IL-23R gene polymorphism rs2201841 is associated with psoriatic arthritis.

We examined rs2201841 within IL-23R gene in Serbian patients with psoriasis and healthy controls. G allele frequency was significantly increased in the group of patients with psoriatic arthritis compared with controls (0.481 vs 0.308). Carriage of G allele increases risk to develop psoriatic arthritis (P = 0.009, OR = 3.311, 95% CI 1.29-8.70).

Cell-type dependent response of melanoma cells to aloe emodin.

Intrinsic characteristics of melanoma cells such as expression of inducible nitric oxide synthase (iNOS), redox status, and activity of signaling pathways involved in proliferation, differentiation and cell death define the response of the cells to the diverse treatments. In this context we compared the effectiveness of herbal antaquinone aloe emodin (AE) against mouse B16 melanoma and human A375, different in initial activity of ERK1/2, constitutive iNOS expression and basal level of reactive oxygen species (ROS). Both cell lines are sensitive to AE treatment. However, while the agent induces differentiation of B16 cells toward melanocytes, in A375 cells promoted massive apoptosis. Differentiation of B16 cells, characterized by enhanced melanin production and tyrosinase activity, was mediated by H(2)O(2) production synchronized with rapid p53 accumulation and enhanced expression of cyclins D1 and D3. Caspase mediated apoptosis triggered in A375 cells was accompanied with Bcl-2 but not iNOS down-regulation. In addition, opposite regulation of Akt-ERK1/2 axis in AE treated B16 and A375 cells correlated with different outcome of the treatment. However, AE in a dose-dependent manner rescued both B16 and A375 cells from doxorubicin- or paclitaxel-induced killing. These data indicate that caution is warranted when AE is administrated to the patients with conventional chemotherapy.

Antiproliferative effect of vitamin A and D analogues on adult human keratinocytes in vitro.

BACKGROUND: Vitamin A and D analogues play an important role in epidermal homeostasis and are used in the treatment of various skin diseases. The failure of retinoid and vitamin D treatments is sometimes difficult to explain. METHODS: We analyzed the effect of all-trans retinoic acid (all-trans RA), 13-cis retinoic acid (13-cis RA), ergocalciferol and cholecalciferol in keratinocyte cultures established from adult donors, on the cell proliferation by means of [(3)H]thymidine incorporation and apoptosis after fluorescein diacetate/trypan blue staining. RESULTS: All tested agents exerted a dose-dependent inhibition of keratinocyte proliferation in the concentration range of 1.25-5 microM. Based on IC(50) values, the antiproliferative efficiency was as follows: cholecalciferol > ergocalciferol = all-trans RA > 13-cis RA. The observed effect of cholecalciferol and ergocalciferol, but not retinoids, involved the induction of apoptotic cell death. Combining vitamins A and D did not further increase the proliferation block and even displayed an antagonistic effect. CONCLUSION: The susceptibility of keratinocytes to the antiproliferative action of vitamins A and D was markedly different in cell cultures derived from different donors, indicating a possible predictive value of the in vitro testing for the efficiency of the clinical response to these agents.

Chloramphenicol induces in vitro growth arrest and apoptosis of human keratinocytes.

Chloramphenicol (CAP) is a broad-spectrum antibacterial drug that is widely used for topical application in ophthalmology and dermatology. In the present study we investigated the influence of CAP on human keratinocyte proliferation and apoptosis in vitro. CAP significantly inhibited proliferation and induced apoptosis of cultivated human keratinocytes, as revealed by incorporation of radioactive thymidine and flow cytometry analysis of intracellular esterase activity in fluorescein diacetate-stained cells, respectively. CAP-induced keratinocyte apoptosis was associated with activation of caspases and increased production of reactive oxygen species. The pro-apoptotic action of CAP was antagonized by the antioxidant agent N-acetylcysteine, the protein synthesis inhibitor cycloheximide, and PD98059, a selective inhibitor of extracellular signal-regulated kinase (ERK) activation. Taken together, these data indicate that CAP inhibits keratinocyte proliferation through induction of oxidative stress and ERK-mediated caspase-dependent apoptosis.

Induction of experimental autoimmune encephalomyelitis in Dark Agouti rats without adjuvant.

Experimental autoimmune encephalomyelitis (EAE) is a well-recognized model for multiple sclerosis (MS) in humans. However, adjuvants used with encephalitogens to induce EAE produce non-specific effects interfering with the mechanisms involved in the autoimmune response to the central nervous system (CNS) tissue. It is therefore important to establish a more suitable model of EAE for analysis of autoimmune phenomena resembling those operative in MS. Here we report that EAE can be induced regularly in Dark Agouti (DA) strain of rats with spinal cord tissue without any adjuvant, as judged by both clinical and histological parameters. The incidence and severity of EAE depended on the origin of the encephalitogen, the rat versus guinea pig spinal cord homogenate being more efficient. Furthermore, EAE could be reinduced in animals which had recovered from disease that had been induced actively with encephalitogen alone, suggesting the role of adjuvant-generated non-specific mechanisms in resistance to reinduction of EAE. Thus, EAE induced in DA rats with encephalitogen alone provides a reproducible model for defining pathogenically relevant events in CNS autoimmunity devoid of the potentially misleading effects of adjuvants.

Interleukin-17 stimulates inducible nitric oxide synthase activation in rodent astrocytes.

The effect of interleukin-17 (IL-17) on production of nitric oxide (NO) in rodent astrocytes was investigated. While IL-17 by itself did not induce NO production, it caused a dose-dependent enhancement of IFN-gamma-triggered NO synthesis in both mouse and rat primary astrocytes. In contrast, IL-17 was unable to stimulate NO synthesis in either murine or rat macrophages. IFN-gamma-triggered expression of mRNA for iNOS, but not for its transcription factor interferon regulatory factor-1 (IRF-1), was markedly elevated in IL-17-treated astrocytes. The induction of iNOS mRNA by IL-17 in IFN-gamma-pretreated astrocytes was abolished by antagonists of nuclear factor-kappaB (NF-kappaB) activation--a proteasome inhibitor MG132 and an antioxidant agent PDTC, as well as with specific p38 MAP kinase inhibitor SB203580. While IL-17 stimulated both IL-1beta and IL-6 production in astrocytes, only IL-1 was partly responsible for IL-17-induced NO release. Finally, IL-17 synergized with exogenous IL-1beta and TNF-alpha for astrocyte NO production. Having in mind a well-known neurotoxic action of NO, these results suggest a possible role for IL-17 in the inflammatory diseases of the CNS.

Muramyl dipeptide potentiates cytokine-induced activation of inducible nitric oxide synthase in rat astrocytes.

We investigated the influence of muramyl dipeptide (MDP), a cell wall component of Gram-positive bacteria, on cytokine-induced nitric oxide (NO) production in rat primary astrocytes. MDP alone did not induce NO release in astrocyte cultures. However, MDP increased astrocyte NO production and subsequent nitrite accumulation triggered by IFN-gamma. IFN-gamma-activated expression of mRNA for inducible NO synthase (iNOS) and iNOS transcription factor interferon regulatory factor-1 (IRF-1) was markedly enhanced in astrocytes treated with MDP. The potentiating effect of MDP on IFN-gamma-induced NO production in astrocytes was completely blocked with protein tyrosine kinase (PTK) inhibitor genistein or mitogen activated protein kinase (MAPK) inhibitor PD98059. In contrast, protein kinase C (PKC) inhibitor calphostin C did not affect ability of MDP to augment IFN-gamma-triggered astrocyte NO synthesis. These results suggest that MDP synergizes with IFN-gamma in the induction of iNOS gene in astrocytes through mechanisms involving PTK and MAPK, but not PKC activation. Finally, MDP also augmented NO production and iNOS mRNA expression in astrocytes treated with IL-1beta.

Relationship between IL-1beta and TNFalpha production by PBMC and clinical features of hemodialysis patients.

The relationship between production of IL-1beta and TNFalpha by peripheral blood mononuclear cells (PBMC) of hemodialysis (HD) patients and clinical disorders characteristic for HD patients was examined. The study included 28 HD patients divided in the 4 groups: group 1--6 patients with malnutrition; group 2--6 patients with secondary hyperparathyroidism; group 3--6 patients with eosinophilia; group 4--10 stable HD patients without clinical complication. The control group consisted of 9 healthy volunteers. All patients were dialyzed with cuprophane membrane more than one years. Blood samples were taken immediately before the beginning of hemodialysis and PBMC were isolated by centrifugation on the density gradient. Concentrations of IL-1beta and TNFalpha were measured in the supernatants of the cultures by ELISA tests. The results showed marked individual differences in cytokine production both in healthy controls and in HD patients. Spontaneous and LPS stimulated production of IL-1beta by PBMC of HD patients was significantly higher compared to PBMC of healthy controls. There were no significant differences between group of patients with different clinical complications in cytokine production. However, a positive correlation was found between IL-1beta concentration and body mass index for patients with malnutrition and between concentration of IL-1alpha and parathyroid hormone (PTH) for patients with secondary hyperparathyroidism. The results suggest that PBMC of HD patients are chronically stimulated to produce IL-1beta, which may contribute to the development of particular chronic complications.

Altered immunoregulation in otitis media with effusion in children: presence of serum immuno-inhibitory factors.

In the present study serum immuno-inhibitory factors in children with otitis media with effusion (OME) were evaluated. The study included 23 children with OME and 23 healthy age-and-sex-matched controls. Serum inhibitory activity (SIA) was expressed as the inhibition index of proliferative response to T-cells lectins. In almost all the children with OME the indices reflecting T-cell proliferation were below the range of those in the control group. The results suggest that the activity of the factor(s) which affect T-cell proliferation may be included in mechanisms contributing to the persistence of OME in children. Further studies are needed in order to establish the cellular and molecular aspects of serum inhibitory activity, as well as its significance in the pathogenesis of OME.

[Spontaneous proliferative response in patients with chronic renal allograft insufficiency due to chronic rejection]. / Spontana proliferacija limfocita kod bolesnika s hronicnim odbacivanjem i smanjenom funcijom transplantisanog bubrega.

Proliferative response of peripheral blood lymphocytes in patients with chronic renal allograft insufficiency was due to aetiological deterioration factors. The highest values of sponntaneous lymphocyte blastogenesis were found in chronic rejection of renal allografts, but on the similar level detected during acute renal rejection attacks. Significant raise of proliferative response was absent in cyclosporine nephropathy, as well as in patients with excellent renal allograft function, maintained on proliferative healthy control levels.

[Mechanisms of the effect of interleukin-1 inhibitor activators]. / Mehanizam delovanja inhibitora aktivnosti interleukina-1.

Interleukin-1 (IL-1) has a broad range of effects on physiologic processes, including immunologic phenomena. In maintaining the tissue homeostasis the existence of feed-back mechanisms regulating down the IL-1 activity would be beneficial for the host. We and other investigators have provided evidence that such a regulatory circuit exists and may be altered by agents such as glucocorticoids and as well as by the UV irradiation. Here we provide evidence describing the mode of action of an accessory cell derived inhibitor of the IL-1 activity. This inhibitor is a product of radioresistant cells, it does not represent a nonspecific inhibitor of the DNA synthesis and appears to be specific for IL-1 since it does not interfere with the IL-2 dependent T cell proliferation. It affects both the IL-2 production and the induction of IL-2 receptor expression. As indicated in our previous work the immunoinhibitory factors affecting T cell proliferation are present in the sera of patients suffering from chronic renal and hepatic diseases. Further analyses have shown that these serum inhibitors have the same mode of action as macrophagederived glucocorticoid-induced inhibitors. Thus, it appears that the production of an IL-1 receptor antagonist may be enhanced in a pathological condition and during chronic inflammations in particular. Its significance for the disease pathogenesis remains to be elucidated.

[The role of T-lymphocyte subsets in susceptibility and resistance to induction of autoimmunity in the central nervous system]. / Uloga subpopulacija T limfocita u osetljivosti i otpornosti na indukciju autoimunosti u centralnom nervnom sistemu.

The importance of two major subsets of T cells (CD4 and CD8) for the induction of EAE was analysed in two strains of rats differing in susceptibility to EAE and the level of IL 2 production. It was found that CD8 cells do not play a role in the induction or in the inherent resistance to EAE. Additionally, the elimination of CD8 cells does not increase the IL 2 production in lymphoid cell cultures. Based on these and previous results showing that a low dose of cyclophosphamide readily abrogates the resistance to EAE and enhances the IL 2 production. Thus it was concluded that genetically determined differences in susceptibility to EAE are governed by immunoregulatory genes whose effect appears to be mediated at the level of cellular interaction of different subsets of CD4 T cells.

Hematopoietic growth factors in anemia of Belgrade laboratory (b/b) rats.

In this study, the extent to which growth factor production and microenvironment might be responsible for defective erythropoiesis and granulopoiesis in anemic b/b rats is investigated. Radioimmunoassay-determined serum erythropoietin (Epo) levels are high in b/b rats and closely related to degree of anemia. The low number of erythroid progenitors in b/b rats despite a high Epo level suggested that the defective erythropoiesis could be due to a low level of burst-promoting activity (BPA). A pokeweed mitogen-stimulated medium (PWM-SCM) was prepared with b/b rat spleen cells and used in normal and anemic rat bone marrow and spleen cultures to determine BPA and other growth factor levels. No erythroid burst-forming unit-derived colonies were found but granulocyte-macrophage colony-forming units were counted in significant number, suggesting that the production of growth factors that supports the growth of granulopoietic progenitors is not significantly disturbed. Because BPA is produced mainly by T-lymphocytes, the low BPA level in b/b rat PWM-SCM raised the question of the functional capacity of T-lymphocytes. Investigations showed a decrease in the proliferative activity of b/b rat spleen mitogen-activated T-lymphocytes to about 20% of controls as well as a decrease in interleukin-2 activity in b/b rat spleen cell supernatants. These results point to defective T-lymphocytes. A study of bone marrow fibroblastoid cell colonies (CFU-F) revealed significantly lower CFU-F counts in the b/b rats. This finding is indicative of a disturbed microenvironment, which could also to some extent be responsible for decreased growth factor production and depressed hematopoiesis in the b/b rat.

An unusual T-cell surface phenotype in vivo correlates with the failure to proliferate and produce IL-2 in vitro in a patient with common variable immunodeficiency.

Stimulation of T-lymphocytes derived from some patients with common variable immunodeficiency (CVID) syndrome results in defective proliferation. The underlying mechanism is related to the inability of stimulated cells to secrete IL-2 while the expression of IL-2 receptor (IL-2R) is normal. We have identified a patient whose peripheral T-cells failed to proliferate and secrete IL-2 upon stimulation. The addition of recombinant IL-2 restored proliferation. The defect did not seem to be caused by accessory cell failure since the patient's adherent cells produced IL-1 and IL-6, and addition of allogeneic irradiated cells did not induce proliferation. Stimulation of CVID T-cells with phorbol esters and Ca2+ ionophore induced both IL-2 secretion and proliferation, indicating the absence of a defect in the transcription and/or translation of the IL-2 gene. The patient's T-cells expressed high levels of CD3. The majority of T-cells expressed the CD38 molecule which is normally found on thymocytes or activated T-cells but not peripheral blood T-cells and HLA-DR, another activation marker. However, CD25 (the IL-2R) and CD1, a marker of more immature thymocytes, were not expressed. Finally, the patient's cells were sensitive to an in vitro corticosteroid treatment. The possibilities that this patient's T-cells represent anergic T-cells or not fully matured thymocytes are discussed.

Evidence for target tissue regulation of resistance to the induction of experimental allergic encephalomyelitis in AO rats.

A myelin basic protein (MBP)-specific T cell line derived from F1 hybrids between experimental allergic encephalomyelitis (EAE)-susceptible DA (RT1avl) strain and EAE-resistant AO (RT1u) strain was capable of inducing clinical EAE in F1 hybrids and DA, but not in AO rats. In vitro restimulation with MBP presented by AO antigen-presenting cells (APC) resulted in the generation of a MBP-specific subline restricted by RT1u MHC products which induced clinical EAE in F1 hybrids but not in the AO parental strain. Deletion of hosts' leukocytes using sublethal irradiation and cytotoxic drugs did not abrogate the resistance of AO rats, which argues against the involvement of hosts' lymphoid cells in the regulation of autoagression. Thus, mechanism(s) regulating the activity of autoagressive T cells on functional elements in the target tissue might be responsible for differences in susceptibility to EAE.

Circulating inhibitor of interleukin 1 activity in patients with chronic renal diseases.

The effects of sera derived from patients suffering from chronic renal diseases (endemic Balkan nephropathy, glomerulonephritis and pyelonephritis) on T cell proliferative response was studied. It was found that these sera contained factors which affect interleukin 1 (IL-1) dependent events in T cell proliferative response. The factors prevent costimulatory effects of IL-1 on T cells but do not bind to IL-1, nor do they affect interleukin 2 (IL-2) dependent T cell proliferation. These findings indicate that immuno-suppression observed in some kidney disorders may be partially due to serum immunoinhibitory factors affecting IL-1 activity.

Glutathione and its associated enzymes in peripheral blood cells in different stages of chronic renal insufficiency.

Reduced glutathione (GSH) levels and glutathione reductase (GR) and glutathione S-transferase (GST) activities were investigated in the erythrocytes and lymphocytes of non-dialyzed patients with varying degrees of chronic renal insufficiency, and also of patients on regular hemodialysis treatment. GSH, GR and GST levels were higher in erythrocytes and lymphocytes of examined patients as compared to their corresponding age-matched healthy controls. A correlation was found between the degree of renal insufficiency and the above parameters tested. A routine hemodialysis did not significantly affect erythrocyte and lymphocyte GSH content and activities of its associated enzymes. The increased GSH levels as well as GSH-linked enzyme activities of blood cells in uremia may be a protective mechanism for the cells due to the accumulation of toxic, oxidizing, wastes in the blood as a result of the uremic state. This view is supported by the results ofin vitro experiments, which have shown that GR and GST activities of normal human lymphocytes are increased when incubated with plasma from uremic patients.

[Immunosuppressive factors in the blood in patients during the development of various kidney diseases]. / Imunospresivni cinioci u serumu bolesnika tokom evolucije razlicitih bubreznih oboljenja.

Sera of patients suffering from Balkan nephropathy, pyelonephritis and glomerulonephritis inhibit lectin-induced T-cell proliferation in vitro. Immunosuppressive factors were registered in the patients' sera during the early stage of the existing disease, and their activity was not in correlation with the degree of renal insufficiency. Serum inhibitors revealed their activity during the early phase of T-cell activation and had no effect on T-cell proliferation and DNK synthesis. Inhibitor activity was registered even if sera were not present in cell culture continuously but only 6 hours of preincubation; then their action was irretrievable.

Analysis of T cell subsets after induction of experimental autoimmune encephalomyelitis in susceptible and resistant strains of rats.

T cell subsets in the peripheral blood, draining lymph node (DLN) and spinal cord lesions were analysed after the induction of experimental autoimmune encephalomyelitis (EAE) in susceptible (DA) and relatively resistant (AO) rats. In DA rats, a significantly higher number of CD4+ cells were generated in the DLN, in response to both nervous tissue antigens and complete Freund's adjuvant (CFA), compared to AO rats. In the peripheral blood of DA rats, the percentage as well as absolute number of CD4+ cells increased in the preclinical phase of EAE, but declined as the disease developed. The percentage of CD8+ cells decreased in both these phases of EAE. In resistant AO rats, however, there were no significant changes in the T lymphocyte subset percentages after EAE induction, although the absolute number of peripheral blood CD4+ cells again increased in the preclinical stage of EAE. In the CFA-treated control DA rats, the absolute number of CD4+ cells was increased in the preclinical phase. However, no decline comparable to that seen in diseased animals followed. It is concluded that the generation of CD4+ cells in response to this antigen is strain specific and, since the cells are released into the circulation, will affect the balance between the T cell subsets in the peripheral blood during the development of EAE.

Quantitative analysis of the immunocompetent cells in periapical granuloma: correlation with the histological characteristics of the lesions.

Granuloma formation includes an immune response in oral tissues to various microorganisms and their products. The immunocompetent cells of both series (T and B) are present in the periapical lesions. In order to further analyze the relative contribution and pathophysiological significance of the T cell subsets in granuloma formation, we undertook the quantitative analysis of the CD3-positive, CD4-positive, CD8-positive and Ig-positive cells in these lesions by using indirect immunofluorescence. Evidence is provided showing predominance of T cells in diffuse and B cells in focal mononuclear infiltrates. CD8-positive cells were more frequent in diffuse infiltrates and in particular in granulomas with distinct epithelium while CD4-positive cells were more numerous in focal infiltrates. It appears that the presence and ratios of different subsets of immunocompetent cells reflects the pathogenesis of granuloma and transformation to cyst.