The possible involvement of sema3A and sema4A in the pathogenesis of multiple sclerosis.

BACKGROUND: Immune semaphorins are widely accepted to have functional impact on autoimmune diseases. OBJECTIVES: To assess the status of sema3A and sema4A in the pathogenesis of Multiple Sclerosis (MS). RESULTS: Sema3A expression on (T regulatory cells)Tregs was decreased in MS patients, compared to healthy controls (35.85 ± 16.7% vs 88.27 ± 3.8%; p ≤ 0.001). Serum levels of sema3A were decreased in MS patients 2.95 ± 0.43 vs 18.67 ± 5.7 ng/ml in healthy individuals; p ≤ 0.001. Sema4A serum levels were increased in MS patients compared to healthy individuals (12.99 ± 8.6 vs 5.83 ± 3.91 ng/ml; p ≤ 0.001). Sema3A and sema4A serum levels were found to be in negative/positive correlation with MS disease severity (rs = 0.62, rs = -0.49, respectively). CONCLUSION: We show that sema3A is a regulatory molecule in MS, whereas sema4A is a stimulatory one. Targeting sema3A and sema4A could become a potential therapeutic approach in MS.

Interleukin-17 is a potential player and treatment target in severe chronic spontaneous urticaria.

BACKGROUND: Chronic spontaneous urticaria (CSU) is considered an autoimmune disorder in 50% of cases at least, in which T- and mast cell mediators are considered to be the primary cause of symptoms. However, H1 -antihistamines, cyclosporine A, and omalizumab fail to achieve complete symptom amelioration in up to 70% of patients. This suggests that other inflammatory pathways are involved and that additional and more effective treatments need to be developed. OBJECTIVE: This preliminary report examines the possibility that interleukin-17 (IL-17), a cytokine involved in the pathogenesis of many autoimmune diseases, may contribute to CSU and its inhibition may offer a relevant therapeutic target. METHODS: The expression of IL-17A in skin biopsies of 20 CSU patients and 10 healthy controls was determined by quantitative histomorphometry. We also assessed the response to secukinumab (anti-IL-17A) treatment patients of eight severe CSU (7-day urticaria activity score UAS7 32-40) who were H1 -antihistamine and omalizumab-resistant. RESULTS: Increased numbers of CD4+ T cells and mast cells were present in both lesional and non-lesional skin of CSU patients compared with healthy controls. Both types of cells were strongly positive for IL-17A and found to be in close proximity to each other. All eight patients treated with the anti-IL-17A antibody, secukinumab, showed significant improvement in CSU disease activity. The action of secukinumab was shown to be relatively slow in onset. The significant reduction in disease activity from baseline UAS7 was demonstrated to be 55% and 82% at 30 and 90 days, respectively. CONCLUSIONS: These findings suggest that IL-17 is involved in the pathogenesis of CSU and that IL-17 should be investigated as a therapeutic target in future studies with larger numbers of patients.

Low levels of the immunoregulator Semaphorin 4D (CD100) in sera of HIV patients.

INTRODUCTION: Semaphorin-4D (CD100), generated by CD4/CD8 T-cells and its receptor on B cells - CD72, play a role in immune regulation. Both have soluble forms - sCD100/sCD72. METHODS: sCD100 and sCD72 levels were determined by ELISA (MyBioSource, USA). RESULTS: 28 chronic HIV patients and 50 matched healthy volunteers participated in our study. Before treatment, CD4 T-cells counts were 267 ±â€¯216 cells/mcl and viral load (VL) was 586,675 ±â€¯1897,431 copies/ml. Two years following HAART, CD4 T-cells counts rose to 475 ±â€¯264 cells/mcl and VL dropped to 2050 ±â€¯10,539 copies/ml. CD8 T-cells counts were stable. sCD72 levels prior (4.13 ±â€¯2.03 ng/ml) and following HAART (3.53 ±â€¯2.01 ng/ml) were similar to control levels (4.51 ±â€¯2.66 ng/ml). sCD100 levels before (40.47 ±â€¯31.4 ng/ml) and following HAART (37.68 ±â€¯29.44 ng/ml) were significantly lower compared to controls (99.67 ±â€¯36.72 ng/ml) despite the significant increase in CD4 T-cells counts. CONCLUSIONS: The permanent low levels of the immunoregulator sCD100 suggest a role for CD100 in the immune dysfunction and T cells exhaustion of HIV.

Increased killer B cells in chronic HCV infection may lead to autoimmunity and increased viral load.

Regulatory B (Breg ) cells are characterized by various membrane markers and the secretion of different inhibitory cytokines. A new subset of Breg cells was identified as CD5hi Fas-ligand (FasL)hi . Their main reported role is to suppress anti-viral and anti-tumour immune responses, and, hence they have been dubbed 'killer' B cells. In this study, we aim to assess the role of these cells in chronic hepatitis C virus (HCV) infection, and determine if they contribute to the increased viral load and persistence of HCV and its related autoimmunity. (i) FasL expression on CD5hi B cells is increased significantly in HCV-infected patients compared to healthy individuals [28·06 ± 6·71 mean fluorescence intensity (MFI) ± standard error of the mean (s.e.m.), median = 27·9 versus 10·87 ± 3·97 MFI ± s.e.m., median = 10·3, respectively, P <  0·0001]. (ii) Killer B cells from HCV patients increased autologous CD4+ T cell apoptosis compared to the apoptosis in healthy individuals [39·17% ± 7·18% mean ± standard deviation (s.d.), median = 39·6 versus 25·92 ± 8·65%, mean ± s.d., median = 24·1%, P <  0·0001, respectively]. A similar increase was observed in CD8+ T cell apoptosis (54·67 ± 15·49% mean ± s.d., median = 57·3 versus 21·07% ± 7·4%, mean ± s.d., median = 20%, P = 0·0006, respectively). (iii) By neutralizing FasL with monoclonal anti-FasL antibodies, we have shown that the induction of apoptosis by killer B cells is FasL-dependent. (iv) Increased expression of FasL on CD5hi B cells is correlated positively with an increased viral load and the presence of anti-nuclear antibodies and rheumatoid factor in HCV. This is the first study in which killer B cells have been suggested to play a pathogenic role in HCV. They seem to be involved in HCV's ability to escape efficient immune responses.

The prevalence of patients suffering from chronic spontaneous urticaria, in whom omalizumab cannot be stopped even after six years.

Background: Omalizumab (OMA) was the first FDA-approved biological drug for severe chronic spontaneous urticaria (CSU), and until today is the only beneficial and truly safe one. The objectives were: To assess the prevalence of CSU patients in whom OMA cannot be stopped over time. We also asked if biomarkers (e.g., anti-TPO antibodies and total IgE) could assist in anticipating this issue. Methods: We used our prospective registry of 93 patients, which included CSU disease duration, the onset of OMA treatment, Urticaria Activity Score (UAS7) during follow-up, co-morbidities, serum IgE levels and the presence of anti-TPO antibodies. Finally, we assessed the response to OMA during a period of six years. Results: Out of the 93 treated CSU patients, OMA was stopped in ten patients after six months being defined as failures. In another ten patients, OMA was discontinued after 2-4 years of therapy, achieving a remission. Seventy-three patients are still treated between 2 and 6 years, having different degrees of response. Of these, in thirty-eight (52%) patients, we could not stop OMA even after six years due to CSU relapses. The prevalence of lower serum IgE levels and anti-TPO antibody positivity was significantly higher in CSU patients in whom OMA could not be stopped. Conclusion: This is the first study where OMA-treated CSU patients were followed up to six years. In half of them, long-term therapy of six years is still required.

The expression of lysyl-oxidase gene family members in myeloproliferative neoplasms.

Myeloproliferative neoplasms (MPNs) are malignant disorders originating from clonal expansion of a single neoplastic stem cell and characteristically show an increase in bone marrow reticulin fibers. Lysyl oxidases (LOXs) are copper-dependent amine oxidases that play a critical role in the biogenesis of connective tissue by crosslinking extracellular matrix proteins, collagen and elastin. Expression of LOX gene family members is increased in disorders associated with increased fibrosis. To evaluate involvement of LOX gene family in various MPNs. In-situ hybridization was used to detect Lysyl-Oxidase family members in bone marrow biopsies from patients with different MPNs. We compared normal bone marrows and those from patients with polycythemia vera, essential thrombocythemia, chronic myeloid leukemia, and primary myelofibrosis (PMF). Serum levels of lysyl-oxidase from patients with PMF and healthy controls were also examined. LOX gene family was not detected in normal bone marrows. All members of the LOX gene family were over expressed in PMF. In other MPNs a differential pattern of expression was observed. Differences in gene expression were statistically significant (P < 0.010). The medianserum LOX levels in normal controls was 28.4 ± 2.5 ng\ml and 44.6 ± 9.44 ng\ml in PMF (P = 0.02). The varying pattern of expression of LOX genes may reflect differences in the pathophysiology of bone marrow fibrosis in these MPNs. These observations could be used as the basis for future targeted therapy directed against bone marrow fibrosis.

Toll-like receptor-4 expression in infants with pertussis infection.

PURPOSE: Our aim was to evaluate the expression of Toll-like receptor-4 (TLR-4) and the level of interferon-gamma (IFN-γ) and interleukin-10 (IL-10) in young infants infected by Bordetella pertussis and B. parapertussis. METHODS: Twenty-two infants under the age of 3.5 months with the clinical suspicion of pertussis were enrolled in the study. Nasopharyngeal secretions were obtained for laboratory testing, and blood samples were obtained for flow cytometry and cytokine level analysis. RESULTS: Six infants had positive PCR results for pertussis; the other 16 infants had infections attributable to another causal agent and were used as the control group. The mean fluorescence index, used as a measure of TLR-4 expression by monocytes, was significantly lower in infants with pertussis than in the control group infants (34.32 ± 18.58 vs. 63.14 ± 28, respectively; p = 0.041). The serum IFN-γ level was also significantly lower in infants with pertussis than in the control group patients (0.41 ± 0.58 vs. 1.36 ± 1.87, respectively; p = 0.04). No differences were found in the levels of IL-10. CONCLUSIONS: Based on these results, we suggest that TL4 expression by monocytes and serum INF-γ levels are lower in infants with positive PCR results for pertussis than in infants with a non-pertussis upper respiratory tract infection.

The autoreactivity of B cells in hereditary angioedema due to C1 inhibitor deficiency.

Patients with hereditary angioedema (HAE) tend to produce autoantibodies and have a propensity to develop immunoregulatory disorders. We characterize the profile of autoantibodies in a group of HAE patients and investigate their memory B cells' phenotype and activation status. We studied the activity status phenotype, Toll-like receptor (TLR)-9 expression and total phosphotyrosine in B cells isolated from HAE patients. Additionally, the following autoantibodies were assessed in the serum of 61 HAE patients: anti-nuclear, rheumatoid factor, anti-cardiolipin, anti-tissue transglutaminase, anti-endomysial, anti-Saccharomyces cerevisiae, anti-thyroid and anti-neutrophil cytoplasmic antibodies. In 47·5% of HAE patients we detected at least one of the tested autoantibodies. Expression of CD69, CD5 and CD21 was found to be significantly higher on memory B cells from HAE patients compared to healthy controls (4·59 ± 4·41 versus 2·06 ± 1·81, P = 0·04, 8·22 ± 7·17 versus 3·65 ± 3·78, P = 0·05, 2·43 ± 0·54 versus 1·92 ± 0·41, P = 0·01, respectively). Total phosphotyrosine in B cells from HAE patients was significantly higher compared to healthy controls (4·8 ± 1·1 versus 2·7 ± 1·3, P = 0·0003). Memory B cells isolated from the HAE group contained higher amounts of TLR-9 compared to healthy controls (8·17 ± 4·1 versus 4·56 ± 1·6, P = 0·0027). Furthermore, the expression of TLR-9 in memory B cells from HAE patients with autoantibodies was significantly higher than the control group (10 ± 4·7 versus 4·56 ± 1·6, P = 0·0002) and from that in HAE patients without autoantibodies (10 ± 4·7 versus 5·8 ± 0·9, P = 0·036). HAE patients have enhanced production of autoantibodies due most probably to the increased activation of B cells, which was found to be in association with a high expression of TLR-9.

Semaphorin 3A - a marker for disease activity and a potential putative disease-modifying treatment in systemic lupus erythematosus.

Semaphorin 3A (sema3A) and neuropilin-1 (NP-1) play a regulatory role in immune responses and have a demonstrated effect on the course of collagen-induced arthritis. Sema3A was also found to be involved in other immune-mediated diseases, e.g. psoriasis and allergic rhinitis. In this review we concentrated on the involvement of sema3A and NP-1 in the pathogenesis of systemic lupus erythematosus (SLE) and on the specific effect of sema3A on the auto-reactive properties of B cells in SLE patients. We demonstrated the expression of sema3A in renal biopsies from lupus glomerulonephritis patients. This expression was found to be inversely correlated with proteinuria and kidney function tests. Sema3A serum levels in SLE patients were found to be significantly lower than in RA patients (disease control) and lower yet than in normal individuals. Altered serum sema3A levels were found to be in inverse correlation with SLE disease activity, mainly with renal damage and the presence of anti-cardiolipin antibodies. The expression of both sema3A and NP-1 on B cells from SLE patients was significantly different in comparison with normal healthy individuals. Finally, we demonstrated that when sema3A was co-cultured with CpG-ODN-stimulated memory B cells of SLE patients, their TLR-9 expression was significantly reduced by almost 50% (p = 0.001). These findings, along with the observation of sema3A being reduced in SLE patients in correlation with disease severity and autoimmunity, and memory B cells being beneficially responsive to sema3A, suggest this regulatory molecule may be considered as a potential therapy for SLE. Such focused therapies will help in achieving the maintenance of self-tolerance and alter pro-inflammatory status in lupus.

The involvement of immune semaphorins and neuropilin-1 in lupus nephritis.

BACKGROUND AND OBJECTIVES: Neuropilin-1 (NP-1), a functional vascular endothelial growth factor (VEGF) receptor, is important in the priming of resting T cells and contributes to the development of peripheral tolerance. Semaphorins, a family of axon guidance molecules, has been found to be involved in regulating the immune system. The aim of this study was to explore the involvement of NP-1 and semaphorins in lupus glomerulonephritis (LGN). METHODS: Twelve kidney biopsies from LGN patients and five normal biopsies were examined in this study. In addition, eight biopsies from patients with primary nephropathy and proteinuria were included serving as a disease control group. Biopsies were stained with anti-VEGF, NP-1, and semaphorins. The Image Pro-Plus software was used to measure the intensity and extent of staining. The correlation with clinico-pathological parameters was evaluated. RESULTS: VEGF expression was slightly higher in LGN. NP-1 and semaphorins were stained with significantly higher intensity in LGN when compared with both the normal and the disease control groups. NP-1 deposits were found only in damaged glomerulus areas and positively correlated with clinico-pathological parameters of renal disease (a statistical trend). However, the semaphorins were found in inverse correlations. DISCUSSION: Being present in normal and slightly increased in diseased glomeruli, VEGF is considered protective during inflammation. Increased NP-1 expression in LGN may intensify the possible protective effect of VEGF, thereby preventing endothelial damage. However, one should consider the possibility that increased NP-1 expression is harmful and could play a role in the damage of LGN. NP-1 is suggested to be a reliable marker differentiating focal versus diffuse LGN. Semaphorin 3A can serve as a histological marker for tubular damage. The altered ability of kidneys to secrete semaphorins during advanced renal damage may in part explain its inverse correlation with renal function. Further work is needed in order to better understand the role of NP-1 and semaphorins in LGN.

Synthesis of gonadotropin-releasing hormone III analogs. Structure-antitumor activity relationships.

Following the observation that the activity of gonadotropin-releasing hormone III (GnRH-III) in the suppression of growth of MDA-MB-231 and MCF-7 breast cancer cells surpasses that of GnRH and other analogs thereof, analogs of GnRH-III were synthesized to investigate the structural basis for the improved antitumor activity. Compounds synthesized include analogs with changes in the central sequence in which GnRH-III differs from GnRH and in the C- and N-terminal regions. The results indicate that a salt bridge between Asp6 and Lys8 stabilizes the active conformation of GnRH-III and show the importance of the Trp7. Replacement of the C-terminal Gly-NH2 with D-Ala-NH2 was not well tolerated, but replacement with ethylamide was. Replacement of pGlu1 with Ac-D-Trp appears to have a significantly deleterious effect on a unique conformation of GnRH-III which is responsible for its binding to the receptors on cancer cell lines and the resultant antitumor activity.

Novel antitumor peptide hormones and their effect on signal transduction.

A series of novel gonadotropin releasing hormone (GnRH) and Somatostatin analogs have been developed in our laboratory and were screened for antiproliferative and signal transduction inhibitory effect. Our GnRH analog Folligen, had significant antitumor activity on DMBA induced mammary carcinomas in rats without blocking ovarian functions. The direct effect of Folligen and Buserelin has been compared on the human breast cancer cell line MDA-MB-231. Folligen was found to be more effective in inhibiting cell proliferation and significant differences were found in the signal transduction pathways activated by these analogs. Our novel Somatostatin analogs were screened for tyrosine kinase inhibition and for antiproliferative effect on human colon tumor cells and for growth hormone (GH) release inhibition in vitro and in vivo. The analog TT-2-50 was significantly more active inhibiting GH release in superfused rat pituitary cells and in vivo than native Somatostatin and it strongly inhibited tyrosine kinase and proliferation while it stimulated protein kinase C activity.

The contribution of proangiogenic factors to the progression of malignant disease: role of vascular endothelial growth factor and its receptors.

Vascular endothelial growth factor is a major inducer of angiogenesis and a vascular permeability inducing factor. Its expression is upregulated in many types of tumors and it is thought to be a major inducer of tumor angiogenesis. This article focuses on the role of vascular endothelial growth factor in tumor progression and on current efforts aimed at the inhibition of tumor progression through the inhibition of vascular endothelial growth factor activity.

Sensitivity analysis of risk assessment programs to various input parameters.

HESP 2.b risk assessment program was studied in detail concerning the effect of changing different input parameters for the output ADI values calculated by the program. We used the standard Netherlands 1.0 scenario offered by the program. With this we fixed a lot of input parameters which define the area, human and animal recipient parameters etc. The remaining 31 unfixed parameters were fixed at first to "BASE" input values and the BASE output values were calculated by HESP. Later we chose only one parameter at a time and changed it to an another value. The calculated ADI values were then compared to BASE output values. Seven parameters (soil type, soil usage, site length, soil pH, groundwater fraction in drinking water, basement floor type and Qev) were studied. We found, that changing soil pH or Qev have not any influence on the output ADI values in case of any contaminant. Soil type change has not any effect on the output ADI value in case of Pb or Cd but it seems to play important role in all cases of the four organic material we investigated. Changing soil usage have influence on the output ADI value almost in every case. It seems to be linear relation between the maximal concentration of contaminant and calculated ADI. Changing the site length and basement floor type gave in some cases different ADI values compared to BASE values. If we alter the groundwater fraction in drinking water we got usually different ADI values. With Risc Human risk assessment program we got similar results: nor the changes in soil type, site diameter or soil pH gave any changes in output ADI values. Our results hint that using HESP and Risc Human requires enhanced caution.

Neuropilins and semaphorins - from angiogenesis to autoimmunity.

Angiogenesis, the growth of new blood vessels from preexisting ones, is an important process in health and disease. The persistence of neovascularization in inflammatory diseases, such as rheumatoid arthritis (RA), might facilitate the entrance of inflammatory cells into the synovium and stimulate pannus formation. Several potent pro-angiogenic cytokines have been implicated in inflammatory angiogenesis. Of these, vascular endothelial growth factor (VEGF) and its receptors (VEGFRs) have been demonstrated to play a central role in RA, systemic lupus erythematosus (SLE) and multiple sclerosis (MS). Increased serum levels of VEGF were found to correlate with disease activity and severity of these diseases whereas, remission was associated with decreased levels. In the last few years, other molecules, initially found in neurodevelopment, were found to be involved in angiogenesis and recently also in the immune system and autoimmunity. Neuropilins (NPs) are VEGF receptors, while some of the semaphorins (SEMAs) are neuropilins' ligands. Their involvement in the development of autoimmune diseases and the various mechanisms by which they may induce autoimmunity will be discussed in this review.