Main Research Findings

1) Thymalin was found to elicit antiviral effects and acts as a potential immunoprotective compound for the prevention of COVID-19.

2) Treatment with Thymalin in patients with COVID-19 was found to reduce the concentration of pro-inflammatory cytokines and reduce the risk of blood clots.

 

Selected Data

1) The study performed by Khavinson et al aimed to elucidate the influence of the peptide thymalin on the differentiation of human hematopoietic stem cells (HSCs) and the expression of the CD28 molecule, particularly in the context of antiviral immunity relevant to COVID-19. The experimental design primarily relied on in vitro cell culture of human HSCs, followed by immunofluorescence and statistical analysis to quantify molecular changes [1].

The foundational material for the study was human hematopoietic stem cells (HSCs), which were isolated from umbilical cord blood. The isolation protocol commenced with diluting the umbilical cord blood 1:1 with Dulbecco’s Phosphate Buffered Saline (DPBS) before transferring it to a tube. Subsequently, a 3 ml aliquot of Ficoll-Paque Plus was introduced, and the sample was subjected to centrifugation at 400 rpm for 30 minutes. Following this density gradient centrifugation, the upper layer of plasma was carefully discarded. The layer containing mononuclear cells, which includes HSCs, was then precisely collected and transferred to a new tube. This mononuclear cell fraction was then mixed 1:3 with DPBS and centrifuged again at 400 rpm for 10 minutes. After this second centrifugation, the supernatant was discarded, and the isolated cells were resuspended in 5 ml of RPMI-1640 culture medium. To ensure an environment conducive to HSC culture, these cells were transferred to a vial with a non-adhesive surface and incubated in a thermostat under standard cell culture conditions [1].

The cultured HSCs were then divided into two main experimental groups: a control group and an experimental group treated with thymalin. The control group received only nutrient medium, maintaining baseline conditions. The experimental group received thymalin at a specific concentration of 100 ng/ml. Thymalin was added to the HSC cultures during each passage, ensuring continuous exposure throughout the culturing period. The cells were cultured for an extended duration, up to passages 2 and 7, to observe both short-term and longer-term effects of thymalin on differentiation.

To assess the expression of various signaling molecules on the HSCs, an immunofluorescence technique was employed. After the designated culture periods, the cells were fixed with 4% paraformaldehyde for 10 minutes. Following fixation, the cells were treated with 0.5% Triton X-100 for 10 minutes at room temperature. This permeabilization step is essential for allowing antibodies access to intracellular targets if present, although the primary targets here (CD44, CD117, CD28) are surface molecules. To minimize non-specific antibody binding, the HSCs were then incubated with Protein Block for 10 minutes. The primary antibodies used were specific for key differentiation markers: CD44 (1:50 dilution), CD28 (1:50 dilution), and CD117 (1:150 dilution) [1].

Cells were incubated with these primary antibodies for 60 minutes in a thermostat at 37°C. Following primary antibody incubation, secondary antibodies conjugated with Alexa Fluor 488 or 647 (1:1000 dilution) were applied for 30 minutes at room temperature in the dark, allowing for fluorescent visualization. To identify cell nuclei and provide a morphological context, DAPI was used as a counterstain. Finally, the prepared samples were embedded in a Dako Fluorescent Mounting Medium under coverslips for microscopic examination.

Microscopic analysis was performed using a confocal laser scanning microscope, specifically an Olympus FV 1000, operating at ×200 magnification. The FW10 software was utilized to acquire micrographs, with 5 fields of view captured from each sample to ensure representative data. ImageJ software was then used for quantitative analysis of the micrographs. Two key parameters were quantified: the relative expression area (%) and the mean brightness (arbitrary units). The relative expression area was calculated as the ratio of the area of immunopositive cells to the total area of the preparation, providing an indication of the number of immunostained cells. The mean brightness, on the other hand, reflected the concentration of the marker within one cell [1].

2) The study conducted by Khavinson et al investigated the efficacy of the peptide drug thymalin as an activator of HSC differentiation in complex therapy for patients with COVID-19. This was conducted as a single-center, open-label, prospective, randomized, controlled clinical trial involving human patients.

Patients all had a clinical diagnosis of COVID-19 with further inclusion criteria specifically targeting individuals with moderate to severe forms of the disease. Key indicators for inclusion were lymphopenia, defined as absolute lymphocyte count below 1.2 x 10^9/L or relative lymphocyte count below 19% in clinical blood tests. Additionally, all included patients exhibited bilateral polysegmental pneumonia confirmed by spiral computed tomography, with a CT1-CT3 lesion index, accompanied by symptoms of respiratory failure indicated by an SpO2 ≤ 95%. Patients were randomized into two groups using the envelope method: a main group and a control group. The main group consisted of 42 patients who received standard treatment augmented with thymalin. The control group comprised 50 patients who received standard treatment alone [2].

Control and experimental groups were randomly assigned to different treatment groups. Patients in the main group received thymalin. The administration protocol involved a daily intramuscular injection of 10 mg of thymalin for 5 days. Both groups received a standard treatment regimen which included several categories of drugs such as antibacterial and antiviral therapeutic agents. If necessary, patients received antibacterial treatment. In the main group, 8 patients received levofloxacin, 33 received ceftriaxone, and one received both. In the control group, all patients received ceftriaxone. Antivirals were prescribed to 27 patients (64%) in the main group and 35 patients (70%) in the control group. Specific antiviral drugs used included hydroxychloroquine, lopinavir and ritonavir 200/50 and interferon alpha-2b. Hydroxychloroquine was given to 8 patients in the main group (19%) and 5 in the control group (10%), following a scheme of 800 mg (400 mg twice a day) initially, then 400 mg per day (200 mg twice a day) for 6 days. Lopinavir and ritonavir were administered to 17 patients (40.5%) in the main group and 28 patients (56%) in the control group, with a regimen of 400 mg + 100 mg per os every 12 hours for 14 days. Interferon alpha-2b was administered to 2 patients in the main group (4.8%) during the first 5 days, 3 instillations per nasal passage [2].

Additionally, patients also received glucocorticosteroid therapy utilizing dexamethasone at a dosage of 12 mg per day. 10 patients in the main group and 12 patients in the control group received dexamethasone, accounting for 24% of the total in both groups. The average duration of dexamethasone use was 2.5 days in the main group and 4 days in the control group. Anticoagulant therapy was also provided using low molecular weight heparin sodium enoxaparin that was administered daily in a prophylactic dose of 4000 IU (40 mg) subcutaneously once a day or as an intermediate dose of 4000 IU (40 mg) twice a day, depending on disease severity. Low molecular weight heparin was used throughout the hospital stay. The overall course of treatment in both groups ranged from 10 to 14 days, with an average of 12 ± 4 days.

Patients in both groups underwent a comprehensive series of laboratory studies both before and after therapy. Blood samples were collected by venipuncture from the cubital vein after an overnight fast, ensuring adherence to preanalytical stage requirements. Clinical blood tests included a complete blood count. Hematological analyses, including determination of CD3, CD4, and CD8 lymphocytes, were performed using an automatic hematological analyzer. Erythrocyte sedimentation rate (ESR) was measured on a Test 1 analyzer. Parameters such as fibrinogen, prothrombin time, and D-dimer were analyzed on an automatic coagulometer. Levels of C-reactive protein (CRP), glucose, and ferritin were determined using a biochemical analyzer. Ferritin concentration was specifically measured on an immunochemiluminescence analyzer. Finally, interleukin-6 (IL-6) levels were determined using equipment designed for ELISA, with specialized reagents [2].

 

Discussion

1) The results of the study conducted by the research team of Khavinson et al demonstrated that thymalin profoundly influences the differentiation trajectory of human HSCs, particularly by downregulating early differentiation markers and significantly upregulating CD28, a crucial marker for mature T lymphocytes. These findings suggest thymalin’s potential as an immunoprotective agent, especially relevant in conditions of immune suppression, such as severe viral infections like COVID-19 [1].

A primary observation concerned the expression of CD44, a stem cell marker and ligand for selectins, involved in maintaining bone marrow stem cell populations and modulating cytokine effects. At passage 2, thymalin did not significantly alter the relative area or mean brightness of CD44 expression in HSCs. However, by passage 7, the relative area of CD44 expression in thymalin-treated HSCs significantly decreased by 2.76 times compared to control cultures (from 5.47% to 1.98%). The mean brightness of CD44 remained unaffected at both passages. This reduction in CD44 expression during long-term culture suggests that thymalin prompts HSCs to differentiate, moving them past a stage where CD44 is highly expressed.

The effects of thymalin on CD117 expression, a marker for an intermediate stage of HSC differentiation and a neutrophil growth factor receptor, were dynamic and passage-dependent. At passage 2, thymalin significantly increased the mean brightness of CD117 expression by 2.16 times compared to controls. This initial upregulation suggests that thymalin stimulates the early stages of HSC differentiation, promoting cell proliferation and the synthesis of CD117. However, by passage 7, the relative area of CD117 expression under the influence of thymalin significantly decreased by 2.2 times (from 4.33% to 1.97%) compared to controls. This decrease in CD117 during long-term cultivation, coupled with the initial increase, indicated that thymalin first stimulates the expression of CD117 as HSCs begin to differentiate, but then, as differentiation progresses, cells move beyond the CD117+ stage and consequently lose their expression. This implies a stimulation of HSC differentiation through the CD117+ stage under thymalin’s influence [1].

The most striking and clinically relevant finding was thymalin’s effect on CD28 expression, a crucial marker for mature T lymphocytes and essential for their activation and antiviral immunity. At passage 2, the relative area of CD28 expression in thymalin-treated HSCs significantly increased by 2.98 times compared to controls (from 1.24% to 3.69%). This positive effect became even more pronounced by passage 7, where the relative area of CD28 expression increased by a remarkable 6.93 times compared to controls (from 2.01% to 14.56%). Concurrently, the mean brightness of CD28 expression also showed significant increases at both passages 2 (2.03 times) and 7 (2.05 times) under thymalin treatment. This substantial upregulation of CD28 expression on HSCs strongly indicates that thymalin actively stimulates the differentiation of CD117+ cells into mature CD28+ T lymphocytes.

The researchers interpret these results as evidence that thymalin promotes the differentiation of HSCs towards a mature T lymphocyte phenotype. The sequential changes in CD44, CD117, and CD28 expression suggest a step-wise stimulation of the differentiation process. Initially, thymalin encourages cells into the CD117+ stage, followed by their further differentiation into CD28+ T lymphocytes, leading to a decrease in the more primitive markers and a significant increase in the mature T-cell marker [1].

The study posits that this compensatory stimulation of HSC differentiation into CD28+ T lymphocytes by thymalin could be highly beneficial in scenarios of severe immune suppression, such as those observed in severe COVID-19 patients. In such cases, a decreased number of CD28+, CD4+, and CD8+ T lymphocytes is a hallmark of compromised antiviral immunity. By promoting the generation of these crucial immune cells, thymalin could counteract the immune suppression and support the body’s defense mechanisms. Therefore, the study concludes that thymalin can be considered an immunoprotective peptide drug, offering a potential strategy for the prevention and complex therapy of viral infections, including SARS-CoV-2. Its mechanism, which involves activating the differentiation of HSCs into mature, functional immune cells, positions it as a valuable therapeutic candidate in conditions requiring immune modulation and regeneration [1].

2) The study completed by Khavinson et al evaluated the impact of adding thymalin to standard COVID-19 therapy on immune parameters, inflammatory markers, and hemostasis, finding significant improvements attributable to thymalin. Critically, no deaths occurred in either group during the observation period, and pathological progression, as assessed by spiral computed tomography, was less frequent in the thymalin-treated group (2 cases) compared to the control group (5 cases) [2].

In the control group receiving standard therapy, there was a decrease in IL-6 concentration by 1.41 times, but no significant improvement in T-cell system parameters. In contrast, the addition of thymalin to standard therapy in the main group led to substantial improvements in T-cell counts. The number of lymphocytes increased by 55% after thymalin treatment, reaching normal values. T lymphocytes increased by 63.8% in the thymalin group, approaching the lower limits of the normal range, a statistically significant increase compared to both baseline and the control group. T helper cells increased by 88.9%, also approaching the lower normal limit and showing statistical significance. This CD4/CD8 ratio, indicative of immune balance, increased by a factor of 1.7 in the thymalin group (from 1.5 to 2.55), a statistically significant improvement compared to both baseline and the control group. Finally, the concentration of IL-6, a key proinflammatory cytokine, significantly decreased by 5.5 times in the thymalin-treated group, a highly significant reduction compared to both baseline and the control group.

The thymalin group also demonstrated significant improvements in markers of inflammation and coagulation, which are critical in COVID-19 pathology. CRP levels, a general marker of inflammation, decreased by 9.7 times in the thymalin group, a highly significant reduction compared to both baseline and the control group. In the control group, CRP decreased only marginally. D-dimer levels, an indicator of thrombotic activity and disease severity, decreased by 5.7 times in the thymalin group, a highly significant reduction compared to both baseline and the control group, with the control group showing a less pronounced decrease. Fibrinogen, another coagulation factor, decreased by 1.5 times in the thymalin group, a significant change compared to baseline. Ferritin levels, associated with inflammation and iron metabolism, decreased by 1.6 times in the thymalin group, a significant reduction compared to baseline and compared to the control group. Finally, prothrombin time also significantly decreased in the thymalin group, reaching normal range and showing a statistically significant change [2].

Thymalin treatment also modulated peripheral blood cell counts, contributing to an overall healthier physiological state. There was a notable 2 time decrease in eosinophils and 3 time decrease in basophils in the thymalin group, with statistically significant reductions compared to baseline and the control group. Monocyte counts increased by 1.3 times in the thymalin group, a significant increase compared to baseline and the control group.This ratio between neutrophils and lymphocytes decreased by a factor of 2 in the thymalin group from 5.30 to 2.67, indicating an improved immune balance. This ratio between platelets and leukocytes increased by a factor of 1.3 in the thymalin group from 35.59 to 44.93, showing a significant change. The absolute number of platelets and plateletcrit (PCT) significantly increased in the thymalin group, which is important for maintaining normal hemostasis. Finally, the erythrocyte sedimentation rate (ESR) decreased in the thymalin group, a statistically significant reduction compared to baseline and the control group.

In conclusion, the inclusion of thymalin in the complex therapy for COVID-19 patients led to substantial improvements across multiple physiological systems. It effectively normalized T-cell counts, significantly reduced key inflammatory markers IL-6, CRP, and ferritin, and attenuated coagulopathy indicators D-dimer and fibrinogen. These comprehensive positive effects underscore thymalin’s ability to modulate both the immune system and hemostasis, thereby reducing the risk of cytokine storm and thrombotic complications often associated with severe COVID-19. The findings provide a strong rationale for incorporating thymalin as an immunomodulatory and immunoprotective peptide in the treatment of coronavirus infection [2].

 

Disclaimer

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

 

Citations

[1] Khavinson VK, Linkova NS, Kvetnoy IM, et al. Thymalin: Activation of Differentiation of Human Hematopoietic Stem Cells. Bull Exp Biol Med. 2020;170(1):118-122. doi:10.1007/s10517-020-05016-z

[2] Khavinson VK, Kuznik BI, Trofimova SV, et al. Results and Prospects of Using Activator of Hematopoietic Stem Cell Differentiation in Complex Therapy for Patients with COVID-19. Stem Cell Rev Rep. 2021;17(1):285-290. doi:10.1007/s12015-020-10087-6

Effects of Thymalin and Immune Function

Thymalin is a polypeptide that can be used for various dysfunctions within the immune system such as immune depression and infections caused by bacteria and viruses. Thymalin was first synthesized by researchers Morozov et. Al from the thymus of calves and has since shown promise in combating apoptosis and regulating regeneration and hematopoiesis. Additionally, the chemicals immunoprotective success is based on the restoration of T and B lymphocytes and the resulting increase in activity. In a study conducted by Khavinson et. Al, researchers examined the effects of thymalin as well as its peptide components, EW peptide, the dipeptide KE, and the tripeptide EDP.
In the study conducted, Khavinson et. Al found that the short peptide components EW, KE, and EDP were most likely to activate and differentiate the immune response, decrease rates of apoptosis, and increase the viability of immune cells.

Thymalin, Thymogen and Postoperative Healing

First, the short peptide, EW, also referred to as thymogen, was shown to be quite similar to thymalin and could be considered very beneficial in preoperative and postoperative situations. Various studies conducted with thymogen found that preoperative treatment restored both functional and structural parameters of immunity. Postoperative treatment led to a drastic decrease in various types of postoperative complications. Thymogen was also shown to have “geroprotective properties” in numerous animal experiments, for example, treatment with thymogen in rats led to a 1.5-fold decrease in tumor and an overall increase in survival rates (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8365293/).

Next, the short dipeptide, KE, also referred to as Vilon, was shown to activate macrophages, blood lymphocytes, thymocytes, and neutrophils. It was also shown to stimulate cellular immunity. One of the main benefits of treating with the KE dipeptide is that it assists the expression of CD4 and CD5 on thymus cells, in turn, this stimulates the differentiation of T helper cells. Additionally, a 2009 study conducted by Khavinson et. Al was cited and described how introducing the KE dipeptide to transgenic mice suppressed HER-2/neu oncogene expression by 2-fold. Overall, the KE dipeptide displayed many benefits such as increasing proportions of euchromatic, decreasing heterochromatin levels, and regulating telomere length (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8365293/).

Finally, the EDP tripeptide was tested in the thymic cells of rats exposed to gamma irradiation, leading to accelerated aging. Following the administration of EDP, the rats maintained the cortex-medulla division which indicates the animal was not subject to aging. Additionally, administration of EDP led to an increase in mast cells and macrophages as well as an increase in the proliferation of thymocytes. Overall, the study found that EDP stimulated various cells of the immune system, led to the proliferation of epithelial cells, and prohibited the synthesization of tumor K-562 cells (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8365293/).

Comparative studies were performed examining the effects thymalin and the short peptide complexes had on immunogenesis by taking spleen tissue cultures of both young and old mice. Following administration of the short peptides EW, KE, and EDP there were notable changes in both apoptosis levels and proliferation rate. Overall the short peptide complexes led to an increase in cell proliferation, a 20-50% increase in the growth index, and a 29-42% reduction in apoptosis. Thymalin supplementation led to even more drastic results potentially due to the presence of all three of the short peptides (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8365293/).

 

Peptides Prefer the Cold
In order to reduce peptide breakdown, keep peptides refrigerated at all times but DO NOT FREEZE.
Swab the top of the vial with a 95% alcohol wipe before accessing.
Only Mix with Sterile Bacteriostatic Water
Bacteriostatic water is vital to preventing contamination and preserving the stability of the compound.
Push the needle through the stopper at an angle in order to direct the stream to the side of the vial.
Reconstituted peptide solution should be stored at around 4 degrees Celsius but not frozen, while lyophilized peptide solution should be kept at -20 degrees Celsius.

Thymalin is sold for laboratory research use only. Terms of sale apply. Not for human consumption, nor medical, veterinary, or household uses. Please familiarize yourself with our Terms & Conditions prior to ordering.