Enumeration of lymphokine-secreting cells as a quantitative measure for cellular immune responses in rhesus macaques

Abstract: We investigated whether enumeration of lymphokine-secreting T cells can be used as a quantitative measure to determine the immunogenicity of foreign proteins in rhesus monkeys. In addition, it was assessed whether this approach can supplement and/or substitute for the well-established lymphoproliferation assay. Two candidate vaccine proteins (e.g., HIV-1 gp120 and HSV-2gD) were used as model antigens for immunization. PBMCs from immunized animals were antigenically stimulated and evaluated on their proliferative capacity and lymphokine release at the single cell level. The experiments showed a close quantitative correlation between antigen-triggered proliferative responses and the antigen-induced generation of Il-2 and IFN-gamma producing cells (pc). Il-4pc were found to appear relatively late after the initiation of antigen exposure. The data indicate that ELISPOT assays provide valuable tools for the assessment of the antigenicity of foreign proteins in vivo.     

Comparative analysis of natural killer cell activity, lymphoproliferation and lymphocyte surface antigen expression in nonhuman primates housed at the CIRMF Primate Center, Gabon

Six different species of nonhuman primates housed at the CIRMF Primate Center, cynomolgus monkeys (Macaca fascicularis), rhesus monkeys (Macaca mulatta), mandrills (Mandrillus sphinx), vervets (Cercopithecus aethiops pygerythrus), chimpanzees (Pan troglodyte) and baboons (Papio hamadryas), were evaluated for their natural killer cell activity and for the ability of their peripheral blood mononuclear cells to proliferate in response to known mitogens (concanavalin A, phytohemagglutinin and staphylococcal enterotoxin A) and to react with a panel of mouse monoclonal antibodies directed against human leukocyte surface antigens. Basic information on normal immune functions in these primates is important because of their use as experimental animal models for the study of human diseases such as acquired immunodeficiency syndrome (AIDS), hepatitis, loiasis and malaria.     

A comparative study of phagocytic activity and lymphoproliferative response in five varieties of tilapia Oreochromis spp.

The immunological status of three native species of tilapia Oreochromis spp. and two hybrids were compared, showing a greater potential of the hybrid strain O. niloticus Rocky Mountain for aquaculture and a better capacity to resist the stressful conditions of a fish farm.     

Cellular immune response of patients with chromoblastomycosis undergoing antifungal therapy

One of the most characteristic features of the chromoblastomycosis is its unresponsiveness to treatment. In order to analyzed whether during therapy could be observed a change of cellular immune response pattern, we evaluated the production of IL-10, TNF-α and IFN-γ, as well as proliferation of peripheral blood mononuclear cell (PBMC) from patients in different periods of chemotherapy treatment. Our results showed that after 6 months of treatment cells from patients proliferated to fungal antigens and produced a significant level of IFN-γ. However, after 1 year of treatment a low proliferation of T cells and production of IFN-γ accompanied by an increase of IL-10 were observed when compared with 6 months of treatment.     

Correlation of T-cell response and lymphokine profile with RESA peptides of Plasmodium falciparum containing a universal T-cell epitope and an immunopotentiator, polytuftsin.

Two RESA repeat sequences, (EENVEHDA)2 and (DDEHVEEPTVA)2, were chemically linked to a universal T-cell epitope, CS.T3 and polytuftsin, and a natural immunopotentiator, was physically mixed with these conjugates. The immunogens were studied for in vitro antigen-induced T-cell proliferation, and cytokine levels were measured in the culture supernatants. The RESA peptide(s)-CS.T3 conjugate containing polytuftsin showed the highest stimulation index (SI) as compared to the RESA peptide-CS.T3 conjugates or RESA peptides alone. Spleen cells from mice primed with either RESA peptide(s)-CS.T3 conjugate or RESA peptide-CS.T3 conjugate containing polytuftsin, when pulsed in vitro with the respective RESA peptide, showed a higher proliferation index as compared to spleen cells primed and pulsed in vitro with the respective RESA peptides. This observation has an important relevance during natural reinfection for boosting the immune response. The culture supernatants from the cells primed and pulsed in vitro with RESA peptide-CS.T3 conjugate and RESA peptide-CS.T3 conjugate containing polytuftsin showed higher IL-2 and IFN-gamma levels as compared to the RESA peptides alone. Very low IL-4 levels were detected with the above formulations. The cytokine profile is suggestive of a CD4+ TH1 type of immune response, which is ideal for the killing of intracellular pathogens like the malarial parasite.     

Role of mannose binding lectin gene variants on its protein levels and macrophage phagocytosis with live Mycobacterium tuberculosis in pulmonary tuberculosis

Mannose-binding lectin (MBL) plays an important role in innate immunity. Functional mutant homozygotes of the MBL gene affect the serum MBL levels and have been correlated with disease susceptibility. We have studied the regulatory role of variant MBL genotypes on serum MBL level and macrophage phagocytosis with live Mycobacterium tuberculosis, and the lymphoproliferative response to M. tuberculosis culture filtrate antigen in pulmonary tuberculosis (PTB) patients (n = 48) and normal healthy subjects (NHS) (n = 58). The total serum MBL level was higher in PTB patients than in NHS (P = 0.0085). Patients and NHS with AA genotype (homozygotes of MBL - common alleles) showed a very high serum MBL level, and those with OO genotype (functional mutant homozygotes of MBL - less frequent alleles) showed a very low MBL level (AA vs. OO: NHS, P = 3.3 x 10(-9); PTB, P = 3.1 x 10(-9)). A significantly lower phagocytosis was observed in NHS with AA genotype than in NHS with AO (heterozygotes) genotype (P = 0.046). In PTB patients, no such difference was observed. A negative correlation of macrophage phagocytosis with MBL level was seen in patients and NHS (P = 0.019). Antigen-induced lymphoproliferative response was significantly decreased in PTB patients with AA genotype as compared with NHS with AA genotype (P = 0.036). The present study suggests that AA genotype with its associated higher serum MBL levels plays a regulatory role in immunity to tuberculosis than functional mutant homozygotes (OO genotype) with its associated lower level of MBL.     

The potentially insecticidal Narcissus pseudonarcissus lectin demonstrates age-related mitogenicity

Lectins from monocotyledonous plants such as Narcissus pseudonarcissus (NPA) possess insecticidal properties and have the potential to increase pest resistance in transgenic crops. Therefore it is of interest to investigate the mitogenic properties of such lectins. Mononuclear cells purified from human umbilical cord and adult peripheral blood samples were stimulated with NPA and compared to phytohaemagglutinin as an example of a lectin from a dicotyledonous plant. Here we report that NPA is slightly mitogenic for adult human lymphocytes but mitogenicity is increased more than sevenfold for lymphocytes from umbilical cord blood. Similarly, NPA was found to be mitogenic for peripheral blood mononuclear cells (PBMC) from lambs and not adult sheep, supporting the age-related mitogenicity and indicating that further examination of the younger human population is warranted.     

Effects of feed restriction and supplementary folic acid and vitamin B12 on immune cell functions and blood cell populations in dairy cows

Cows undergoing a negative energy balance (NEB) often experience a state of immunosuppression and are at greater risk of infectious diseases. The present study aimed to evaluate the impact of a folic acid and vitamin B₁₂ supplement and feed restriction on several immune parameters. Sixteen cows at 45 ± 3 days in milk were assigned to 8 blocks of 2 cows each according to each cow’s milk production in the previous week, and within each block, the cows randomly received weekly intramuscular injections of either saline or 320 mg of folic acid and 10 mg of vitamin B₁₂ for 5 weeks. During week 5, the cows were fed 75% of their ad libitum intake for 4 days. Blood samples were taken before the beginning of the experiment, just before feed restriction and after 3 days of feed restriction, in order to evaluate blood cell populations, the phagocytosis capacity and oxidative burst of polymorphonuclear leukocytes (PMNs), the proliferation of peripheral blood mononuclear cells (PBMCs) and concentrations of non-esterified fatty acids (NEFAs) and β-hydroxybutyrate. The vitamin supplement did not affect any of the tested variables except milk fat and lactose content. Feed restriction reduced milk production and increased the concentration of NEFAs. Feed restriction did not affect blood cell populations but did reduce the percentage of PMN positive for oxidative burst after stimulation with phorbol 12-myristate 13-acetate. The proliferation of PBMCs was reduced when the cell culture medium was supplemented with sera collected during the feed restriction. In conclusion, feed restriction affected the functions of PMN and PBMC and this effect was not prevented by the folic acid and vitamin B₁₂ supplement. These results support the hypothesis that the greater risk of infectious diseases in cows experiencing a NEB is related to impaired immune cell functions by high circulating concentration of NEFAs.     

Modulatory effects of interferon‐γ and interleukin‐4 on cellular immune responses against Hypoderma lineatum antigens

This study investigates the in vitro modulatory effects of interferon‐γ (IFN‐γ) and interleukin‐4 (IL‐4) on both proliferative bovine T cell responses and IL‐10 production induced by different antigens [crude larval extract and the purified fractions hypodermin A, B and C (HyA, HyB, HyC)] obtained from first instars of Hypoderma lineatum (Diptera: Oestridae), alone or in the presence of the mitogen concanavalin A. Incubation with the different parasitic antigens resulted in significant inhibition of T cell proliferation and IL‐10 production, which, in general, did not revert after the addition of IFN‐γ and IL‐4. In the absence of antigens, IL‐4 induced significant inhibition of mitogen‐induced T cell responses. Exogenous IFN‐γ exhibited an inhibitory effect on cell proliferation in the presence of the purified fractions HyB and HyC. These in vitro data suggest that far from neutralizing the effects of larval antigens, the addition of IFN‐γ potentiates their anti‐proliferative activity; by contrast, IL‐4 had no consistent effects on proliferative responses to Hypoderma. IL‐4 provoked an increment of IL‐10 levels in supernatants of HyB‐stimulated cells. In conclusion, exogenous IFN‐γ and IL‐4 were unable to counteract the suppressor effects of H. lineatum antigens.