Spontaneous Lymphocyte Proliferation Research Articles

The histone lysine methyltransferase MLL1 regulates the activation and functional specialization of regulatory T cells

The activation and specialization of regulatory Tcells (Tregs) are crucial for maintaining immune self-tolerance; however, the regulation of these processes by histone modifications is not fully understood. Here, we show that Tcell-specific deletion of the lysine methyltransferase MLL1 results in a spontaneous lymphocyte proliferation phenotype in aged mice without disturbing the development of conventional Tcells and Tregs. Treg-specific MLL1 ablation leads to a systemic autoimmune disease associated with Treg dysfunction. Moreover, RNA sequencing demonstrates that the induction of multiple genes involved in Treg activation, functional specialization, and tissue immigration is defective in MLL1-deficient Tregs. This dysregulation is associated with defects in H3K4 trimethylation at these genes' transcription start sites. Finally, using a T-bet fate-mapping mouse system, we determine that MLL1 is required to establish stable Th1-type Tregs. Thus, MLL1 is essential in optimal Treg function by providing a coordinated chromatin context for activation and specialization.

Photoperiodic regulation of the splenocyte immune responses in the fresh water snake, Natrixpiscator

Photoperiod and melatonin are important regulators of immunity. We hypothesized that these two factors play an important role in the regulation of immune responses in the Natrix piscator. Animals were kept in either short or long days and splenocyte immune responses were studied. Respiratory burst activity of splenocytes was assessed through reduction of nitrobluetetrazolium salt while production of nitric oxide was assessed indirectly by nitrite assay. Density gradient centrifugation was used to isolate splenic lymphocytes which were utilized to study proliferation with and without mitogens. Super oxide production by splenocytes was reduced significantly in the cultures obtained from animals kept either in short or long days. Nitrite release was decreased when animals were subjected to long days. The photoperiodic alterations acted differentially on proliferations of the splenic lymphocytes. Spontaneous and mitogen-induced proliferation of splenic lymphocytes were enhanced in cultures obtained from snakes maintained in short days when compared with cultures from snakes obtained either from long day or natural day length conditions. In vitro melatonin significantly enhanced the splenic lymphocyte proliferation of the cultures obtained from animals kept in long days when compared with splenic lymphocyte proliferations of the cultures obtained from long day animals or the animals kept in natural day length conditions. We found evidence which suggest that photoperiod may influence seasonal energy budgets and induce adjustments which optimize energy allocation for costly physiological processes such as immune function. In seasonally breeding animals such as Natrix piscator, the pineal hormone melatonin assists in the suppression of reproduction and elevation of immunity, which are the crucial adaptation for perpetuation of species.

Methyl mercury (MeHg) in vitro exposure alters mitogen-induced lymphocyte proliferation and cytokine expression in Steller sea lion (Eumetopias jubatus) pups

Steller sea lions (Eumetopias jubatus, SSLs) are managed as two distinct population segments within U.S. waters: the endangered western distinct population segment and the recently delisted eastern distinct population segment. Recent studies reported concentrations of mercury in several tissues collected from young SSLs in the Aleutian Islands that were at or above concentrations found to negatively impact health in other fish-eating mammals. However, there are limited studies which have investigated the range of mercury concentrations that may negatively influence the SSL immune system. This study assessed relationships between methyl mercury (MeHg+) concentrations and two immune functions, lymphocyte proliferation and cytokine expression. Peripheral blood mononuclear cells (PBMCs) were isolated and cryopreserved from pups on three rookeries within the western distinct population segment: Chiswell Island, Ulak, and Agattu Islands. Lymphocyte proliferation and cytokine expression were assessed in vitro using thawed PBMCs with exposure to MeHg+ (unexposed control, 0.001, 0.01, and 0.1 μg/ml). Lymphocyte proliferation was measured without and with stimulation with a T cell mitogen (ConA) and B cell mitogen (LPS) and the concentration of cytokines was measured in the cell culture supernatant (with and without ConA or LPS). Spontaneous lymphocyte proliferation was significantly increased at 0.01 and 0.1 μg/ml. T lymphocyte proliferation was significantly increased at 0.001 μg/ml and 0.1 μg/ml, while B lymphocyte proliferation was decreased at 0.1 μg/ml. Cytokine concentrations for INFγ, IL-10, IL-6, and TNFα were reduced at 0.1 μg/ml upon either T or B cell mitogen stimulation, with the exception for IL-10, where 0.1 μg/ml reduced IL-10 concentration compared to unstimulated cells. These data suggest immune functions were affected by MeHg+ exposure requiring in vivo follow up investigations. The observed modulation of immune functions is of concern as any toxicant-induced modulation may adversely affect the health of individuals, particularly younger animals undergoing periods of critical development.

Immunostimulating Activity of Gold-modified Nanodiamond Particles

Background: The fight against infectious diseases includes two main components – immediate direct anti-infective action and stimulation of one's own immunity. Objective: In this study, we investigated the properties of diamond nanoparticles modified with gold. The use of such gold nanoparticles as indirect anti-infectious agents and immunostimulators has certain prospects. Materials and Methods: Gold hydrosols were synthesized by the reduction in an aqueous solution of gold (III) with sodium citrate (Na3Cit) under heating. Modification procedure of nanodiamond by gold requires incubation, a small sample of nanodiamond in a defined volume of a gold sol for about 24 hours in a dark place. We use human blood cells as test objects. The reaction of blastic transformation of lymphocytes was applied here as a test of biological actions of modified nanodiamond. Results: Modified nanodiamond do not have a toxic influence on blood cells. Modified nanodiamond possesses stimulation effects on spontaneous proliferation of lymphocytes and does not significantly affect phytohemagglutinin-induced proliferation. Nanodiamond slightly increases phagocytosis parameters of neutrophil leucocytes. Conclusion: Thus, results showed that the nanodiamond modified by gold possesses immunostimulating activity, increases the phagocytic activity of neutrophilic leukocytes and stimulates lymphocytes in the spontaneous proliferation test. Gold-modified nanodiamond could be considered as a non-direct anti-infective agent through immune stimulation.

Polymorphisms in HLA-C and KIR alleles are not associated with HAM/TSP risk in HTLV-1-infected subjects

IntroductionSeveral genetic polymorphisms may be related to susceptibility or resistance to viral disease outcomes. Immunological or genetic factors may act as major triggers of the immune pathogenesis of HAM/TSP. This study investigated the association of immune related genetic polymorphisms with viral and immunological markers. Methods247 HTLV-1-infected volunteers, drawn from a larger group of HTLV-infected subjects followed at the Institute of Infectious Diseases “Emilio Ribas” (IIER) for up to 19 years, participated in this study, which ran from June 2011 to July 2016. The subjects were classified according to their neurological status into two groups: Group 1 (160 asymptomatic individuals) and Group 2 (87 HAM/TSP patients). Samples were tested for spontaneous lymphocyte proliferation (LPA) and HTLV-1 proviral load (PVL) and for IFN-λ4, HLA-C and KIR genotypes using qPCR. ResultsWe found associations between LPA (p=0.0001) with HAM/TSP and confirmed the IFN-λ4 polymorphism rs8099917, allele GG, as a protective factor using a recessive model (OR=3.22, CI=1.10–9.47). Polymorphisms in HLA-C and KIR alleles were not associated with risk of developing HAM/TSP. ConclusionWe demonstrated that age, LPA and an IFN-λ4 polymorphism were associated with progression to HAM/TSP. Understanding HAM/TSP pathogenesis can provide important markers of prognostic value for clinical management, and contribute to the discovery of new therapeutic interventions in the future.

Autologous and Allogeneic Equine Mesenchymal Stem Cells Exhibit Equivalent Immunomodulatory Properties In Vitro.

The use of allogeneic bone marrow-derived mesenchymal stem cells (BMDMSCs) may provide an effective alternative to autologous BMDMSCs for treatment of equine musculoskeletal injuries. However, concerns have been raised regarding the potential safety and effectiveness of allogeneic BMDMSCs. We conducted studies to assess the immunological properties of equine allogeneic BMDMSCs compared with those of autologous BMDMSCs. For assessment of inherent immunogenicity, the relative ability of allogeneic and autologous BMDMSCs to stimulate spontaneous proliferation of equine lymphocytes was compared. The immunosuppressive activity of the two cell types was evaluated by adding autologous or allogeneic BMDMSCs to activated lymphocytes and assessing suppression of lymphocyte proliferation and IFNγ production. Fifty-six allogeneic and 12 autologous combinations were evaluated. Studies were also done to elucidate mechanisms by which equine mesenchymal stem cells (MSCs) suppress lymphocyte function. Potential mechanisms evaluated included production of prostaglandin E2 (PGE2), nitric oxide, transforming growth factor-beta, and indoleamine 2,3-dioxygenase. We found that autologous and allogeneic BMDMSCs both induced mild but equivalent levels of spontaneous lymphocyte activation in vitro. In in vitro assays assessing the ability of BMDMSCs to suppress activated lymphocytes, both allogeneic and autologous BMDMSCs suppressed T cell proliferation and IFNγ production to an equal degree. The primary mechanism of equine BMDMSC suppression of T cells was mediated by PGE2. We concluded that allogeneic and autologous BMDMSCs are equivalent in terms of their immunomodulatory properties, and stimulated peripheral blood mononuclear cells appear to trigger the immunosuppressive properties of MSCs. Therefore, both cell types appear to have equal potency in modulating inflammatory processes related to acute or chronic musculoskeletal injuries in the horse.

IFN-γ Attenuates Spontaneous Lymphocyte Proliferation by Fuelling Regulatory T Cells in HIV-1-Infected Patients.

HIV infection is characterized by a high degree of immune activation. It has an impact on CD4 cell count and populations' distribution and function. T regulatory cells (Tregs) were found to play a controversial role in the course of infection because of their beneficial effect on the degree of immune activation and unfavorable influence on the antigen-specific responses. The goal of the present work was to study the relationship among interferon-γ (IFN-γ), spontaneous lymphocyte proliferation, and regulatory T cells in HIV patients receiving therapy. Three lymphocyte populations, isolated after a stepwise magnetic separation from 17 individuals, were investigated-peripheral blood lymphocytes, CD4+CD45RA- (CD4+TM), and CD4+CD45RA-CD25- (TMCD25depl.) cells. The spontaneous, phytohemagglutinin (PHA) and HIV-1p24Ag-stimulated IFN-γ production and the spontaneous lymphocyte proliferation were evaluated. The potential of Tregs to establish a productive infection was determined by measurement of free HIV-1p24 antigen. Two types of constellations among subsets were found. In the first one (in 11 subjects), the spontaneous INF-γ inversely correlated with the spontaneous proliferation in all fractions (r = -0.9, p < 0.001). Conversely, in the second group (six subjects), no associations between the selected parameters were observed. The overall increase in p24-stimulated IFN-γ from TMCD25depl. cells was weak. Four samples: one in Tregs and three in TMCD25depl. cells were positive for the free p24 antigen. No association with the CD4+ T cell count, percentage of Tregs, and stage of infection was determined. In conclusion, our results demonstrate that IFN-γ could impact the proliferative capacity of non-Treg cells by fuelling Tregs. Furthermore, Tregs may control the spontaneous lymphocyte proliferation, but are less powerful in the suppression of Ag-specific IFN-γ production from non-Treg lymphocytes. The direct viral influence on Treg functions should be also considered.

5‐Amino‐3‐methyl‐4‐isoxazolecarboxylic acid hydrazide derivatives with in vitro immunomodulatory activities

Isoxazoles are an important class of compounds of potential therapeutic value. The aim of this study was to determine immunotropic effects of 5-amino-3-methyl-4-isoxazolecarboxylic acid hydrazide derivatives on spontaneous and mitogen-induced lymphocyte proliferation in young and old mice, cytokine production by peritoneal cells as well as possible mechanism of action in a model of Jurkat cells. Three-month-old and 13-month-old BALB/c mice were used as donors of the cells from a thymus, a spleen, mesenteric lymph nodes, and a peritoneal cavity. Spontaneous and concanavalin A or lipopolysaccharide (LPS)-induced cell proliferation was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric method. IL-1β and TNF-α production induced by LPS in macrophage-enriched peritoneal cell cultures was measured by enzyme-linked immunoassay. 5-amino-3-methyl-4-isoxazolecarboxylic acid hydrazide, 01K (4-phenyl-1-(5-amino-3-methylisoxazole-4-carbonyl)-thiosemicarbazide), and 06K (4-(4-chlorophenyl)-1-(5-amino-3-methylisoxazole-4-carbonyl)-thiosemicarbazide) exhibited regulatory activity in the proliferation tests. Prevailing stimulatory activity of the hydrazide and inhibitory activity of 01K and 06K was observed. Those effects were connected with different influence of the compounds on signaling proteins expression in Jurkat cells. The regulatory effects of the compounds on IL-1β production were more profound than those on TNF-α. Differences in the compound activity in young versus old mice were mainly restricted to 01K. Immunosuppressive isoxazole leflunomide and a stimulatory RM-11 (1,7-dimethyl-8-oxo-1,2H-isoxazole [5,4-e]triazepine) were applied as reference drugs.

Common γ-chain blocking peptide reduces in vitro immune activation markers in HTLV-1-associated myelopathy/tropical spastic paraparesis

Human T-cell lymphotropic virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP) is a progressive inflammatory myelopathy occurring in a subset of HTLV-1-infected individuals. Despite advances in understanding its immunopathogenesis, an effective treatment remains to be found. IL-2 and IL-15, members of the gamma chain (γc) family of cytokines, are prominently deregulated in HAM/TSP and underlie many of the characteristic immune abnormalities, such as spontaneous lymphocyte proliferation (SP), increased STAT5 phosphorylation in the lymphocytes, and increased frequency and cytotoxicity of virus-specific cytotoxic CD8(+) T lymphocytes (CTLs). In this study, we describe a novel immunomodulatory strategy consisting of selective blockade of certain γc family cytokines, including IL-2 and IL-15, with a γc antagonistic peptide. In vitro, a PEGylated form of the peptide, named BNZ132-1-40, reduced multiple immune activation markers such as SP, STAT5 phosphorylation, spontaneous degranulation of CD8(+) T cells, and the frequency of transactivator protein (Tax)-specific CD8(+) CTLs, thought to be major players in the immunopathogenesis of the disease. This strategy is thus a promising therapeutic approach to HAM/TSP with the potential of being more effective than single monoclonal antibodies targeting either IL-2 or IL-15 receptors and safer than inhibitors of downstream signaling molecules such as JAK1 inhibitors. Finally, selective cytokine blockade with antagonistic peptides might be applicable to multiple other conditions in which cytokines are pathogenic.

Immunomodulatory effects of brevetoxin (PbTx-3) upon in vitro exposure in bottlenose dolphins (Tursiops truncatus)

Harmful algal blooms (HAB), commonly referred to as ‘red tides’, involving the dinoflagellate Karenia brevis produce a series of neurotoxins known as brevetoxins (PbTx). Brevetoxins have long been associated with extensive fish kills, adverse human health effects such as neurotoxic shellfish poisoning, and have been associated with mortality events in aquatic mammals such as bottlenose dolphins (Tursiops truncatus). The immunotoxicological effects of these brevetoxins have been studied in manatees, humans, and cell lines, however, the effects in bottlenose dolphins remain unclear. There are increasing concerns that dolphins may be exposed to repeated/chronic, sub-lethal concentrations, which may impact their overall health. The objectives of this study were to measure the changes in innate (phagocytosis, respiratory burst, NK cell activity) and adaptive (mitogen-induced B and T lymphocyte proliferation) immune functions upon in vitro exposure to increasing concentrations of brevetoxin (PbTx-3) (0, 0.01, 0.1, 1, 10, 100, 500, and 1000nM) using bottlenose dolphin peripheral blood immune cells. Brevetoxin significantly increased spontaneous lymphocyte proliferation at 0.1–1000nM compared to the unexposed control. Brevetoxin significantly increased T lymphocyte proliferation upon suboptimal (0.1μg/ml) and optimal (1.0μg/ml) Con-A stimulation at 0.01–100nM and 0.1nM of PbTx-3 respectively, as well as suboptimal (0.05μg/ml) and optimal (5.0μg/ml) LPS-induced B lymphocyte proliferation at 0.01–100nM and 0.01–500nM of PbTx-3, respectively. Both neutrophil and monocyte respiratory burst were significantly increased at 500 and 1000nM. There were no significant effects on neutrophil or monocyte phagocytosis or NK cell activity. Importantly, concentrations that modulated immune functions in vitro were within the range measured in the blood of dolphins during two unusual mortality events, suggesting that naturally exposed dolphins may be at risk for immunomodulation. Brevetoxin-induced immunomodulation may increase an animal's susceptibility to bacterial, viral, or fungal infections. Understanding the risk for immunomodulation upon HAB toxin exposure can contribute in the health assessment and management of marine mammals, as well as guide veterinarians and wildlife rehabilitators in caring for and treating afflicted animals.

Modulation of glutathione intracellular levels alters the spontaneous proliferation of lymphocyte from HTLV-1 infected patients

The human T-cell lymphotropic virus type 1 (HTLV-1) is a retrovirus associated with neoplasias and inflammatory diseases, such as adult T-cell leukemia/lymphoma and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HTLV-1-infected individuals present a spontaneous T lymphocyte proliferation. This phenomenon is related to the HTLV-1-proviral load and the persistence of the infection. Viral proteins induce many cellular mediators, which can be associated with the abnormal cellular proliferation. The intracellular levels of glutathione (GSH) are important to modulate the cellular proliferation. The aim of this study was to investigate the correlation between the modulation of intracellular GSH levels and the spontaneous lymphocyte proliferation during the HTLV-1 infection. Intracellular GSH level can be modulated by using dl-buthionine-[S,R]-sulfoximine (BSO, GSH synthesis inhibitor) and N-acetylcysteine (NAC, peptide precursor). Our results demonstrated that BSO was capable of inducing a decrease in the spontaneous proliferation of PBMC derived from HTLV-1 carriers. On the other hand, the GSH precursor induces an increase in mitogen-stimulated cellular proliferation in infected and uninfected individuals. Similar results were observed by the inhibition of ABCC1/MRP1 protein, augmenting the mitogen-induced proliferation. This effect can be related with an increase in the GSH levels since ABCC1/MRP1 transports GSH to the extracellular medium. There was a significant difference on the expression of CD69 and CD25 molecules during the lymphocyte activation. We did not observe any alterations on CD25 expression induced by BSO or NAC. However, our results demonstrated that NAC treatment induced an increase in CD69 expression on unstimulated CD8+ T lymphocytes obtained from HTLV-1 infected individuals, healthy donors and HTLV carriers. Therefore, our results suggest that the cellular proliferation promoted by the infection with HTLV-1 and the activation phenotype of CD8+ T lymphocytes can be regulated by changing the intracellular GSH levels; suggesting the modulation of these intracellular levels as a new approach for the treatment of pathologies associated with the HTLV-1 infection.

Effect of Tripeptides on Lymphoid and Stem Cells

Tripeptides T-36 and, particularly, T-38 in concentrations of 0.1, 1, and 10 ng/ml inhibited proliferation of primary trypsinized embryonic mesenchymal stem cells, rat transplantable KF-1 fibroblasts, and human erythromyelosis K-562 cells. Inhibition of proliferation in embryonic and immortalized cells under the influence of tripeptides probably reflects antitumor activity of these substances. Tripeptides had no effect on lymphocyte survival and their adhesive, cytotoxic, and induced proliferative activities. T-36 did not modulate the proliferative properties of erythromyelosis K-562 cells. Tripeptides did not change engulfment activity and spontaneous and induced bactericidal activities of granulocytes. T-36 in a concentration of 0.1 ng/ml increased spontaneous proliferation of normal lymphocytes. These data suggest that tripeptides stimulate nontumor immune cells in adult people.

Promoter region polymorphism (−174 G/C) of interleukin-6 gene and SLE; are they associated?

IntroductionSLE is a systemic autoimmune disease with polyclonal B cell hyperactivity, spontaneous lymphocyte proliferation, and the production of pathogenic antibodies to self-antigens. Interleukin-6 is a pleiotropic cytokine with diverse functions including B-cell growth and differentiation. IL-6 levels have been shown to be affected by single nucleotide change from G to C at position −174 in the promoter region of the IL-6 gene. Aim of the workTo find out whether single nucleotide polymorphisms in the promoter region of the IL-6 gene (−174 G/C) constitute a genetic susceptibility for SLE and its association with various disease clinical and immunological features. Patients and methodsForty-two female SLE patients and 40 healthy controls were genotyped for IL-6 gene promoter region (−174 G/C) polymorphism using PCR. SLE patients satisfied the 1982 revised criteria of the American Rheumatism Association for the classification of SLE, with a mean age of 32.4±5.5years and mean disease duration of 5.7±1.5years. The healthy controls were matched for age and sex, with a mean age of 31.7±4.9years. All subjects were recruited from the Rheumatology and Rehabilitation and Internal Medicine Departments, Kasr El Aini Hospitals. SLE clinical and laboratory features were recorded including constitutional, hematological, joint, renal, and neuropsychiatric manifestations, oral ulcers, serositis, malar rash, and photosensitivity and CBC, liver, kidney functions and serum C3 and C4 levels. Positivity for ANAs, Anti-dsDNA and Anti-Sm antibodies were determined. ResultsGenotypic and allelic distributions showed no significant differences between SLE patients and controls. The frequency of G allele was higher than C allele in both patients (83.3% vs. 16.7%) and controls (85% vs. 15%). SLE patients with GG genotype showed significantly higher frequencies and increased risk of; constitutional manifestations at disease onset (P=0.02), OR (95% CI)=6.55 (1.22–35.12), photosensitivity (P=0.03), OR (95% CI)=4.67 (1.11–19.54), hematological disorders (P=0.02), OR (95% CI)=5.5 (1.29–23.39) and positivity of ANAs and Anti-dsDNA [P=0.046, 0.03: OR (95% CI)=7 (1.1–45.44), 6.43 (1.23–33.65), respectively]. Furthermore, those patients had significantly lower mean WBCs counts when compared to SLE patients with (GC and CC) genotypes (4.54±1.31 vs. 5.98±1.04/dl, P=0.002). Twenty-five patients had lupus nephritis (LN) proved by renal biopsy but none of them had CC genotype. LN patients with GG genotype had nearly similar mean 24-h proteinuria to those with GC genotype (2.93±1.07 vs. 2.68±1.06g/24h and P=0.39). No significant difference was found in IL-6 genotype and allele distributions when patients with diffuse proliferative glomerulonephritis (class IV), which has the worst prognostic outcome, were compared to patients with non-class IV glomerulonephritis (classes II and III) [P=0.12, 0.15, respectively]. ConclusionIL-6 promoter region (−174 G/C) polymorphism does not confer susceptibility to SLE but it is related to the presence of distinct clinical and immunological features. Furthermore, the increased frequency of the high-response G allele suggests that a genetically determined high IL-6 response may have a pathogenic role.

Human T cell leukemia virus type 1 infection drives spontaneous proliferation of natural killer cells

Most human T cell leukemia virus type 1 (HTLV-1) infected subjects remain asymptomatic throughout their lives, with a few individuals developing HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) or adult T cell leukemia. Lymphocytes from about half of HTLV-1 infected subjects spontaneously proliferate in vitro, and how this phenomenon relates to symptomatic disease outcome and viral burden is poorly understood. Spontaneous proliferation was measured in lymphocyte subsets, and these findings were correlated with HTLV-1 proviral load and Tax expression in PBMCs. We found that in addition to previously described vigorous CD8+ T cell spontaneous proliferation, natural killer (NK) cells spontaneously proliferated to a similar high level, resulting in expansion of CD56-expressing NK cells. Spontaneous NK cell proliferation positively correlated with HTLV-1 proviral load but not with Tax expression or the presence of HAM/TSP. The strongest correlate with clinical outcome in this cohort was the ability of cells to express Tax, while HTLV-1 proviral load was more closely related to spontaneous NK cell proliferation. These results demonstrate that spontaneous proliferation, Tax expression, and proviral load are inter-related but not equivalent, and that spontaneous lymphocyte proliferation is not restricted to T cells, the targets of HTLV-1 infection.

Correlation between the degree of immune activation, production of IL-2 and FOXP3 expression in CD4+CD25+ T regulatory cells in HIV-1 infected persons under HAART

Chronic immune activation is the leading event in the pathogenesis of HIV-1 infection and is associated with depletion of CD4+ T cells and Th1/Th2 imbalance. The role of Tregs in HIV infection is still controversial as these cells may control both immune activation and HIV-specific T cell responses. Aim of the present study was to correlate the degree of immune activation with FOXP3 expression and production of IL-2 and IL-10. Peripheral blood was obtained from 10 HIV-1-infected subjects with sustained response to HAART. Four cellular fractions were purified and simultaneously used in further experiments: PBMCs, CD4+CD45RO+, CD4+CD45RO+CD25− and CD4+CD45RO+CD25+. The level of immune activation was measured both by flow cytometry and radiometry as short-term 1-hour spontaneous lymphocyte proliferation; IL-2 and IL-10 production were determined by ELISA in supernatants of cultures non-stimulated or stimulated with PHA and p24 antigen. FOXP3 mRNA expression was detected by RT-PCR. A group of healthy subjects was used as control. Statistical analysis was performed by SPSS. The level of immune activation (total CD25 expression) was found correlated to both CD4+ and Tregs (CD4+CD25high+; FOXP3mRNA) and it was found higher in HIV-1 infected subjects in comparison to the healthy control group. The highest SLP-1 h was measured in the CD4+CD4+CD45RO+ CD25− population (T reg depleted), suggesting a role of Tregs in controlling immune activation during HIV-1 infection. In addition, CD4+ cells from HIV-infected individuals remained responsive to the negative regulation by Tregs (measured as IL-10 production) both spontaneously and upon stimulation. It is worth noting, however, that total PBMCs of these patients also responded well to specific antigen (p24) stimulation even though IL-2 release was impaired. The results obtained suggest that Tregs significantly influence the level of immune activation. At the same time, their function appears to be dependent on IL-2 production by the remaining T cells. These findings suggest that the control of Th1/Th2 balance could be an approach to immune-based therapy for HIV infection.

Spontaneous proliferation of memory (CD45RO+) and naive (CD45RO-) subsets of CD4 cells and CD8 cells in human T lymphotropic virus (HTLV) infection: distinctive patterns for HTLV-I versus HTLV-II.

Spontaneous lymphocyte proliferation (SLP) in vitro is a characteristic feature of about 50% of individuals infected with HTLV-I or HTLV-II. Both CD4 cells and CD8 cells contribute to SLP in HTLV-I infection, whereas SLP in HTLV-II infection is usually restricted to CD8 cells. In this study, we asked if SLP was restricted to the memory (CD45RO+) cell subset of CD4 and CD8 cells in HTLV infection. Purified CD4 and CD8 cells were separated into CD45RO+ and CD45RO- populations by a modified panning technique, and spontaneous proliferation (SP) of the cell subsets was assessed. For all five HTLV-I-infected persons whose mononuclear cell cultures were SLP+, only CD45RO+ cells, but not CD45RO- cells, within CD4 and CD8 subsets showed SP. In contrast, five of six SLP+ HTLV-II+ individuals showed SP in both the CD45RO+ and the CD45RO- subsets of CD4 cells, and 10 of 12 SLP+ HTLV-II+ individuals showed SP of both the CD45RO+ and CD45RO- subsets of CD8 cells. Polymerase chain reaction studies showed that proviral genome was generally present in both CD45RO+ and CD45RO- subsets of CD4 and CD8 cells, regardless of HTLV type and SP activity. These findings show that SP of both CD4 and CD8 cells in HTLV-I infection is usually restricted to CD45RO+ memory cells, whereas in HTLV-II infection, both CD45RO+ memory and CD45RO- naive subsets of CD4 and CD8 cells may exhibit SP. It thus appears that HTLV-I infection and HTLV-II infection exhibit distinctive dysregulatory effects on memory and naive T cell subpopulations.

Cellular immunity in tuberculous pleural effusions: evidence of spontaneous lymphocyte proliferation and antigen-specific accelerated responses to purified protein derivative (PPD).

The kinetics of in vitro cellular proliferation against a PPD of Mycobacterium tuberculosis or streptococcal antigen (streptokinase-streptodornase) was evaluated in pleural fluid and peripheral blood mononuclear cells (PBMC) from patients with tuberculous and non-tuberculous pleuritis. The peak proliferative response to PPD by mononuclear cells from pleural fluid occurred earlier (day 3) in 65% of patients with tuberculosis, a finding not seen in non-tuberculous effusions. Spontaneous lymphocyte proliferation of both peripheral blood lymphocytes and pleural effusion lymphocytes was frequently observed, irrespective of etiology. However, 20 of 21 tuberculous patients manifesting spontaneous lymphocyte proliferation had accelerated kinetics of proliferation to PPD, which was antigen-specific. These results suggest that spontaneous lymphocyte proliferation occurs as a response to antigen stimulation at the site of disease, and is not a non-specific response to inflammation. Furthermore, enhanced reactivity against mycobacterial antigen, manifested by accelerated kinetics of proliferation, has diagnostic potential in patients with pleural effusions.

The effects of Plasmodium falciparum malaria on immune control of B lymphocytes in Gambian children

Children living in hyperendemic malarious regions have high immunoglobulin levels and an increased frequency of Burkitt's lymphoma. In a study of Gambian children which endeavours to explain these findings we showed that acute P. falciparum malaria caused spontaneous activation and growth of their B lymphocytes in vitro. A high proportion of these cells contained Epstein-Barr nuclear antigen (EBNA). In ancillary experiments aimed at explaining these findings. CD4 helper cells from adult donors were destroyed with monoclonal antibody and complement. This manoeuvre resulted in loss of cytotoxic T cell control of their B lymphocytes when infected with Epstein-Barr virus (EBV). In children with acute malaria, both spontaneous immunoglobulin and antibody production by B cells was increased yet CD4 helper cell control over these cells, as measured by responses to pokeweed mitogen, was found to be intact. Spontaneous and concanavalin A-driven lymphocyte proliferation was depressed. We infer from these findings that in patients with P. falciparum malaria loss of cytotoxic T cell control of the EBV in B cells, possibly due to destruction or dysfunction of a subset of CD4 cells responsible for induction of suppressor/cytotoxic CD8 cells, leads to activation and proliferation of foci of B cells containing EBV. The expanded pool and rapid turnover of these cells may increase chances of malignant transformation leading to the genesis of Burkitt's tumor. Partial loss of suppressor mechanisms coupled with normal CD4 helper/inducer activity may result in high serum levels of immunoglobulin which are characteristic of persons living in malarious regions.

Effects of Montreal municipal sewage effluents on immune responses of juvenile female rainbow trout ( Oncorhynchus mykiss)

The objective of this study was to examine the immunotoxicity of treated Montreal sewage effluents on juvenile female rainbow trout ( Oncorhynchus mykiss). A comprehensive panel of immunological assays was used to evaluate the effects of exposure for 1 and 4 weeks to 1, 3, 10 and 20% sewage effluent. Phagocytic ingestion of fluorescent latex beads by head kidney macrophages and granulocytes was suppressed following 1-week of exposure, with the highest exposure concentration being the most suppressive. Phagocytic activity returned to control levels after 4 weeks of exposure. The cytotoxic activity of head kidney derived non-specific cytotoxic cells was enhanced after a 1-week exposure, especially at the lowest exposure concentration, and returned to control levels after 4 weeks of exposure. In vitro lymphocyte proliferation in response to LPS and ConA activation was not affected following sewage effluent exposure, but nonactivated, spontaneous proliferation of lymphocytes was suppressed in a dose-dependent manner after 4 weeks of exposure. Plasma lysozyme activity was elevated at lowest exposure concentration after 4 weeks. No changes were noted in either the blood leukocyte/erythrocyte ratio or in the proportion of circulating lymphocytes and thrombocytes. The proportion of circulating granulocytes increased following exposure for 4 weeks to the low effluent concentration. Plasma cortisol levels were not affected by effluent exposure suggesting that mechanisms other than stress influenced the observed immunomodulation. In summary, this study demonstrates that sewage effluent can alter the immune functions of rainbow trout.

Functional development of immune response in rabbits

The aim of our study was to extend knowledge concerning postnatal development of the immune system in rabbits from two aspects. Firstly, capability of lymphocytes from peripheral blood, spleen, mesenteric, and popliteal lymph nodes to respond to Concanavalin A stimulation was investigated. Secondly, changes in the ability to produce antibodies against tetanus toxoid by rabbits during maturation were studied. Proliferation of lymphocytes was reduced in mesenteric lymph nodes in newborns, in PB up to the age of two weeks, and in popliteal lymph nodes up to the age of four weeks when compared to adults. High spontaneous lymphocyte proliferation that lasted up to the age of two weeks was recorded in spleen. The study of antibody response showed that even one-day-old rabbits were able to form specific antibodies of isotype IgM and IgG. Nevertheless, significantly lower formation of both isotypes was noted in one-day and two-week-old rabbits, and commencement of IgG isotype formation was delayed in one-day, two-week, and four-week-old rabbits when compared to adults.