Effect of two thymosin fraction 5 polypeptides on human peripheral blood lymphocytes

Thymosin fraction 5 polypeptides beta 4 and alpha 1 were tested for their ability to affect certain immunological parameters of human peripheral blood lymphocytes (PBL). PBL were cultured with various concentrations of the peptides for 24 hours. Thymosin beta 4 was found to induce a significant decrease in the expression of the Fc alpha receptors of PBL, as well as in their ability to express antibody dependent cellular cytotoxic (ADCC) activity. In addition, this peptide had the ability to increase the percentage of T4 lymphocytes in normal and immunosuppressed donors and to decrease the percentage of T8 positive cells in normal donors. Finally, beta 4 peptide caused a small increase in the capacity of peripheral blood lymphocytes to form sheep red blood cell (SRBC) rosettes (ER). In parallel experiments thymosin alpha 1 was found inactive. The results presented here indicate that thymosin beta 4 may be used as an immunoregulatory molecule in patients with immunodeficiencies.     

Mitogenic response of marmoset lymphocytes: cytokinetics and identification of responsive cells

Isotope (tritiated thymidine, [³H]Tdr) incorporation into lymphocytes from two marmoset species, New World primates which are haemopoietic chimeras, was studied using peripheral blood lymphocyte (PBL) cultures incubated in vitro for 1–5 days with different concentrations of Concanavalin A (Con A), leucoagglutinin (LA), bacterial lipopolysaccharide (LPS) from gram-negative bacteria, rabbit anti-marmoset immunoglobulin (anti-Ig) serum, thymosin or irradiated histoincompatible (xenogeneic) lymphocytes. Only the plant lectins and xenogeneic lymphoid cells stimulated the uptake of [³H]Tdr. Lymphocyte enrichment experiments demonstrated that cells responsive to plant lectins and xenoantigens were primarily thymus-derived (T) lymphocytes. Bacterial endotoxin (LPS), however, enhanced the mitogenic response of PBL to Con A when LPS and plant lectin were added together at culture initiation. Thymosin caused either enhancement or suppression of the response to Con A depending on its time of addition relative to lectin stimulation. Addition of thymosin to lymphocyte cultures with Con A or 24 h later caused a decrease in isotope incorporation, while addition of thymosin 48 h later caused a 12–100% increase in [³H]Tdr uptake. Lymphocyte chimerism did not influence the mitogenic response since single-born, nonchimeric marmosets responded to plant lectins in a manner similar to marmosets with varying proportions of chimeric blood elements. Cytological analysis of stimulated lymphocytes from heterosexual marmosets revealed the percentage chimerism in the polyclonal mitogenic response and the clonal mixed lymphocyte reaction to be similar.     

Low Periconceptional Maternal Serum Thymosin α1 Levels Are Associated With Blighted Pregnancies

OBJECTIVE: The thymus-derived peptides, thymosin α₁ and thymosin (β₄, are believed to contribute to the maintenance of immune homeostasis. They are also associated with the hypothalamic-pituitary-adrenal-gonadal axis and may play a role in reproduction. STUDY DESIGN: Patients were recruited from a university hospital setting. Eligible candidates were 24 to 38 years old who were being seen in an ovulation induction and in vitro fertilization program. Serial maternal serum thymosin α₁ and β₄ levels were assayed preconceptual and then twice in the first trimester by ELISA in 28 women with known ovulation dates who successfully conceived as demonstrated by positive serum β human chorionic gonadotropin (hCG). Thymosin α₁ and β₄ serum levels for viable pregnancies (group I; N = 19) were compared to pregnancies that aborted (group II; N = 9) using repeated measures of multivariate analysis of variance (MANOVA). Periconceptional (preovulatory and early pregnancy) thymosin α₁ and β₄ values between groups I and II were compared using repeated measure one-way ANOVA. RESULTS: Thymosin α₁ levels from pregnancies that remained viable were significantly higher than those from pregnancies that spontaneously aborted. Preovulation thymosin α₁ levels also tended to be lower in pregnancies that subsequently aborted. Thymosin β₄ levels were similar between the two groups. CONCLUSIONS: Decreased maternal serum thymosin α₁ levels may be associated with periconceptional endocrine and/or immune disturbances preceding miscarriage.     

Discrepancy between biological activity of thymosin and its cyclic nucleotide-modulating activity

We have studied cyclic adenosine 3':5'-monophosphate (cAMP) and cyclic 3':5'-guanosine monophosphate (cGMP) concentration in cord blood lymphocytes (CBL) and in adult peripheral blood lymphocytes (PBL) after stimulation with thymosin V-1 and V-2. At concentrations of 100 micrograms/ml, thymosin V-1 produced an 85% increase of cAMP in adult PBL, alterations being demonstrated within 1 min, whereas it did not affect the cAMP level in CBL which have a nine times higher basal level than PBL. On the other hand, thymosin V-1 did not have any effect on cGMP in CBL nor in adult PBL. Thymosin V-2 which inhibits E-rosette formation also increased cAMP in adult PBL like thymosin V-1. The increasing effect of thymosin V-1 on cAMP was observed even when cAMP phosphodiesterase was completely inhibited with isobutyl methylxanthine. From these data it was assumed that thymosin probably acts through the stimulation of adenylate cyclase.     

Amniotic Fluid Thymosin α1 Levels Increase During Gestation

ABSTRACT: Thymosin α₁ is one of several cytokines produced by the thymus that modulates immune function. The presence of elevated serum levels of thymosin α₁ in pregnant women and their newborns has suggested that this peptide may play a role in perinatal immunology. In this investigation, we used a microenzyme-linked immunosorbent assay to assay amniotic fluid for immunoreactive thymosin α₁ and found levels that were remarkably higher than newborn serum levels (P < 10^?4). The increase of thymosin α₁ in amniotic fluid with fetal age was natural logarithmic (r = 0.838, P < 10^?6). Thymosin α₁ in amniotic fluid may account for some of the immunologic properties of this medium.     

Immunogenicity of the ALLAVGATK (gp10017 – 25) peptide in HLA-A3.1 melanoma patients

A T cell line recognizing autologous and allogeneic HLA-A3.1 melanomas was obtained from a disease-free melanoma patient (patient 15392). By transfection of a tumor cDNA library and in vitro sensitization experiments, the ALLAVGATK gp100/Mel17-derived peptide was found to be the epitope recognized by this melanoma-specific T cell line. The role of the ALLAVGATK peptide in the systemic immune response to melanoma of this patient was evaluated. When pulsed on the autologous peripheral blood mononuclear cells, the ALLAVGATK peptide generated tumor-specific HLA-A3-restricted T lymphocytes and a single restimulation in vitro was sufficient to raise gp100-specific T lymphocytes, indicating a high frequency of epitope-specific T cells. gp100-specific T cells were also induced from T lymphocytes purified from tumor-invaded lymph nodes (tumor-associated lymphocytes, TAL). TAL-derived effectors displayed lower peptide affinity and lower tumor recognition than effectors elicited from peripheral blood lymphocytes (PBL). To further evaluate its immunogenicity, ALLAVGATK was used to stimulate PBL derived from six additional HLA-A3.1 melanoma patients and seven healthy donors. After 7 weeks of peptide stimulation in vitro the generation of anti-gp100 and tumor-specific T cell lines was achieved in one out of the six patients analyzed. Taken together these data indicate that an in vivo priming leading to a systemic immunity against gp100 in HLA-A3 melanoma patients may occasionally occur and that the immunogenicity of ALLAVGATK peptide in melanoma patients is comparable to that of other HLA-A2-restricted epitopes derived from gp100/Mel 17 protein.     

Effect of Thymic Hormones on Interleukin 2 Synthesis by Lymphocytes from HIV-Positive PRE-AIDS Subjects

The PHA-induced synthesis of interleukin 2 (IL-2) by peripheral blood lymphocytes (PBL) from 9 normal and 8 pre-AIDS individuals was evaluated. IL-2 content in supernatant fluids of PBL cultures derived from pre-AIDS patients was only around 20% of that found in normal PBL cultures. The addition of two thymic preparations, thymosin faction 5 and TFX-Polfa, to PHA-stimulated PBL cultures from pre-AIDS patients caused significant increase of IL-2 content in cultures. Thymosin alfal was ineffective in this respect. However, thymic factors corrected only partially the defective IL-2 synthesis by PBL from pre-AIDS patients increasing it to ca. 35% of value for normal PBL. The findings suggest the potential of PBL from pre-AIDS patients to respond in vitro to enhancing activity of thymic hormones.     

Differentiation changes in cord blood T lymphocytes induced by synthetic C-terminal peptides of thymosin alpha 1

Thymosin fraction 5 and its component thymosin alpha 1 has been reported to play a regulatory role in the later stages of T lymphocyte differentiation. Previous studies from our group have also demonstrated that thymosin fraction 5 can induce changes in purine degradative enzymes and in surface antigenic markers of cord blood lymphocytes, which have been shown to be immature compared with normal adult lymphocytes. Birr et al., have shown that the C-terminal region of thymosin alpha 1 is essential for the biological activity. In this study, the effects of these synthetic peptide fragments on cord blood T lymphocytes were studied. After incubation with different peptide sequences, cord blood lymphocytes were analysed for surface antigenic markers (OKT4/OKT8) and for purine degradative enzymes (PNP, 5 NT). In the range of 1.3 X 10 to 1.3 X 10(-5)M, one of the eleven peptides examined (sequence 13-19) exhibited activity approximately equal to that of the parent peptide thymosin alpha 1: increase in activity of 5'NT (p less than 0.01) and reduction of cells positive for OKT4 (p less than 0.01). Another sequence (20-28) was able to induce an increase in 5'NT only and a third one (20-25) a reduction of cells positive for OKT4. The results obtained in this study confirm that the C-terminal region contains molecular signals for T cell differentiation.     

Cellular Immunity in Human Milk

ABSTRACT: The responses of human milk lymphocytes (MIL) to a variety of immunogenic stimuli were studied and compared to those of peripheral blood lymphocytes (PBL) from the milk donors. MIL showed a decreased proliferative response to mitogens and allogeneic leukocytes in vitro but displayed the ability to stimulate alloreactivity equivalent to PBL. Neither pretreatment with cell-free autologous milk nor co-cultured MIL were capable of suppressing the proliferative responses of PBL. Moreover, macrophages isolated from milk and pulsed with soluble antigen or allogeneic cells effectively induced proliferation by peripheral blood T cells whereas the response of milk nonadherent cells to antigen presented by peripheral macrophages was very low. MIL respond better to pathogenic enteric E. coli than PBL but not as well as PBL to Yersinia enterocolitica. Treatment of MIL with monoclonal antibodies cytotoxic for T cells abolished their response to bacterial antigens. Application of an anti HLA class II antigen monoclonal antibody to mixed lymphocyte or lymphocyte-bacteria cultures resulted in substantial inhibition of the MIL response similarly to that of PBL. The relevance of these data to the immunological needs of the neonate are discussed.     

In Vitro Effects of Thymosin on T-Cell Subsets in Systemic Lupus Erythematosus

Abstract

The possible immunomodulatory influence of thymosin on lymphocytes from patients with active systemic lupus erythematosus (SLE) has been evaluated. Such patients have decreased numbers of T-suppressor (Tγ) cells and normal numbers of T-helper (Tμ) cells, resulting in an abnormally low Tγ/Tμ ratio. In vitro incubation of lymphocytes from active SLE patients with thymosin resulted in a normalization of the Tγ/Tμ ratio. This occurred because of a decrease in Ty cells rather than an increase in Tγ cells. The normalization of Tγ/Tμ ratios in vitro in the presence of thymosin is compatible with possible in vivo immunomodulatory effects of these peptides.     

Activation of α7nAChR by Nicotine Reduced the Th17 Response in CD4+T Lymphocytes

Background: nAChRs play an important role in the regulation and modulation of immune cell proliferation, differentiation, migration and cell-cell interactions. The present study was to characterize the expression of α7nAChR on human peripheral blood mononuclear cells (hPBMC) and CD4⁺T lymphocytes, and to explore the change of Th17 expression after activation of α7nAChR on human CD4⁺T lymphocytes.

Methods: A Ficoll gradient was used to separate hPBMC from whole blood, and then CD4⁺T lymphocytes were isolated by magnetic bead separation. The expression of α7nAChR on PBMC and CD4⁺T lymphocytes was analyzed using flow cytometry before and after stimulation with phytohemagglutinin (PHA). The effect of α7nAChR stimulation by nicotine or inhibition by α-bungarotoxin (α-BTX), as well as Th17 expression on the phenotype of CD4⁺T cells was evaluated using flow cytometric analysis.

Results: The percentage of CD4⁺T cells in reduced PBMC, while the expression of α7nAChR increased when cells were stimulated by nicotine. This effect vanished when co-treated with nicotine and α-BTX. α7nAChR was found to expressed in about 90% of CD4⁺T cells. However, α7nAChR expression reduced to 80% on CD4⁺T cells after stimulation with PHA for 24?h. Stimulation of α7nAChR with nicotine increased the expression of Th17 cells, and this upregulation reduced when AChRα7 was inhibited by α-BTX.

Conclusion: α7nAChR was ubiquitously expressed by CD4⁺T lymphocytes, which was correlated with the cell activation status. Meanwhile, activation of nAChRα7 by nicotine in CD4 cells reduced the Th17 response.     

肝癌术后早期应用胸腺肽α 1对T淋巴细胞亚群的影响

目的：探讨肝癌患者术后早期应用胸腺肽α1对T淋巴细胞亚群的影响。方法： 46例肝癌手术患者, 随机分治疗组（23例）和对照组（23例）, 治疗组于术后1、3、5 d皮下注射胸腺肽α1 1.6 mg,观察2组术前后第1、4、7 d CD3+、CD4+、CD8+、CD4+/CD8+的变化情况。结果：（1）组内比较：对照组术后CD4+、CD4+/CD8+低于术前（P<0.05）,术后第1、4、7 d CD8+高于手术前（P<0.05）。治疗组CD3+、CD4+、 CD4+/CD8+术后与术前相比无显著差异;CD3+、CD4+/CD8+术后第1、7 d比较有显著差异（P<0.05）。（2）组间比较：CD4+、CD4+/CD8+治疗组高于对照组（术后第1、4、7 d均P<0.05）,治疗组CD8+细胞百分率低于对照组（术后第1、4、7 d均P<0.05）;治疗组CD3+细胞百分率高于对照组（术后第4、7 d P<0.05）。结论： 手术对肝癌患者术后T淋巴细胞免疫功能有抑制作用,胸腺肽α1对T淋巴细胞免疫功能有调节作用。     

Thymosin alpha 1 modulates the expression of high affinity interleukin-2 receptors on normal human lymphocytes

In this report we demonstrate that thymosin alpha 1 (T alpha 1), a synthetic peptide composed of 28 amino acid residues, and thymosin fraction 5 (TF5) enhance the number of high affinity interleukin 2 receptors (IL-2R) expressed by human peripheral blood lymphocytes in response to in vitro stimulation with phytohemagglutinin (PHA). Thymosins did not, however, alter the affinity of the IL-2R for its ligand. Dose-response studies using a wide range of concentrations indicated a bimodal distribution of responsiveness to T alpha 1. In most experiments the high and low concentration peaks of activity were observed at 10(-8) M and 10(-12) M, respectively, although peak responses were observed at different T alpha 1 concentrations in different donors. No effects were elicited by thymosins in the absence of mitogenic stimulation. Thymosin enhancement of PHA-induced high affinity IL-2R expression directly correlated with increased levels of Tac antigen expression, as determined by flow cytometry, and enhanced interleukin 2 (IL-2) production. Since the biological effects of IL-2 are associated with the occupancy of high affinity IL-2R, the findings presented in this report strongly suggest that thymosins play a significant role in the regulation of immune responses through the modulation of high affinity IL-2R expression.     

Peripheral blood CD4+CD8+ lymphocytes in cynomolgus monkeys are of resting memory T lineage

In this study, we analyzed peripheral blood CD4+CD8+ double-positive (DP) lymphocytes in adult cynomolgus monkeys (Macaca fascicularis). Forty of 55 monkeys had > 5% of the peripheral blood DP subpopulation (9.3 +/- 5.9%; mean +/- SD) in peripheral blood lymphocytes (PBL) in contrast to a low percentage of peripheral blood DP cells in humans and mice. In a cross-sectional study, the peripheral blood DP cells were found to increase in proportion with age. To clarify whether peripheral blood DP lymphocytes were immature precursors released from thymus without prior differentiation, the expressions of CD8 chains and CD1b on peripheral blood DP lymphocytes were compared with those on thymocytes. The peripheral blood DP lymphocytes were CD8 alpha + beta- and CD1b-, while thymic DP lymphocytes were CD8 alpha + beta + and CD1b +, suggesting that the peripheral blood DP cells are extrathymic T lymphocytes. Furthermore, the peripheral blood DP lymphocytes exhibited a resting memory T cell phenotype with CD2hiCD3+CD28-CD29hiCD49dhiCD69- CD80lo. Taken together, adult cynomolgus monkeys possess a unique peripheral blood DP T cell subpopulation which expresses a resting memory T cell phenotype. In addition, similar phenotypic properties of DP lymphocytes were distributed in the spleen and lymph nodes, although the proportion was less in the spleen and much less in lymph nodes than in PBL.      

The Capacity of Lymphokine Production by Peripheral Blood Lymphocytes from Aged Humans

The functional capability to produce interleukin 2 (IL2) and interferon gamma (IFNγ) by peripheral blood lymphocytes (PBL) from elderly humans was evaluated. Thirty-nine samples of PBL derived from donors aged from 56 to 79 yr were cultured with phytohaemagglutinin (PHA) as stimulus at 37°C for 48 hours. The IL2 activity in the supernatants was measured by its ability to sustain the IL2-dependent CTLL growth. Cultures of elder human T cells produced low level of IL2 activity, that is on the average of 9.7±9.8 μ/ml, equivalent to 28.1% the IL2 activity in parallel cultures of PBL derived from young donors (34.2±8.3 μ/ml, assessed on the basis of 145 samples). In the elder humans, the IL2 production by PBL decreased progressively as the increase of the age of the donors.

In contrast to the impairment of IL2 production, the amount of IFNγ secretion by elder human T cells was almost at the same level as that by young human T cells. The average IFNγ activity of 39 samples of elder PBL cultures and 128 samples of young PBL cultures was 2,961±736 μ/ml and 3133±950 μ/ml, respectively.

There was no significantly positive correlation between the level of IL2 and IFNγ when comparison was made based on each individual samples. This implies that it may exist an alternative way of IFNγ production which does not require the induction by IL2, and the relatively high level of IFNγ may not impose adverse effect on the IL2 production.     

The Effect of Levamisole on Peripheral T-Lymphocytes of Patients with Malignant Lymphomas

Abstract

The effect of various concentrations (0.015–10 μg/ml) of Levamisole (LMS) on the peripheral lymphocytes of patients with malignant lymphomas (ML) and normal donors was investigated in vitro. The parameters studied include : E rosettes forming cells (total T lymphocytes), active E rosettes (early T lymphocytes) and DNA synthesis induced by pitogens PHA and Con A.

LMS improved significantly lymphocyte response both in patients with ML and normal donors when the cells were stimulated by Con A. In both groups no significant effect was observed on the response to PHA nor on the percentage of E-rosettes, whereas the mean number of active E rosettes was significantly increased on all concentrations of the drug.

While in the normal subjects a positive statistical correlation between active E-rosettes and Con A response was observed, in patients with ML an inverse correlation was found. This latter correlation was partially reversed by LMS.     

Transfer of immune components from rabbit autoimmune cardiomyopathy into severe combined immunodeficiency (SCID) mice induces cardiomyopathic changes

Background: Growing evidence suggests that autoimmune mechanism plays an important role in the pathogenesis of cardiomyopathy. The purpose of this study was to investigate whether passive transfer of IgG and/or lymphocytes from rabbits with autoimmune cardiomyopathy is able to reproduce cardiomyopathic changes in severe combined immunodeficiency (SCID) mice.

Methods and results: SCID mice were injected intraperitoneally with IgG and/or peripheral blood lymphocytes (PBL) from either rabbits immunized with both β1-adrenoceptor peptide and M2-muscarinic receptor peptide (β1+M2 group) or rabbits with adjuvant (N group). Thirty five SCID mice were divided into seven groups; N-IgG, N-PBL, N-IgG &; PBL, (β1+M2)-IgG, (β1+M2)-PBL, (β1+M2)-IgG &; PBL and control groups. Heart weight in three (β1+M2) groups were significantly increased. All mice in three (β1+M2) groups showed high titer of rabbit anti-β1 adrenocepter autoantibodies, and 4 mice in the (β1+M2)-PBL group and 3 mice in the (β1+M2)-IgG &; PBL group showed a significant increase in titer of rabbit anti-M2-muscarinic receptor autoantibodies. Focal infiltration of inflammatory cells in the myocardium was observed in the (β1+M2)-IgG &; PBL group. In the (β1+M2)-PBL group and (β1+M2)-IgG &; PBL group, cardiomyocyte diameters were significantly increased. Some myocytes of the (β1+M2)-IgG &; PBL group exhibited intracellular edema, clumps of Z-band and increased numbers of mitochondria by using electron microscopy.

Conclusion: Transfer of IgG and PBL from rabbits immunized with combined β1 and M2 peptides was able to reproduce the early stage of cardiomyopathic changes in SCID mice     

Role of Bacterial Lipopolysaccharides in the Development of Natural Antibacterial Activity Mediated by Human Peripheral Blood T Lymphocytes

The role of bacterial lipopolysaccharides (LPS) has been evaluated for their influence on the human T cell-mediated anti-Salmonella typhi activity. In nonendemic areas for salmonellosis this activity is exerted by CD4⁺ lymphocytes armed by IgA, whereas in endemic zones besides these cells also CD8⁺ lymphocytes armed by IgG display an elevated anti bacterial activity. These results suggest that, in endemic regions, continuous antigenic challenge and, in particular, that exerted by lipid A (the active moiety of LPS) may play a role in triggering this activity.

In other series of experiments, pretreatment of endemic peripheral blood lymphocytes (PBL) with smooth and rough (Rb and Re) forms of Salmonella LPS leads to the inhibition of antibacterial activity. In this respect, Re-LPS, which contains lipid A covalently linked to the core-chetodeoxyoctonate, gives rise to the maximum of inhibition.

Finally, fractionation of PBL by means of S. minnesota R345 (Rb) cytoadherence has led to the conclusion that anti bacterial activity is present in the Rb-unbound population, thus indicating that bacterial adherence to PBL is a distinct phenomenon from natural anti-S. typhi activity.

The overall results suggest that lipid A is able to modulate the expression of antibacterial activity exerted by human peripheral blood T cells.     

The Influence of Thymosin on Affinity Binding in the E-Rosette Assay

Human peripheral blood lymphocytes (PBL) were analyzed in the E rosette assay with respect to (1) the effect of SRBC/ lymphocyte ratio on rosette number; (2) the effect of this ratio on the presence of lymphocytes with multiple binding sites for SRBC; and (3) the influence of thymosin on increasing the number of lymphocyte receptors for SRBC. An increase in the SRBC/ lymphocyte ratio was associated with an increase in rosette number. The number of multiple binding rosettes was also dependent on the SRBC/lymphocyte ratio. At saturating SRBC concentrations, greater than 30% of the rosettes were in the morula form. Treatment of PBL with thymosin was associated with a slight increase in the number of total rosettes and a large increase in number of morulas. It is suggested that the morula represents a mature form of T cell in contrast to the 2-3 SRBC rosette, which may be induced by thymosin to become a morula form.     

The influence of thymosin on affinity binding in the E-rosette assay.

Human peripheral blood lymphocytes (PBL) were analyzed in the E rosette assay with respect to (1) the effect of SRBC/lymphocyte ratio on rosette number; (2) the effect of this ratio on the presence of lymphocytes with multiple binding sites for SRBC; and (3) the influence of thymosin on increasing the number of lymphocyte receptors for SRBC. An increase in the SRBC/lymphocyte ratio was associated with an increase in rosette number. The number of multiple binding rosettes was also dependent on the SRBC/lymphocyte ratio. At saturing SRBC concentrations, greater than 30% of the rosettes were in the morula form. Treatment of PBL with thymosin was associated with a slight increase in the number of total rosettes and a large increase in number of morulas. It is suggested that the morula represents a mature form of T cell in contrast to the 2 - 3 SRBC rosette, which may be induced by thymosin to become a morula form.