George J. Reclos

Immunoregulatory effects of fraction 5 thymus peptides. I. Thymosin α1 enhances while thymosin β4 suppresses the human autologous and allogeneic mixed lymphocyte reaction

Thymosin alpha 1 and thymosin beta 4, two peptides isolated from preparations of calf thymus fraction 5, were tested in the human mixed lymphocyte reaction (MLR). Thymosin alpha 1 was found capable of enhancing both the allogeneic and autologous MLR. On the contrary, thymosin beta 4 suppressed MLR proliferative responses. Study of the responses of the T cell subpopulations revealed that T4+ (helper/inducer) cells but not T8+ (suppressor/cytotoxic) are responsible for the enhanced, proliferative response to allo- and autoantigens in the presence of thymosin alpha 1. Both the autologous and the allogeneic proliferative responses of either T4+ cells or T8+ cells were not influenced by the addition of thymosin beta 4 in the cultures. However, when T4+ and T8+ subsets were cocultured, thymosin beta 4 was capable of activating T8+ cells to suppress the allogeneic and the autologous proliferative response of T4+ cells. These studies show that thymosin fraction 5 peptides exert immunoregulatory effects on the human MLR proliferative responses in vitro.

Peptides of myelin basic protein stimulate T lymphocytes from patients with multiple sclerosis

Peripheral blood T lymphocytes from patients with multiple sclerosis (MS) and other neurological diseases (OND) were tested for primary in vitro proliferation in response to four synthetic peptides derived from the sequence of human myelin basic protein (HuMBP) and to HuMBP 45-89 peptide fragment, using a [3H]thymidine incorporation assay. The synthetic peptides used corresponded to residues HuMBP 15-31, 75-96, 83-96 and 131-141 of human myelin basic protein. Significant proliferation of T lymphocytes to peptides was noted only in the MS group (with the exception of peptide 131-141): the majority of control subjects and OND patients did not respond to the above-mentioned peptides. The sensitized T lymphocytes in MS patients displayed the inducer/helper phenotype and required autologous monocytes for optimal proliferation. An anti-HLA-DR monoclonal antibody, directed against a monomorphic determinant of DR molecules, was able to block the responses in a dose-dependent fashion. These results suggest that autoimmune inducer/helper T lymphocytes in the peripheral blood of MS patients may initiate and/or regulate the demyelination process in patients with MS. Furthermore, our data demonstrate that monocytes and HLA-DR molecules are essential for activation of these cells. Finally primary in vitro T cell proliferation to HuMBP synthetic peptide may be used as an additional diagnostic test in MS.

Enhancement of human T lymphocyte functions by prothymosin α. I. Augmentation of mixed lymphocyte culture reactions and soluble protein-induced proliferative responses

Prothymosin alpha (ProT alpha), a 115-amino-acid thymic polypeptide, was tested for its effect on soluble antigen, allo- and auto-antigen-induced human T-cell proliferation. ProT alpha enhanced the secondary T-cell proliferative response to ovalbumin (OVA)- and keyhole limpet haemocyanin (KLH)-pulsed antigen-presenting cells (peripheral blood monocytes). Maximum enhancement (20-fold for OVA and 23-fold for KLH) occurred when suboptimal concentrations of either OVA or KLH were employed. Subset depletion experiments showed that the helper/inducer T-cell subpopulation was responsible for the observed enhancement. In the mixed lymphocyte reaction (MLR), ProT alpha enhanced autoantigen- (autoMLR; 9- to 14-fold) as well as the alloantigen- (alloMLR; 8- to 10-fold) induced T-cell proliferation when suboptimal ratios of the participating cells were used. Preincubation of the stimulating (autologous or allogeneic monocytes) with ProT alpha induced significantly higher T-cell proliferation in both primary and secondary MLR responses as compared to that induced by non-treated monocytes. In contrast, T lymphocytes pre-incubated with ProT alpha did not show enhanced proliferative activity when tested subsequently in the MLR. Suboptimal numbers of T cells exhibited high proliferative activity when pre-incubated with ProT alpha in the presence of autologous monocytes. These studies suggest that ProT alpha potentiates T-cell proliferative responses not directly, but via monocytes which are included in the cultures either as antigen-presenting cells or accessory and/or stimulator cells. The importance of ProT alpha in pathologically occurring defective cellular immune response systems discussed.

Prothymosin α Restores the Depressed Autologous and Allogeneic Mixed Lymphocyte Responses in Patients with Systemic Lupus Erythematosus

AbstractSUMMARY Systemic lupus erythematosus (SLE) is characterized by a variety of profound T-cell abnormalities among which are decreased autologous and allogeneic mixed lymphocyte reactions (auto-MLR and allo-MLR, respectively). In a group of 10 patients with SLE, the mean auto-MLR and allo-MLR responses, tested by tritiated thymidine incorporation, were significantly decreased. If optimal doses of highly purified prothymosin a (ProT α) were present during the auto- of allo-MLR, the T-cell proliferative responses of SLE patients were increased to normal levels. ProT α had more pronouced enhancing effect in patients than in normal individuals. Among patients, ProT α was more effective in those who had active disease and low proliferative responses. These results demonstrate that ProT α can fully restore the deficient T-cell proliferative responses in auto- and allo-MLR in patients with SLE. ProT α, or a certain peptidic fragment of it, could prove potentially useful in the treatment of SLE.

Enhancement of Human T Lymphocyte Function by Prothymosin α: Incremed Production of Interleukin-2 and Expression of Interleukin-2 Receptors in Normal Human Peripheral Blood t Lymphocytes

The in vitro incubation of phytohemagglutinin (PHA)- or alloantigen-stimulated peripheral blood T cells with prothymosin alpha (ProT alpha) resulted in a marked and reproducible increase in the production of interleukin-2 (IL-2). Incubation of T cells with ProT alpha, in the absence of PHA or alloantigen, failed to induce any production of IL-2. ProT alpha by itself did not exert any IL-2 activity. Finally, ProT alpha was shown to increase the expression of IL-2 receptors on phytohemagglutinin- or alloantigen-activated T cells. These data provide the basis for understanding the in vitro immunoenhancing effects of ProT alpha in cellular immune systems.

Decreased expression of HLA-DR antigens on monocytes in patients with multiple sclerosis

Immunofluorescence, cell binding assays and enzyme immunoassays were used to investigate the expression of class II major histocompatibility antigens on peripheral blood monocytes in 67 patients with multiple sclerosis. Monocytes from patients with active disease expressed fewer HLA-DR molecules on their surface than normal monocytes; furthermore the percentage of cells which exhibited detectable amounts of surface HLA-DR antigens was decreased in patients with active multiple sclerosis. During the inactive stage of the disease both deficiencies were milder, probably representing secondary pathogenetic phenomena. Quantitation of monocyte surface HLA-DR antigen expression could be valuable in assessing the clinical disease activity. The demonstration of a molecular defect in patients with multiple sclerosis will improve our understanding of the pathogenesis of the disease.

Prothymosin-α enhances HLA-DR antigen expression on monocytes from patients with multiple sclerosis

Abstract Monocytes from patients with multiple sclerosis (MS) express decreased numbers of class II major histocompatibility complex (MHC) antigens in peripheral blood and are poor stimulators in the autologous mixed lymphocyte reaction (autoMLR). We assessed the effect of prothymosin-α (ProTα) on the expression of MHC class II antigens by monocytes. Immediately after isolation, monocytes were analyzed for MHC class II antigen expression using a radiolabelled monoclonal antibody specific for a monomorphic determinant on HLA-DR antigens. After incubation with ProTα we observed significant increases in HLA-DR antigens on MS monocytes (1.5- to 4-fold increase compared to freshly isolated monocytes). Kinetic analysis revealed that enhancement peaked after 2 days of incubation with ProTα. The increase in HLA-DR antigen on MS monocytes resulted in the restoration of the deficient autoMLR in MS patients. This is the first demonstration suggesting a link between HLA-DR antigen expression and cellular immune defects in MS. The significance of low autoMLR responses for T suppressor levels in MS patients is discussed.

Mechanism of Action of Prothynosin α in the Human Autologous Mixed Lymphocyte Response

AbstractProthymosin α(Prota), an immunologically active polypeptide derived initially from rat thymus, and now pig thymus, was tested for its effect on autoantigen-induced human T cell proliferation in vitro. Pig ProTa was found to enhance the autologous mixed lymphocyte response (auto-NLR). Optimum enhancement was achieved at doses which varied among different donors. Treatment of the stimulatory monocytes with ProTa resulted in considerably higher auto-MLR responses as compared to those with non treated monocytes. ProTa was without effect on T lymphocytes. In contrast, T lymphocytes exhibited enhanced proliferative activity when treated with ProTa in the environment of autologous monocytes. Horeover, supernatants from cultures of monocytes incubated with ProTa(ProTa-sup) were also shown to enhance the human auto-NLR either after addition in cultures or after preincubation with responder T lymphocytes. In addition, ProTa-sup did not demonstrate any detectable inter-leukin I (IL 1) or interleukin 2 (1L 2)...

Evaluation of glucose-6-phosphate dehydrogenase activity in two different ethnic groups using a kit employing the haemoglobin normalization procedure.

AIM Correct evaluation of Glucose-6-Phosphate Dehydrogenase (G-6-PD) activity of two ethnic groups using a fully quantitative kit with a simultaneous Hemoglobin Normalization (Hb Normalization) procedure. DESIGN AND METHODS Two groups of mothers and their healthy full term newborns of Greek (n = 1.166) and Albanian (n = 818) origin were tested for their G-6-PD activity employing a direct normalization protocol. RESULTS Greek mothers and newborns showed a higher prevalence for G-6-PD deficiency as compared to those of Albanian origin. Males of G-6-PD deficient mothers confirmed the efficacy of the method. CONCLUSION A fully quantitative G-6-PD kit employing Hb Normalization is essential for the correct classification of G-6-PD activity, both in male and female subjects.

Sister-chromatid exchange in highly purified human CD4+ and CD8+ lymphocytes

Sister-chromatid exchange (SCE) frequencies were determined in human peripheral blood CD4+ and CD8+ T lymphocyte subpopulations which were rapidly and highly purified from pooled T lymphocytes by immunological methods. The purified lymphocytes were stimulated with phytohemagglutinin (PHA) for 4 days. CD4+ lymphocytes showed significantly higher SCE frequencies than autologous CD8+ lymphocytes when measured simultaneously after identical bromodeoxyuridine (BrdU) incubation times. Differences in SCE frequencies between CD4+ and CD8+ lymphocytes were also detected when mitomycin C (MMC) was added to the cultures. Higher SCE frequencies in CD4+ lymphocytes were associated with lower proliferating rate indices (PRI) as compared to autologous CD8+ lymphocytes. Abnormalities in CD4+ T lymphocyte function and number in peripheral blood have been observed in several diseases characterized by immunological disorders. Thus, our data may suggest a link between some immunological disturbances and abnormal SCE frequencies in T lymphocyte subsets.