these patients, an inverse correlation has been observed between lymphocyte counts and disease activity, suggesting a loss of immunological tolerance to intestinal mucosal antigens—a phenomenon that appears to be more pronounced in the elderly [ 32 – 34 ]. B1a lymphocytes can function similarly to regulatory B cells, as they produce high concentrations of anti-inflammatory cytokines such as interleukins − 3, -10, and − 35 [ 2 , 8 ]. The decline in circulating B cells in the peripheral blood of older adults is primarily due to reduced lymphopoiesis in the bone marrow. This reduction in naïve B cells is accompanied by an expansion of memory B cells, which exhibit impaired differentiation into plasma cells, hallmarks of the immunosenescence phenotype, and are associated with the development of age-related diseases [ 21 , 29 ]. In a previous study, elderly Cubans aged 80 years or older showed lower absolute counts of CD19⁺CD20⁺ B lymphocytes [ 21 ]. Treatment with BT has proven effective in older Cuban adults with recurrent infections [ 21 , 22 ] with a significant increase in the percentage of total CD19⁺ B lymphocytes. Ramos HE and colleagues demonstrated that, in elderly individuals treated with BT and the VAMENGOC-BC® vaccine either separately or in combination, BT significantly increased CD19⁺ antigen expression [ 35 ]. Similarly, Hernández et al. reported that BT administration in older Cuban adults led to significant increases in both the percentage and absolute count of total CD19⁺ B lymphocytes six weeks after completing treatment, with this effect persisting for six months [ 21 ]. These findings supported the large-scale intervention carried out in Cuba during the COVID-19 epidemic, in which BT was administered to institutionalized older adults, contributing to reduced morbidity and mortality from SARS-CoV-2 infection [ 21 , 22 ]. In this study, BT administration was associated with a decrease in the percentage of CD5⁺CD19⁺ B lymphocytes at six weeks, although the change did not reach statistical significance. By six months, however, this percentage had increased relative to baseline, showing a trend toward significance (p = 0.09), suggesting a possible delayed immunostimulatory effect in older adults. The initial decline observed at six weeks warrants further investigation, particularly in elderly individuals with polyclonal or monoclonal B lymphoproliferative disorders coexpressing CD5⁺ and CD19⁺ antigens. In contrast, BT significantly increased both the percentage and absolute count of B1a CD5⁺CD20⁺ lymphocytes six weeks after treatment completion. Although these parameters remained elevated at six months, the differences were no longer statistically significant. CD5⁺CD20⁺ cells represent a phylogenetically old B-cell subset responsible for producing IgM antibodies of low specificity and affinity. The persistence of this fetal-like phenotype has been associated with delayed maturation of the humoral immune system, potentially increasing susceptibility to infections [ 36 ]. In the present study, BT administration led to a reduction in both the percentage and absolute count of CD19⁺CD20⁺ B lymphocytes after six weeks of treatment. However, six months after completing therapy, both parameters increased to levels exceeding baseline values. These findings are consistent with previous reports (21,22) regarding the persistence of BT-induced effects on B- and T-lymphocyte populations in older adults for up to at least six months, suggesting that additional treatment cycles may be required depending on individual clinical needs. Aging reduces the diversity of the B-cell repertoire by influencing the selection process during affinity maturation. Age-related changes typically include a decline in naïve B cells and an expansion of memory B cells, which compromise the immune system’s ability to respond to novel antigens. Moreover, certain aged B-cell subsets exhibit increased mitochondrial mass and elevated mitochondrial reactive oxygen species, reflecting mitochondrial dysfunction. These alterations result in reduced energy production and impairments in one-carbon metabolism—processes essential for amino acid and nucleotide synthesis, as well as for antibody production and B-cell activation [ 37 ]. B1a cells are recognized for their protective role against viral, bacterial, and parasitic infections through the production of natural IgM antibodies. Increasing evidence also suggests that these cells, particularly the highly polyspecific subsets, may participate in autoimmune pathogenesis. Their involvement in controlling several bacterial infections has been demonstrated [ 38 ]. In the present study, none of the elderly participants developed infections during the follow-up period. Although the absence of infections could suggest improved resistance and a lower tendency toward inflammatory responses, this observation should be interpreted with caution. It may be consistent with the regulatory role attributed to B1a lymphocytes, but further studies are needed to confirm this relationship. Overall, the findings suggest that BT may exert a modulatory influence on different B-cell subpopulations, CD5⁺CD19⁺, CD5⁺CD20⁺, and CD19⁺CD20⁺, with the direction and magnitude of these changes appearing to depend on the duration of treatment. To contextualize our findings, Table 2 summarizes results from previous Cuban studies evaluating the immunomodulatory role of BT in different clinical settings. Table 2 Immunomodulatory effect of Biomodulin T® on different cell populations and clinical scenarios in Cuban subjects Study design Human subjects Dosage Outcomes p value Prospective, controlled. n = 34 [ 14 ] Pediatric patients with recurrent infections. 3 mg (one vial), IM, twice a week for 8 weeks. Increase of the Thymus area. Decrease in the frequency and severity of infections. Increase of the postural parameters. < 0.005 n.s. n.s. Presentation of a clinical case [ 16 ] Pediatric patient with cystic fibrosis and severe thymic hypoplasia. 3 mg (one vial), IM, three times a week for 8 weeks. Increase of the Thymus area. Normalization of the pondoestatural parameters. Decrease in the frequency and severity of infections. Not evaluated Prospective, uncontrolled, multicenter, clinical trial. n = 60 [ 17 ] Pediatric patients with thymic hypoplasia. 3 mg (one vial), IM, *frequency according to the thymic area for 8 weeks. Increase of the Thymus area. Increase in T CD3 + CD4 + lymphocytes. Increase in IgA. Decrease in the frequency and severity of infections. < 0.0001 < 0.05 0.013 0.0001 Prospective, uncontrolled. n = 31 [ 18 ] Older adult patients with recurrent respiratory infections. 3mg (one vial), IM n = 15 once a week n = 16 two times a week, both cases for 6 weeks. Increase in T Naïve CD3+/CD4 + lymphocytes. Increase CD4 + recent thymic emigrants. Increase CD8 + recent thymic emigrants. Increase CD8 + stem cells-like memory. Decreased expression of CD4 + PD-1 + T cells. Decreased expression of CD8 + PD-1 + T cells. Increase of Ki67 on CD4 + T lymphocytes. Increase of Ki67 on CD8 + T lymphocytes. Increase in intracellular expression of INF-γ in CD4 + T lymphocytes. (< 0.05) ' (< 0.05) '/'' (< 0.05) ' (< 0.05) ' [< 0.05] ' (< 0.05) ' (< 0.0001) ' (< 0.05) '/'' (< 0.05) '' (< 0.05) Prospective, uncontrolled. n = 80 [ 19 ] Adult patients with advanced lung cancer after finishing first-line platinum-based chemotherapy (4–6 cycles). 3 mg (one vial), IM, three times a week for 4 weeks. Decrease of terminally differentiated CD45RA effector T cells (EMRA) CD4+. Decrease of terminally differentiated CD45RA effector T cells (EMRA) CD8+. Decreased expression of CD4 + PD-1 + T cells. Decreased expression of CD8 + PD-1 + T cells. p = 0.0031 p = 0.0372 0.0005 p < 0.0001 Prospective, uncontrolled. n = 80 [ 20 ] Adult patients with advanced lung cancer after finishing first-line platinum-based chemotherapy (4–6 cycles). 3 mg (one vial), IM, three times a week (4–6 weeks). Decrease of T CD3 + CD4 + CD8 + CD57+ Lymphocytes. Decrease of CD57 + NK cells. n.s. 0.042 Prospective, uncontrolled, non-randomized, experimental clinical trial. n = 30 [ 21 ] Elderly adults institutionalized. 3 mg (one vial), IM, twice a week for 6 weeks. Increase in T Naïve CD3+/CD4+/CD27+/CD45RA + lymphocytes. Increase T central memory CD3+/CD4+/CD27+/CD45RA-. Increase in of T Naïve CD3+/CD8+/CD27+/CD45RA + lymphocytes. Increase in T central memory CD3+/CD8+/CD27+/CD45RA-. Increase in NK CD3-CD16+/CD56 + cells. Increase in CD3+/HLA-/DR+. Increase in % of CD3+/CD25+. Increase in B lymphocytes. (0.023**) (0.040***) (0.002***) [0.017***] (0.031***) [0.031***] (0.029***) [0.024***] (< 0.001***) [0.001***] (< 0.001**) [< 0.001**] (< 0.001**) [< 0.001**] (< 0.001***) [< 0.001***] (0.002**) [0.005**] (< 0.001***) [0.003***] Prospective, controlled, randomized, experimental clinical trial. n = 60l [ 35 ] Elderly adults. 3 mg (one vial), IM, three times a week one week. Increase in B lymphocytes. Increase of CD3 + CD56 + T lymphocytes. 0.005 0.007 Abbreviations : n: studied subjects; n.s: no significant statistical difference; IM:intramuscular; ( ): percentage; [ ]: cells per µL; p < 0.05: value statistically significant differences between the groups before and after treatment, NK: natural killer; * thymus area 500 800 ≤ 100 mm 2 once a week; **: after 6 weeks of treatment; ***: after 6 months of treatment; INF-γ: interferon gamma; ': a dose; ': two doses. These investigations have consistently demonstrated immunomodulatory effects of BT in diverse clinical contexts, including pediatric patients with recurrent infections, individuals with thymic hypoplasia, older adults with chronic respiratory diseases, and cancer patients after chemotherapy. Our findings add to this body of evidence by specifically addressing the behavior of B1a lymphocyte subpopulations in institutionalized elderly adults. Unlike earlier reports that focused predominantly on T-cell and global B-cell responses, this study provides new insights into the modulation of B1a subsets (CD5⁺CD19⁺, CD5⁺CD20⁺, CD19⁺CD20⁺), highlighting both the short-term increases and the long-term persistence of certain changes after BT treatment. The potential mechanisms underlying these immunomodulatory effects may include the enhancement of CD4⁺ TH1 T-cell number and function, the inhibition of CTLA-4 and PD-1 expression on B1a cells, and the reduction of CD4⁺FOXP3⁺ regulatory T cells, which collectively could favor increased IFN-γ production. Similar pathways have been proposed in previous studies of BT and other thymic peptides, suggesting that modulation of checkpoint molecules and regulatory populations may play a central role in counteracting immunosenescence. Further mechanistic studies will be necessary to confirm these hypotheses and to clarify the molecular pathways through which BT modulates B1a lymphocytes in the context of aging. At the same time, some limitations of this study should be acknowledged. From the total population of institutionalized older adults in the selected nursing home, only 44.1% met the predefined inclusion and exclusion criteria and were enrolled. This limited recruitment restricts the generalizability of the findings to broader elderly populations. Future studies should aim to replicate these results in larger and more diverse cohorts, ideally across multiple institutions, to confirm the reproducibility and external validity of the observed effects. 5. Conclusions BT demonstrated immunomodulatory effects on B1a lymphocytes in elderly Cuban adults, with changes partially sustained for up to six months after a six-week treatment regimen. This effect was observed in a population particularly susceptible to immunosenescence, highlighting the potential of BT as an immune-supportive intervention. The findings suggest possible clinical applications in the prevention and management of infections, autoimmune diseases, and B-cell lymphoproliferative disorders in older adults. Despite these encouraging findings, confirmation in multicenter, randomized controlled trials with larger and more diverse populations is required to validate these results, explore long-term outcomes, and establish optimal dosing strategies. In parallel, mechanistic studies are recommended to further elucidate the pathways through which BT modulates B1a lymphocyte activity in the context of aging. Declarations Funding: This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Author Contributions: Vianed Marsán Suárez: conceptualization of the research, immunophenotyping by flow cytometry, analysis, interpretation of the results, writing of the manuscript and approval of the final report. Imilla Casado Hernández: analysis of the results, review of the manuscript, and approval of the final report. Elizabeth Hernández Ramos: immunophenotyping by flow cytometry, statistical analysis, and approval of the final report. Yenisey Triana Marrero, Yaneisy Duarte Pérez, Eduardo Alfonso Hernández Marsán, and Mibelys Santana García: immunophenotyping by flow cytometry and approval of the final report. Alexis Labrada Rosado, Gisela María SuárezFormigó, and Yendry Ventura Carmenate: manuscript review, editing, and approval of the final report. Declaration of competing interest: The authors declare that they have no conflicts of interest regarding the research, authorship, and/or publication of this article References Rastogi I, Jeon D, Moseman JE, et al. 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The role of CD5 B cells in the presentation of antigen to antigen-specific T cells. Arch Immunol Ther Exp. 1994;42(5–6):349–53. Berland R, Wortis HH. Origins and functions of B-1 cells with notes on the role of CD5. Annu Rev Immunol. 2002;20:253–300. Kroese FG, Butcher EC, Stall AM, et al. Many of the IgA producing plasma cells in murine gut are derived from self-replenishing precursors in the peritoneal cavity. Int Immunol. 1989;1:75–84. Griffi DO, Holodick NE, Rothstein TL. Human B1 cells in umbilical cord and adult peripheral blood express the novel phenotype CD20 + CD27 + CD43 + CD70-. J Exp Med. 2011;208(1):67–80. https://doi:10.1084/jem.20101499 . Qorbani A, Gao G, Dwyre DM. Polyclonal CD5+/CD19 + B1a lymphocytes after allogeneic stem cell transplantation: a potential diagnostic pitfall. Autops Case Rep. 2020;10(2):e2020147. https://doi.org/10.4322/acr.2020.147 . Tabibian-Keissar H, Hazanov L, Schiby G, et al. Aging affects B‐cell antigen receptor repertoire diversity in primary and secondary lymphoid tissues. Eur J Immunol. 2016;46(2):480–92. Centro para el. control estatal de medicamentos, e. y. d. m. C. Biomodulina t®. RESUMEN de Las CARACTERISTICAS DEL PRODUCTO; 2015. Christian LD, Rodríguez RR, Rabassa J, et al. Efecto de la Biomodulina T ® 1000 sobre el timo en niños con infecciones recurrentes. Revista Cubana de Pediatría. 2000;72(1):3–9. García M, Capdevila V, Suárez R. Infecciones respiratorias altas y la polifarmacia del anciano. Revista Habanera de Ciencias Médicas. 2014;13(3):425–36. de la Guardia O, Ustariz C, García MA et al. Hipoplasia tímica en un niño con fibrosis quística. Rev Cubana Hematol Inmunol Hemoter [Internet]. 2014 [citado 12/06/2018];30(1):74–80. Disponible en: http://scielo.sld.cu/pdf/hih/v30n1/hih10114 de la Guardia OM, Labrada A, Macías C et al. Seguridad de la Biomodulina T en niños con hipoplasia del timo. Revista Cubana de Hematología, Inmunología y Hemoterapia. 2025;41: e2088. Disponible en: https://creativecommons.org/licenses/by-nc/4.0/deed.es_ES Saavedra D, Fuertes S, Suarez GM, et al. Biomodulina T partially restores immunosenescent CD4 and CD8 T cell compartments in the elderly. Exp Gerontol. 2019;124:110633. https://doi.org/10.1016/j.exger.2019.110633 . Suárez GM, Catalá M, Peña Y, et al. Thymic Polypeptide Fraction Biomodulina T Decreases Exhausted and Terminally Differentiated EMRA T Cells in Advanced Lung Cancer Patients Treated With Platinum-Based Chemotherapy. Front Oncol. 2022;12:823287. https://doi:10.3389/fonc.2022.823287 . Suárez GM, Catalá M, Peña Y, et al. Assessment of non-classical lymphocyte populations in patients with advanced lung cancer treated with Biomodulina T following platinum-based chemotherapy. Explor Immunol. 2024;4:433–45. https://doi.org/10.37349/ei.2024.00150 . Hernández IC, Suárez VM, Ramos EH, et al. BIOMODULINA T® Modulates Lymphocyte Compartments in Institutionalized Cuban Geriatric Patients. J Cell Immunol. 2022;4(2):79–91. Reyes MC, Almarales SR, Iglesias IR, et al. Seguridad de la Biomodulina T ® en la población cubana antes y durante su uso en la prevención de la COVID-19. Rev Habanera de Ciencias Médicas. 2023;22(2):5078. Kokuina E, Breff C, Villegas CA, et al. Normal values of T, B and NK lymphocytes subpopulations in peripheral blood of healthy Cuban adults. MEDICC Rev. 2019;21(2–3):16–21. Shapiro S, Wilk MB, Chen HJ. A Comparative Study of Various Tests of Normality. J Am Stat Assoc. 1968;63:1343–72. https://doi.org/10.1080/01621459.1968.10480932 . Subirana I, Sanz H, Vila J. Building Bivariate Tables: The compare Groups Package for R. J Stat Softw. 2014;57(12):1–16. https://doi.org/10.18637/jss.v057.i12 . Declaración de Helsinki de la AMM. Principios éticos para las investigaciones médicas en seres humanos. 64ª Asamblea Gen [Internet] Fortaleza, Brasil; 2013. Disponible en: http://www.wma.net/es/30publications/10policies/b3/ Ariev A, Safonova Y, Diachkova-Gerceva D. Geriatric syndromes: the importance of the problem, controversial issues of terminology. Adv Gerontol. 2024;37(1–2):60–6. Gibson KL, Chang WY, Barnett Y et al. B-cell diversity decreases in old age and is correlated with poor health status Aging Cell. 2009; 8:18–25 10.1111/j.1474-9726.2008 . 00443.x. Bulatti M, Caruso C, Colonma-Romano G. From lymphopoiesis to plasma cells differentiation, the age-related modifications of B cell compartment are influenced by Inflamm-Ageing. Ageing Res Rev. 2017;36:125–36. http://dx.doi.org/10.1016/j.arr.2017.04.001 S1568-1637(17)30037-5 . Diponible en:. Marsán V, Casado I, Hernández E, et al. Immunophenotypic characterization of B1a lymphocytes in Cuban older adults. Exp Gerontol. 2022;167:111900. García B, Saavedra D, Lorenzo P, et al. Immunosenescence and gender: a study in healthy Cubans. Immun Ageing. 2013;10:16. Disponible en:. http://www.immunityageing.com/content/10/1/16 . Polese L, Boetto R, De Franchis G, et al. B1a lymphocytes in the rectal mucosa of ulcerative colitis patients. World J Gastroenterol. 2012;18(2):144–9. Disponible en: URL:. http://www.wjgnet.com/1007-9327/full/v18/i2/144 . Gil R, García C, Benet C et al. B1a Lymphocytes (CD19 + CD5+) Deficiency in Patients with Crohn’s Disease and Its Relationship with Disease Severity. Dig Dis 2018:1–8. 10.1159/000486893 Marks DJ, Segal AW. Innate immunity in inflammatory bowel disease: a disease hypothesis. J Pathol. 2008;214:260–6. Ramos EH, Suárez VM, Hernández IC, et al. Effect of Biomodulina-T ® and VA-MENGOC-BC ® on lymphocyte subpopulations in older adults. Exp Gerontol. 2021;153:111497. Wasik M, Kaczorowska M, Demkow U. Altered expression of immune surface markers in children with recurrent infections of respiratory tract. J Physiol Pharmacol. 2005;56(Suppl 4):237–43. PMID: 16204799. Goyani P, Christodoulou R, Vassiliou E, Immunosenescence. Aging and Immune System Decline. Vaccines. 2024;12:1314. https://doi.org/10.3390/vaccines12121314 . Baumgarth N. The double life of a B-1 cells self-reactivity selects for protective effector functions. Nat Rev Immunol. 2011;11:34–46. 10.1038/nri2901 . [DOI] [PubMed]. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8649614","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":579751225,"identity":"77a97f87-281d-4c79-99df-24f83ff1463e","order_by":0,"name":"Vianed Marsan Suarez","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8klEQVRIiWNgGAWjYDACZhDBBuNVMDA2kKjlDDFaGJC1MLYRocXgOPOzBx/KGOwNjp99+PDnPDvZ+e2HHzD8qMCj5TCbueGMcwyJG86kGxtIbks23nAmzYCx5wxuLZLNDGbSvG0MCQYH0tgkDLcxJ26Q4GFgAIrg0cL+TfpvG9Bh55+x/0icU584fwYPA+Pff7i18DPzmEmDfL3hRhobw8GGw4kNN3gYmHnxBAJQS5lkzzmJxJk3njFLNhw7DvbLYZljuLWw8R/fJvGjzMae73wa48cfNdWgEHv48E0Nbi1QIIHKPUBQwygYBaNgFIwCvAAA1l1NJAfHj1oAAAAASUVORK5CYII=","orcid":"","institution":"Abu Dhabi Stem Cells Center","correspondingAuthor":true,"prefix":"","firstName":"Vianed","middleName":"Marsan","lastName":"Suarez","suffix":""},{"id":579751226,"identity":"39cfa3ba-46ca-478c-8740-130951b2c79d","order_by":1,"name":"Imilla Casado Hernandez","email":"","orcid":"","institution":"Abu Dhabi Stem Cells Center","correspondingAuthor":false,"prefix":"","firstName":"Imilla","middleName":"Casado","lastName":"Hernandez","suffix":""},{"id":579751227,"identity":"bb920138-c62f-40de-bf00-e11a5b7ec59a","order_by":2,"name":"Elizabeth Hernández Ramos","email":"","orcid":"","institution":"Institute of Hematology and Immunology","correspondingAuthor":false,"prefix":"","firstName":"Elizabeth","middleName":"Hernández","lastName":"Ramos","suffix":""},{"id":579751228,"identity":"c7f06371-c0a3-4b89-aa0a-add783be1710","order_by":3,"name":"Yenisey Triana Marrero","email":"","orcid":"","institution":"Institute of Hematology and Immunology","correspondingAuthor":false,"prefix":"","firstName":"Yenisey","middleName":"Triana","lastName":"Marrero","suffix":""},{"id":579751229,"identity":"5544b80e-b051-4b88-9965-07ab7949c8e7","order_by":4,"name":"Yaneisy Duarte Pérez","email":"","orcid":"","institution":"Institute of Hematology and Immunology","correspondingAuthor":false,"prefix":"","firstName":"Yaneisy","middleName":"Duarte","lastName":"Pérez","suffix":""},{"id":579751230,"identity":"4258e9c8-d6b6-4c5d-9815-e7014df66236","order_by":5,"name":"Eduardo Alfonso Hernández Marsán","email":"","orcid":"","institution":"Faculty of Medical Sciences “Manuel Fajardo","correspondingAuthor":false,"prefix":"","firstName":"Eduardo","middleName":"Alfonso Hernández","lastName":"Marsán","suffix":""},{"id":579751231,"identity":"a32b4f37-8b23-4cff-9e05-6f87dbe4d4fd","order_by":6,"name":"Mibelys Santana García","email":"","orcid":"","institution":"Institute of Hematology and Immunology","correspondingAuthor":false,"prefix":"","firstName":"Mibelys","middleName":"Santana","lastName":"García","suffix":""},{"id":579751232,"identity":"f5e87722-43bd-4a83-9afa-e42bd3f347f4","order_by":7,"name":"Alexis Labrada Rosado","email":"","orcid":"","institution":"National Center for Biopreparations (BioCen)","correspondingAuthor":false,"prefix":"","firstName":"Alexis","middleName":"Labrada","lastName":"Rosado","suffix":""},{"id":579751233,"identity":"db368c09-c3f0-4f22-a62e-fa6d83bb6533","order_by":8,"name":"Gisela María Suárez Formigó","email":"","orcid":"","institution":"Abu Dhabi Stem Cells Center","correspondingAuthor":false,"prefix":"","firstName":"Gisela","middleName":"María Suárez","lastName":"Formigó","suffix":""},{"id":579751234,"identity":"514a85f5-d2f9-4b89-b7f0-acc65ab93631","order_by":9,"name":"Yendry Ventura Carmenate","email":"","orcid":"","institution":"Abu Dhabi Stem Cells Center","correspondingAuthor":false,"prefix":"","firstName":"Yendry","middleName":"Ventura","lastName":"Carmenate","suffix":""}],"badges":[],"createdAt":"2026-01-20 13:30:59","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8649614/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8649614/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":101201278,"identity":"e2d54b58-cba5-434a-835b-d759e46a8127","added_by":"auto","created_at":"2026-01-27 09:14:49","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":284299,"visible":true,"origin":"","legend":"\u003cp\u003eGate analysis strategy for the selection of B1a lymphocytes. A): FSC-H/FSC-A dot plot; singlet selection. B): Dot plot SSC-A/CD45PerCP-A; selection of lymphocytes. C): CD19APC-A/CD5PE-A dot plot; selection of B1a CD19+CD5+ lymphocytes (1.15%). D): Dot plot CD20FITC/CD5PE; selection of B1a CD20+CD5+ lymphocytes (0.21%) and E): Dot plot CD19APC/CD20FITC; selection of CD19+CD20+ B lymphocytes (3.81%).\u003c/p\u003e","description":"","filename":"Figure0.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8649614/v1/902ff347ae52adb0adcc9312.jpg"},{"id":101513935,"identity":"7746ef7a-9cd8-4748-821e-202615618701","added_by":"auto","created_at":"2026-01-30 15:40:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1130002,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8649614/v1/ae672f6a-687b-4e1c-b272-3655a77662c0.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Counteracting Immunosenescence: Effects of Biomodulina T® on B-1a Lymphocytes in Older Cuban Adults","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eIn humans, B lymphocytes develop in the bone marrow and, after selection and maturation, migrate to peripheral lymphoid organs such as the spleen, lymph nodes, and mucosa-associated lymphoid tissue. Their primary functions include antibody production and antigen presentation, but they can also mediate cell-based immunity by activating antigen-specific CD4⁺ and CD8⁺ T cells, as well as exert regulatory and cytotoxic effects. B lymphocytes are generally classified into three main subsets: B-1 cells, B-2 cells, and regulatory B cells (Breg). Among these, B-1a lymphocytes are of particular interest in aging, as they contribute to innate immunity and display unique phenotypic and functional characteristics that may be altered during immunosenescence. [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eB-2 lymphocytes are the most abundant subset in humans. Their development and maturation are characterized by the generation of a specific surface immunoglobulin receptor (IgM or IgD) and the production of immunoglobulin isotypes IgM, IgG, IgA, and IgE [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Regulatory B cells (Breg) represent less than 1% of peripheral blood mononuclear cells. They secrete interleukin (IL)-10, IL-35, and transforming growth factor beta (TGF-β), inhibit antigen presentation by monocytes and macrophages, and exert immunomodulatory functions by suppressing inflammation [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eB-1a lymphocytes were first described by Lee Herzenberg in mice [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. They originate from Lin-CD34\u0026thinsp;+\u0026thinsp;CD38\u003csup\u003elow\u003c/sup\u003e hematopoietic stem cells [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In humans, they are found mainly in the peritoneal and pleural cavities, where they exhibit features of activated lymphoid cells, including larger size and greater cytoplasmic complexity [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. During fetal and neonatal life, B-1a cells predominate in umbilical cord blood, representing 60\u0026ndash;80% of total B cells, but their proportion decreases progressively with age [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eB-1a lymphocytes are distinguished from B-1b cells by expression of the CD5 antigen, which is absent in the latter [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. They contribute to innate immunity, constitutively express antigen-presentation and co-stimulatory molecules such as MHC-II, CD80, and CD86, and can perform phagocytosis similarly to macrophages via clearance receptors, complement receptors, Fc receptors, and integrins [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. They provide a first line of defense against infectious microorganisms and are considered the sole immune protection against encapsulated bacteria. Zimecki et al. reported that B-1 cells may surpass conventional B-2 lymphocytes in antigen presentation, eliciting greater proliferation of antigen-specific T cells [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eB-1a lymphocytes produce natural antibodies in a T-independent manner and contribute to barrier immunity by promoting isotype switching to IgA, thereby aiding in the control of microorganisms at mucosa-associated lymphoid tissues. They also cross-react with epitopes on apoptotic and senescent cells, contributing to their clearance and maintaining immune homeostasis [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGriffin et al. identified a novel B-1a phenotype in peripheral and umbilical cord blood, characterized by coexpression of CD20⁺CD27⁺CD43⁺ antigens and the absence of CD70; notably, 70% of these cells also expressed CD5 [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. B-1a lymphocytes maintain their population through self-renewal of pre-existing cells, a capacity that declines with age, as part of the phenomenon known as immunosenescence. This age-related change is associated with increased susceptibility to infections and chronic non-communicable diseases [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eImmunosenescence as a dynamic process can be modified by nutritional and pharmacological interventions. Components of both innate and adaptive immunity may be renewed or activated through cytokines and other immunomodulatory agents [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBiomodulina T\u003csup\u003e\u0026reg;\u003c/sup\u003e (BT) is a Cuban biological immunomodulator, registered in 1994 product from the Biopreparations Center (BioCen) registered in 1994 for the treatment of recurrent infections in older adults. It is a biological immunomodulator of natural origin, not derived from blood and composed of specific fractions of natural bovine thymus polypeptides [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003ePrevious studies have reported its benefits in reducing the frequency and severity of infections in both children and adults [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], including patients with thymic hypoplasia, chronic obstructive pulmonary disease, and immune system deterioration associated with cancer [\u003cspan additionalcitationids=\"CR17 CR18 CR19\" citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Recently, BT demonstrated safety and efficacy in older adults during the COVID-19 epidemic in Cuba [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis study aimed to evaluate the effect of BT on B-1a lymphocyte subpopulations in older Cuban adults. To address this objective, we carried out a clinical investigation in a cohort of institutionalized older adults.\u003c/p\u003e"},{"header":"2. Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Design and subjects\u003c/h2\u003e \u003cp\u003eThis study was derived from an uncontrolled, non-randomized, experimental clinical trial entitled \u0026ldquo;Evaluation of the efficacy and safety of a new dosage regimen of Biomodulina T\u0026reg; for the prevention of infections, including COVID-19, in older adults in Cuba\u0026rdquo;, sponsored by the Biopreparations Center (BioCen) and coordinated by the National Clinical Trials Coordinating Center (CENCEC). The trial is publicly registered at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://registroclinico.sld.cu/ensayos/RPCEC00000319-Sp\u003c/span\u003e\u003cspan address=\"http://registroclinico.sld.cu/ensayos/RPCEC00000319-Sp\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eFor the present analysis, conducted in March 2020, the source population comprised 68 residents of the \u0026ldquo;Alfredo G\u0026oacute;mez Gendra\u0026rdquo; Nursing Home in Havana, Cuba. Based on predefined inclusion and exclusion criteria, 30 older adults aged 60\u0026ndash;99 years were selected.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Inclusion criteria\u003c/h2\u003e \u003cp\u003eOlder adults aged 60 years or above, of any sex and skin color, who provided written informed consent to participate in the study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Exclusion criteria\u003c/h2\u003e \u003cp\u003eOlder adults were excluded if they had received BT in the previous two months; had known hypersensitivity to any component of the formulation; presented with acute allergic conditions or a history of severe allergic reactions; or had uncontrolled intercurrent illnesses, including acute infections with fever, symptomatic heart failure, unstable angina, or were receiving immunosuppressive treatment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Treatment\u003c/h2\u003e \u003cp\u003eParticipants received intramuscular BT at a dose of 3 mg (one vial) twice weekly for six weeks. Each participant served as their control, with baseline measurements obtained before BT administration to evaluate the potential immunomodulatory effect on the selected lymphocyte populations.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Obtaining and procedure of the biological sample\u003c/h2\u003e \u003cp\u003eFrom each participant, 3 mL of peripheral blood was collected in EDTA-containing Vacutainer\u0026reg; tubes at three time points: before BT treatment, one week after completing the six-week regimen (week 7), and six months later. B-1a lymphocyte subpopulations (CD19⁺CD5⁺, CD20⁺CD5⁺, and CD19⁺CD20⁺) were quantified by flow cytometry at the Department of Immunochemistry and Immunology, Institute of Hematology and Immunology, Havana, Cuba.\u003c/p\u003e \u003cp\u003eFor staining, 50 \u0026micro;L of whole blood were placed in 15 mL tubes, and 5 \u0026micro;L of fluorochrome-conjugated monoclonal antibodies were added: anti-CD20-FITC, anti-CD5-PE, anti-CD19-APC (DaKo Multi Mix\u0026trade;), and anti-CD45-PerCP (Miltenyi Biotec). Samples were incubated at room temperature (RT) for 20 minutes, protected from light. Red blood cells were lysed with lysing solution for 10 minutes at RT, then washed twice with 0.9% sodium chloride and centrifuged for 10 minutes at 1500 rpm\u003c/p\u003e \u003cp\u003eData acquisition was performed on a Gallios\u0026trade; Beckman Coulter flow cytometer, collecting 100,000 events per tube. Analysis was carried out using Kaluza Analysis\u0026reg; software version 1.2. The lymphocyte subpopulations were compared against previously established reference values: \u0026le;12% for CD19⁺CD5⁺ and CD20⁺CD5⁺ B lymphocytes, and 5\u0026ndash;15% for CD19⁺CD20⁺ cells [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eGating strategy included initial singlet selection using FSC-H versus FSC-A to exclude aggregates (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), followed by lymphocyte identification on SSC-A versus CD45-PerCP-A plots (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Subsequent analyses of CD45⁺ lymphocytes included: (C) CD19-APC-A/CD5-PE-A for B-1a CD19⁺CD5⁺ cells, (D) CD20-FITC-A/CD5-PE-A for B-1a CD20⁺CD5⁺ cells, and (E) CD19-APC-A/CD20-FITC-A for CD19⁺CD20⁺ cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Statistical processing\u003c/h2\u003e \u003cp\u003eA database was created in Microsoft Excel to store the counts and percentages of CD5⁺CD19⁺, CD5⁺CD20⁺, and CD19⁺CD20⁺ B-1a lymphocytes. The Shapiro\u0026ndash;Wilk test was used to assess the normality of data distribution [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDescriptive statistics included the median and standard deviation (SD) at three points: baseline (before treatment), six weeks after treatment initiation, and six months after completing BT therapy. Binary logistic regression was applied to estimate the odds of observing an increase (yes/no) in the percentage or absolute count of CD5⁺CD19⁺ lymphocytes after treatment, compared to baseline values. Odds ratios (OR) and 95% confidence intervals (CI) were calculated using the compare Groups package in the R statistical environment [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. A significance threshold of p\u0026thinsp;\u0026le;\u0026thinsp;0.05 was adopted. All statistical analyses were performed using GraphPad Prism software, version 9.5.0.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Bioethical aspects\u003c/h2\u003e \u003cp\u003eThe study was conducted in accordance with the principles of the Declaration of Helsinki of the World Medical Association, last updated at the 64th General Assembly, Fortaleza, Brazil, 2013 [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe protocol was reviewed and approved by the Research Ethics Committee of the Center for Research on Longevity, Aging, and Health (CITED). All participants\u0026rsquo; data were kept confidential, and no conflicts of interest were declared among the investigators.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003eThe most common comorbidities among participants were cardiovascular disease (n\u0026thinsp;=\u0026thinsp;19, 63.3%), dementia (46.7%), cerebrovascular disease (30%), diabetes mellitus (26.7%), chronic obstructive pulmonary disease (20%), bronchial asthma (10%), and neoplasms (6.7%).\u003c/p\u003e \u003cp\u003eAnalysis of the percentage of B1a CD5⁺CD19⁺ lymphocytes showed a slight decrease in the median value (5.48 vs 4.83; SD 7.77 vs 4.24) after six weeks of BT treatment, whereas the absolute count increased (57.87 vs 79.78; SD 193.7 vs 70.91), without reaching statistical significance. At six months post-treatment, both the percentage and absolute count were higher than baseline (5.48 vs 8.38; SD 7.77 vs 7.58, and 57.87 vs 139.9; SD 193.7 vs 128.5, respectively), although these differences were not statistically significant (Table).\u003c/p\u003e \u003cp\u003eAnalysis of B1a CD5⁺CD20⁺ lymphocytes showed an increase in both percentage and absolute count after six weeks of BT treatment (0.96 vs 2.11; SD 6.02 vs 2.38, and 17.02 vs 40.58; SD 80.09 vs 39.70), with statistical significance (p\u0026thinsp;=\u0026thinsp;0.026 and p\u0026thinsp;=\u0026thinsp;0.014, respectively). These values remained elevated at six months post-treatment (0.96 vs 2.12; SD 6.02 vs 4.28, and 17.02 vs 38.91; SD 80.09 vs 62.86), although without statistical significance.\u003c/p\u003e \u003cp\u003eIn contrast, the percentage and absolute count of lymphocytes co-expressing CD19⁺ and CD20⁺ antigens decreased after six weeks of BT treatment (4.87 vs 1.94; SD 4.69 vs 4.57, and 88.48 vs 31.03; SD 62.14 vs 65.54), with statistical significance (p\u0026thinsp;=\u0026thinsp;0.014 and p\u0026thinsp;=\u0026thinsp;0.018, respectively). Six months after treatment, both parameters were higher than baseline (4.87 vs 8.47; SD 4.69 vs 26.69, and 88.48 vs 173.5; SD 62.14 vs 323.8), with statistical significance (p\u0026thinsp;=\u0026thinsp;0.012 and p\u0026thinsp;=\u0026thinsp;0.011, respectively) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eDistribution of B1a lymphocytes in older adults before and after treatment with Biomodulina T\u0026reg;\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eImmunophenotype\u003c/p\u003e \u003cp\u003e% /cells/\u0026micro;L\u003c/p\u003e \u003cp\u003e(RRV)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTimepoint\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eMedian before\u003c/p\u003e \u003cp\u003e(SD)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eMedian after\u003c/p\u003e \u003cp\u003e(SD)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAbsolute Δ\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eOR (95% CI)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCD5⁺CD19⁺ %\u003c/p\u003e \u003cp\u003e(\u0026le;\u0026thinsp;12%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6 weeks\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e5.48 (7.77)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e4.83 (4.24)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026minus;\u0026thinsp;0.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.96 [0.88\u0026ndash;1.04]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.976\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6 months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8.38 (7.58)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e+\u0026thinsp;2.9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.05 [0.97\u0026ndash;1.12]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.09\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCD5⁺CD19⁺\u003c/p\u003e \u003cp\u003e(cells/\u0026micro;L)\u003c/p\u003e \u003cp\u003e(26.2\u0026ndash;40.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6 weeks\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e57.87 (193.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e79.78 (70.91)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e+\u0026thinsp;21.91\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.00 [0.99\u0026ndash;1.00]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.701\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6 months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e139.9 (128.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e+\u0026thinsp;82.03\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.00 [1.00\u0026ndash;1.00]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.121\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCD5⁺CD20⁺ %\u003c/p\u003e \u003cp\u003e(\u0026le;\u0026thinsp;12%)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6 weeks\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0.96 (6.02)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2.11 (2.38)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e+\u0026thinsp;1.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.97 [0.87\u0026ndash;1.09]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.026*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6 months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2.12 (4.28)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e+\u0026thinsp;1.16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.02 [0.92\u0026ndash;1.12]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.131\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCD5⁺CD20⁺\u003c/p\u003e \u003cp\u003e(cells/\u0026micro;L)\u003c/p\u003e \u003cp\u003e(26.2\u0026ndash;40.1)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6 weeks\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e17.02 (80.09)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e40.58 (39.70)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e+\u0026thinsp;23.56\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.00 [0.99\u0026ndash;1.01]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.014*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6 months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e38.91 (62.86)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e+\u0026thinsp;21.89\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.00 [0.99;1.01]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.299\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCD19⁺CD20⁺ %\u003c/p\u003e \u003cp\u003e(5.4\u0026ndash;49.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6 weeks\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e4.87 (4.69)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.94 (4.57)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026minus;\u0026thinsp;2.93\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.91 [0.81\u0026ndash;1.02]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.014*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6 months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e8.47 (26.69)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e+\u0026thinsp;3.6\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.10 [1.02\u0026ndash;1.18]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.012*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCD19⁺CD20⁺\u003c/p\u003e \u003cp\u003ecells/\u0026micro;L\u003c/p\u003e \u003cp\u003e(114\u0026ndash;1491)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6 weeks\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e88.48 (62.14)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e31.03 (65.54)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e\u0026minus;\u0026thinsp;57.45\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e0.99 [0.98\u0026ndash;1.00]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.018*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6 months\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e173.5 (323.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e+\u0026thinsp;85.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1.01 [1.00\u0026ndash;1.01]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.011*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"7\"\u003e\u003cb\u003eAbbreviations\u003c/b\u003e: RRV: range of normal reference values; SD: standard deviation; OR: odds ratios; CI: confidence interval; * statistically significant differences between the groups before and after treatment.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eOlder adults are more susceptible to recurrent infections, neurodegenerative disorders, cardiovascular diseases, and neoplasms of mature B cells, such as small lymphocytic lymphoma, diffuse large B-cell lymphoma, and mantle cell lymphoma [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWith advancing age, both the composition and function of the immune system undergo significant changes. One of the most evident effects on the B-cell compartment is the marked reduction in circulating B lymphocytes [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Several studies have reported a decline in B1a CD5⁺CD19⁺ and CD5⁺CD20⁺ lymphocytes in older adults, particularly in those over 80 years of age [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA deficiency in CD5⁺CD19⁺ B lymphocytes has also been reported in the peripheral blood of patients with inflammatory bowel diseases, including ulcerative colitis and Crohn\u0026rsquo;s disease. In these patients, an inverse correlation has been observed between lymphocyte counts and disease activity, suggesting a loss of immunological tolerance to intestinal mucosal antigens\u0026mdash;a phenomenon that appears to be more pronounced in the elderly [\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eB1a lymphocytes can function similarly to regulatory B cells, as they produce high concentrations of anti-inflammatory cytokines such as interleukins \u0026minus;\u0026thinsp;3, -10, and \u0026minus;\u0026thinsp;35 [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe decline in circulating B cells in the peripheral blood of older adults is primarily due to reduced lymphopoiesis in the bone marrow. This reduction in na\u0026iuml;ve B cells is accompanied by an expansion of memory B cells, which exhibit impaired differentiation into plasma cells, hallmarks of the immunosenescence phenotype, and are associated with the development of age-related diseases [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn a previous study, elderly Cubans aged 80 years or older showed lower absolute counts of CD19⁺CD20⁺ B lymphocytes [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. Treatment with BT has proven effective in older Cuban adults with recurrent infections [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] with a significant increase in the percentage of total CD19⁺ B lymphocytes.\u003c/p\u003e \u003cp\u003eRamos HE and colleagues demonstrated that, in elderly individuals treated with BT and the VAMENGOC-BC\u0026reg; vaccine either separately or in combination, BT significantly increased CD19⁺ antigen expression [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Similarly, Hern\u0026aacute;ndez et al. reported that BT administration in older Cuban adults led to significant increases in both the percentage and absolute count of total CD19⁺ B lymphocytes six weeks after completing treatment, with this effect persisting for six months [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. These findings supported the large-scale intervention carried out in Cuba during the COVID-19 epidemic, in which BT was administered to institutionalized older adults, contributing to reduced morbidity and mortality from SARS-CoV-2 infection [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn this study, BT administration was associated with a decrease in the percentage of CD5⁺CD19⁺ B lymphocytes at six weeks, although the change did not reach statistical significance. By six months, however, this percentage had increased relative to baseline, showing a trend toward significance (p\u0026thinsp;=\u0026thinsp;0.09), suggesting a possible delayed immunostimulatory effect in older adults. The initial decline observed at six weeks warrants further investigation, particularly in elderly individuals with polyclonal or monoclonal B lymphoproliferative disorders coexpressing CD5⁺ and CD19⁺ antigens.\u003c/p\u003e \u003cp\u003eIn contrast, BT significantly increased both the percentage and absolute count of B1a CD5⁺CD20⁺ lymphocytes six weeks after treatment completion. Although these parameters remained elevated at six months, the differences were no longer statistically significant.\u003c/p\u003e \u003cp\u003eCD5⁺CD20⁺ cells represent a phylogenetically old B-cell subset responsible for producing IgM antibodies of low specificity and affinity. The persistence of this fetal-like phenotype has been associated with delayed maturation of the humoral immune system, potentially increasing susceptibility to infections [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the present study, BT administration led to a reduction in both the percentage and absolute count of CD19⁺CD20⁺ B lymphocytes after six weeks of treatment. However, six months after completing therapy, both parameters increased to levels exceeding baseline values. These findings are consistent with previous reports (21,22) regarding the persistence of BT-induced effects on B- and T-lymphocyte populations in older adults for up to at least six months, suggesting that additional treatment cycles may be required depending on individual clinical needs.\u003c/p\u003e \u003cp\u003eAging reduces the diversity of the B-cell repertoire by influencing the selection process during affinity maturation. Age-related changes typically include a decline in na\u0026iuml;ve B cells and an expansion of memory B cells, which compromise the immune system\u0026rsquo;s ability to respond to novel antigens. Moreover, certain aged B-cell subsets exhibit increased mitochondrial mass and elevated mitochondrial reactive oxygen species, reflecting mitochondrial dysfunction. These alterations result in reduced energy production and impairments in one-carbon metabolism\u0026mdash;processes essential for amino acid and nucleotide synthesis, as well as for antibody production and B-cell activation [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eB1a cells are recognized for their protective role against viral, bacterial, and parasitic infections through the production of natural IgM antibodies. Increasing evidence also suggests that these cells, particularly the highly polyspecific subsets, may participate in autoimmune pathogenesis. Their involvement in controlling several bacterial infections has been demonstrated [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the present study, none of the elderly participants developed infections during the follow-up period. Although the absence of infections could suggest improved resistance and a lower tendency toward inflammatory responses, this observation should be interpreted with caution. It may be consistent with the regulatory role attributed to B1a lymphocytes, but further studies are needed to confirm this relationship.\u003c/p\u003e \u003cp\u003eOverall, the findings suggest that BT may exert a modulatory influence on different B-cell subpopulations, CD5⁺CD19⁺, CD5⁺CD20⁺, and CD19⁺CD20⁺, with the direction and magnitude of these changes appearing to depend on the duration of treatment.\u003c/p\u003e \u003cp\u003eTo contextualize our findings, Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e summarizes results from previous Cuban studies evaluating the immunomodulatory role of BT in different clinical settings.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eImmunomodulatory effect of Biomodulin T\u0026reg; on different cell populations and clinical scenarios in Cuban subjects\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"13\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c12\" colnum=\"12\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c13\" colnum=\"13\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eStudy design\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eHuman subjects\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eDosage\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eOutcomes\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003ep value\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colspan=\"4\" nameend=\"c13\" namest=\"c10\"\u003e\u0026nbsp;\u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProspective, controlled.\u003c/p\u003e \u003cp\u003en\u0026thinsp;=\u0026thinsp;34 [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePediatric patients with recurrent infections.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3 mg (one vial), IM, twice a week for 8 weeks.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eIncrease of the Thymus area.\u003c/p\u003e \u003cp\u003eDecrease in the frequency and severity of infections.\u003c/p\u003e \u003cp\u003eIncrease of the postural parameters.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.005\u003c/p\u003e \u003cp\u003en.s.\u003c/p\u003e \u003cp\u003en.s.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c13\" namest=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003ePresentation of a clinical case [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePediatric patient with cystic fibrosis and severe thymic hypoplasia.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3 mg (one vial), IM, three times a week for 8 weeks.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eIncrease of the Thymus area.\u003c/p\u003e \u003cp\u003eNormalization of the pondoestatural parameters. Decrease in the frequency and severity of infections.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eNot evaluated\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c13\" namest=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProspective, uncontrolled, multicenter,\u003c/p\u003e \u003cp\u003eclinical trial. n\u0026thinsp;=\u0026thinsp;60 [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePediatric patients with thymic hypoplasia.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3 mg (one vial), IM, *frequency according to the thymic area for 8 weeks.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eIncrease of the Thymus area.\u003c/p\u003e \u003cp\u003eIncrease in T CD3\u0026thinsp;+\u0026thinsp;CD4\u0026thinsp;+\u0026thinsp;lymphocytes.\u003c/p\u003e \u003cp\u003eIncrease in IgA.\u003c/p\u003e \u003cp\u003eDecrease in the frequency and severity of infections.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003cp\u003e\u0026lt;\u0026thinsp;0.05\u003c/p\u003e \u003cp\u003e0.013\u003c/p\u003e \u003cp\u003e0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c13\" namest=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProspective, uncontrolled.\u003c/p\u003e \u003cp\u003en\u0026thinsp;=\u0026thinsp;31 [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eOlder adult patients with recurrent respiratory infections.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3mg (one vial), IM\u003c/p\u003e \u003cp\u003e\u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15 once a week \u003cem\u003en\u003c/em\u003e\u0026thinsp;=\u0026thinsp;16 two times a week, both cases for 6 weeks.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eIncrease in T Na\u0026iuml;ve CD3+/CD4\u0026thinsp;+\u0026thinsp;lymphocytes.\u003c/p\u003e \u003cp\u003eIncrease CD4\u0026thinsp;+\u0026thinsp;recent thymic emigrants.\u003c/p\u003e \u003cp\u003eIncrease CD8\u0026thinsp;+\u0026thinsp;recent thymic emigrants.\u003c/p\u003e \u003cp\u003eIncrease CD8\u0026thinsp;+\u0026thinsp;stem cells-like memory.\u003c/p\u003e \u003cp\u003eDecreased expression of CD4\u0026thinsp;+\u0026thinsp;PD-1\u0026thinsp;+\u0026thinsp;T cells.\u003c/p\u003e \u003cp\u003eDecreased expression of CD8\u0026thinsp;+\u0026thinsp;PD-1\u0026thinsp;+\u0026thinsp;T cells.\u003c/p\u003e \u003cp\u003eIncrease of Ki67 on CD4\u0026thinsp;+\u0026thinsp;T lymphocytes.\u003c/p\u003e \u003cp\u003eIncrease of Ki67 on CD8\u0026thinsp;+\u0026thinsp;T lymphocytes.\u003c/p\u003e \u003cp\u003eIncrease in intracellular expression of INF-γ in CD4\u0026thinsp;+\u0026thinsp;T lymphocytes.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003e(\u0026lt;\u0026thinsp;0.05) '\u003c/p\u003e \u003cp\u003e(\u0026lt;\u0026thinsp;0.05) '/''\u003c/p\u003e \u003cp\u003e(\u0026lt;\u0026thinsp;0.05) '\u003c/p\u003e \u003cp\u003e(\u0026lt;\u0026thinsp;0.05) '\u003c/p\u003e \u003cp\u003e[\u0026lt;\u0026thinsp;0.05] '\u003c/p\u003e \u003cp\u003e(\u0026lt;\u0026thinsp;0.05) '\u003c/p\u003e \u003cp\u003e(\u0026lt;\u0026thinsp;0.0001) '\u003c/p\u003e \u003cp\u003e(\u0026lt;\u0026thinsp;0.05) '/''\u003c/p\u003e \u003cp\u003e(\u0026lt;\u0026thinsp;0.05) ''\u003c/p\u003e \u003cp\u003e(\u0026lt;\u0026thinsp;0.05)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c9\" namest=\"c8\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"4\" nameend=\"c13\" namest=\"c10\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProspective, uncontrolled.\u003c/p\u003e \u003cp\u003en\u0026thinsp;=\u0026thinsp;80 [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAdult patients with advanced lung cancer after finishing first-line platinum-based chemotherapy (4\u0026ndash;6 cycles).\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3 mg (one vial), IM, three times a week for 4 weeks.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eDecrease of terminally differentiated CD45RA effector T cells (EMRA) CD4+.\u003c/p\u003e \u003cp\u003eDecrease of terminally differentiated CD45RA effector T cells (EMRA) CD8+.\u003c/p\u003e \u003cp\u003eDecreased expression of CD4\u0026thinsp;+\u0026thinsp;PD-1\u0026thinsp;+\u0026thinsp;T cells.\u003c/p\u003e \u003cp\u003eDecreased expression of CD8\u0026thinsp;+\u0026thinsp;PD-1\u0026thinsp;+\u0026thinsp;T cells.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c8\" namest=\"c6\"\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.0031\u003c/p\u003e \u003cp\u003ep\u0026thinsp;=\u0026thinsp;0.0372\u003c/p\u003e \u003cp\u003e0.0005\u003c/p\u003e \u003cp\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.0001\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c10\" namest=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c13\" namest=\"c11\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProspective, uncontrolled.\u003c/p\u003e \u003cp\u003en\u0026thinsp;=\u0026thinsp;80 [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAdult patients with advanced lung cancer after finishing first-line platinum-based chemotherapy (4\u0026ndash;6 cycles).\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3 mg (one vial), IM, three times a week\u003c/p\u003e \u003cp\u003e(4\u0026ndash;6 weeks).\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eDecrease of T CD3\u0026thinsp;+\u0026thinsp;CD4\u0026thinsp;+\u0026thinsp;CD8\u0026thinsp;+\u0026thinsp;CD57+\u003c/p\u003e \u003cp\u003eLymphocytes.\u003c/p\u003e \u003cp\u003eDecrease of CD57\u0026thinsp;+\u0026thinsp;NK cells.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c8\" namest=\"c6\"\u003e \u003cp\u003en.s.\u003c/p\u003e \u003cp\u003e0.042\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"3\" nameend=\"c11\" namest=\"c9\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c13\" namest=\"c12\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProspective, uncontrolled, non-randomized, experimental clinical trial.\u003c/p\u003e \u003cp\u003en\u0026thinsp;=\u0026thinsp;30 [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eElderly adults institutionalized.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3 mg (one vial), IM, twice a week for 6 weeks.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIncrease in T Na\u0026iuml;ve CD3+/CD4+/CD27+/CD45RA\u0026thinsp;+\u0026thinsp;lymphocytes.\u003c/p\u003e \u003cp\u003eIncrease T central memory CD3+/CD4+/CD27+/CD45RA-.\u003c/p\u003e \u003cp\u003eIncrease in of T Na\u0026iuml;ve CD3+/CD8+/CD27+/CD45RA\u0026thinsp;+\u0026thinsp;lymphocytes.\u003c/p\u003e \u003cp\u003eIncrease in T central memory CD3+/CD8+/CD27+/CD45RA-.\u003c/p\u003e \u003cp\u003eIncrease in NK CD3-CD16+/CD56\u0026thinsp;+\u0026thinsp;cells.\u003c/p\u003e \u003cp\u003eIncrease in CD3+/HLA-/DR+.\u003c/p\u003e \u003cp\u003eIncrease in % of CD3+/CD25+.\u003c/p\u003e \u003cp\u003eIncrease in B lymphocytes.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e(0.023**)\u003c/p\u003e \u003cp\u003e(0.040***)\u003c/p\u003e \u003cp\u003e(0.002***)\u003c/p\u003e \u003cp\u003e[0.017***]\u003c/p\u003e \u003cp\u003e(0.031***)\u003c/p\u003e \u003cp\u003e[0.031***]\u003c/p\u003e \u003cp\u003e(0.029***)\u003c/p\u003e \u003cp\u003e[0.024***]\u003c/p\u003e \u003cp\u003e(\u0026lt;\u0026thinsp;0.001***)\u003c/p\u003e \u003cp\u003e[0.001***]\u003c/p\u003e \u003cp\u003e(\u0026lt;\u0026thinsp;0.001**)\u003c/p\u003e \u003cp\u003e[\u0026lt;\u0026thinsp;0.001**]\u003c/p\u003e \u003cp\u003e(\u0026lt;\u0026thinsp;0.001**)\u003c/p\u003e \u003cp\u003e[\u0026lt;\u0026thinsp;0.001**]\u003c/p\u003e \u003cp\u003e(\u0026lt;\u0026thinsp;0.001***)\u003c/p\u003e \u003cp\u003e[\u0026lt;\u0026thinsp;0.001***]\u003c/p\u003e \u003cp\u003e(0.002**)\u003c/p\u003e \u003cp\u003e[0.005**]\u003c/p\u003e \u003cp\u003e(\u0026lt;\u0026thinsp;0.001***)\u003c/p\u003e \u003cp\u003e[0.003***]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"7\" nameend=\"c13\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eProspective, controlled, randomized, experimental clinical trial.\u003c/p\u003e \u003cp\u003en\u0026thinsp;=\u0026thinsp;60l [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eElderly adults.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3 mg (one vial), IM, three times a week one week.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eIncrease in B lymphocytes.\u003c/p\u003e \u003cp\u003eIncrease of CD3\u0026thinsp;+\u0026thinsp;CD56\u0026thinsp;+\u0026thinsp;T lymphocytes.\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c6\" namest=\"c5\"\u003e \u003cp\u003e0.005\u003c/p\u003e \u003cp\u003e0.007\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"6\" nameend=\"c12\" namest=\"c7\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colspan=\"1\" nameend=\"c13\" namest=\"c13\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"13\"\u003e\u003cb\u003eAbbreviations\u003c/b\u003e: n: studied subjects; n.s: no significant statistical difference; IM:intramuscular; ( ): percentage; [ ]: cells per \u0026micro;L; p\u0026thinsp;\u0026lt;\u0026thinsp;0.05: value statistically significant differences between the groups before and after treatment, NK: natural killer; * thymus area\u0026thinsp;\u0026lt;\u0026thinsp;500 mm\u003csup\u003e2\u003c/sup\u003e three times a week, \u0026gt;\u0026thinsp;500\u0026thinsp;\u0026lt;\u0026thinsp;800 mm\u003csup\u003e2\u003c/sup\u003e twice a week, \u0026gt;\u0026thinsp;800\u0026thinsp;\u0026le;\u0026thinsp;100 mm\u003csup\u003e2\u003c/sup\u003e once a week; **: after 6 weeks of treatment; ***: after 6 months of treatment; INF-γ: interferon gamma; ': a dose; ': two doses.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThese investigations have consistently demonstrated immunomodulatory effects of BT in diverse clinical contexts, including pediatric patients with recurrent infections, individuals with thymic hypoplasia, older adults with chronic respiratory diseases, and cancer patients after chemotherapy. Our findings add to this body of evidence by specifically addressing the behavior of B1a lymphocyte subpopulations in institutionalized elderly adults. Unlike earlier reports that focused predominantly on T-cell and global B-cell responses, this study provides new insights into the modulation of B1a subsets (CD5⁺CD19⁺, CD5⁺CD20⁺, CD19⁺CD20⁺), highlighting both the short-term increases and the long-term persistence of certain changes after BT treatment.\u003c/p\u003e \u003cp\u003eThe potential mechanisms underlying these immunomodulatory effects may include the enhancement of CD4⁺ TH1 T-cell number and function, the inhibition of CTLA-4 and PD-1 expression on B1a cells, and the reduction of CD4⁺FOXP3⁺ regulatory T cells, which collectively could favor increased IFN-γ production. Similar pathways have been proposed in previous studies of BT and other thymic peptides, suggesting that modulation of checkpoint molecules and regulatory populations may play a central role in counteracting immunosenescence. Further mechanistic studies will be necessary to confirm these hypotheses and to clarify the molecular pathways through which BT modulates B1a lymphocytes in the context of aging.\u003c/p\u003e \u003cp\u003eAt the same time, some limitations of this study should be acknowledged. From the total population of institutionalized older adults in the selected nursing home, only 44.1% met the predefined inclusion and exclusion criteria and were enrolled. This limited recruitment restricts the generalizability of the findings to broader elderly populations. Future studies should aim to replicate these results in larger and more diverse cohorts, ideally across multiple institutions, to confirm the reproducibility and external validity of the observed effects.\u003c/p\u003e"},{"header":"5. Conclusions","content":"\u003cp\u003eBT demonstrated immunomodulatory effects on B1a lymphocytes in elderly Cuban adults, with changes partially sustained for up to six months after a six-week treatment regimen. This effect was observed in a population particularly susceptible to immunosenescence, highlighting the potential of BT as an immune-supportive intervention. The findings suggest possible clinical applications in the prevention and management of infections, autoimmune diseases, and B-cell lymphoproliferative disorders in older adults.\u003c/p\u003e \u003cp\u003eDespite these encouraging findings, confirmation in multicenter, randomized controlled trials with larger and more diverse populations is required to validate these results, explore long-term outcomes, and establish optimal dosing strategies. In parallel, mechanistic studies are recommended to further elucidate the pathways through which BT modulates B1a lymphocyte activity in the context of aging.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eVianed Mars\u0026aacute;n Su\u0026aacute;rez: conceptualization of the research, immunophenotyping by flow cytometry, analysis, interpretation of the results, writing of the manuscript and approval of the final report.\u003c/p\u003e\n\u003cp\u003eImilla Casado Hern\u0026aacute;ndez: analysis of the results, review of the manuscript, and approval of the final report.\u003c/p\u003e\n\u003cp\u003eElizabeth Hern\u0026aacute;ndez Ramos: immunophenotyping by flow cytometry, statistical analysis, and approval of the final report.\u003c/p\u003e\n\u003cp\u003eYenisey Triana Marrero, Yaneisy Duarte P\u0026eacute;rez, Eduardo Alfonso Hern\u0026aacute;ndez Mars\u0026aacute;n, and Mibelys Santana Garc\u0026iacute;a: immunophenotyping by flow cytometry and approval of the final report.\u003c/p\u003e\n\u003cp\u003eAlexis Labrada Rosado, Gisela Mar\u0026iacute;a Su\u0026aacute;rezFormig\u0026oacute;, and Yendry Ventura Carmenate: manuscript review, editing, and approval of the final report.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest:\u003c/strong\u003e The authors declare that they have no conflicts of interest regarding the research, authorship, and/or publication of this article\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eRastogi I, Jeon D, Moseman JE, et al. 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[DOI] [PubMed].\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"B-1a lymphocytes, Flow cytometry, Immunosenescence, Immunomodulation, Thymic peptides","lastPublishedDoi":"10.21203/rs.3.rs-8649614/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8649614/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eIntroduction:\u003c/h2\u003e \u003cp\u003eB1a lymphocytes are components of the innate immune response that maintain their population through self-renewal, a process that declines with age.\u003c/p\u003e\u003ch2\u003eObjective\u003c/h2\u003e \u003cp\u003eTo evaluate the effect of Biomodulina T\u0026reg; (BT) on B1a lymphocyte subpopulations in older Cuban adults.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eIn this non-randomized clinical trial, 30 institutionalized adults aged\u0026thinsp;\u0026ge;\u0026thinsp;60 years received intramuscular BT (3 mg) twice weekly for six weeks. B1a lymphocyte subpopulations (CD19⁺CD5⁺, CD20⁺CD5⁺, and CD19⁺CD20⁺) were quantified by flow cytometry at baseline, six weeks, and six months post-treatment.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe median percentages and absolute counts of B1a CD5⁺CD20⁺ lymphocytes increased after six weeks of treatment with BT (0.96 vs. 2.11, SD 6.02 vs. 2.38) and (17.02 vs. 40.58, SD 80.09 vs. 39.70), with statistical significance (p\u0026thinsp;=\u0026thinsp;0.026 and p\u0026thinsp;=\u0026thinsp;0.014, respectively). In contrast, the percentage and absolute count of lymphocytes coexpressing CD19⁺ and CD20⁺ antigens decreased after six weeks of BT treatment (4.87 vs. 1.94, SD 4.69 vs. 4.57) and (88.48 vs. 31.03, SD 62.14 vs. 65.54), with statistical significance (p\u0026thinsp;=\u0026thinsp;0.014 and p\u0026thinsp;=\u0026thinsp;0.018, respectively). Six months after completing the treatment, both the percentage and absolute count of these cells continued to increase (4.87 vs. 8.47, SD 4.69 vs. 26.69) and (88.48 vs. 173.5, SD 62.14 vs. 323.8), reaching statistical significance (p\u0026thinsp;=\u0026thinsp;0.012 and p\u0026thinsp;=\u0026thinsp;0.011, respectively).\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eBT exerts an immunomodulatory effect on B1a lymphocytes in older adults, with partial persistence six months after treatment, suggesting potential benefits in preventing age-related immune decline.\u003c/p\u003e","manuscriptTitle":"Counteracting Immunosenescence: Effects of Biomodulina T® on B-1a Lymphocytes in Older Cuban Adults","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-27 09:14:40","doi":"10.21203/rs.3.rs-8649614/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"af76511c-4ff2-4848-9893-2437f1e87290","owner":[],"postedDate":"January 27th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-30T15:40:21+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-27 09:14:40","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8649614","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8649614","identity":"rs-8649614","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}