Pathogenic role of anti-endothelial cell antibodies in autoimmune rheumatic diseases

Anti-endothelial cells antibodies have been detected in numerous autoimmune and inflammatory diseases, including systemic lupus erythematous, rheumatoid arthritis, vasculitis and sarcoidosis. Anti-endothelial cells antibodies bind to endothelial cell antigens and induce endothelial damage. Their effects on the endothelial cell have been considered responsible, at least in part, by the vascular injury which occurs in these pathological conditions. Lupus (2009) 18, 1233-1238.

Novel humanized anti-CD3 antibodies induce a predominantly immunoregulatory profile in human peripheral blood mononuclear cells

Strategies to minimize the immunogenicity and toxicity of murine anti-CD3 antibodies (e.g. OKT3) are of special interest for organ transplantation and for the treatment of autoimmune diseases. In the present work, we have developed two humanized anti-CD3 antibodies. These molecules were shown to bind to human CD3, though less efficiently, and display less mitogenic activity than CKT3. These results prompted us to investigate whether this reduced mitogenic potential was associated with the development of anti-inflammatory properties. Indeed, in peripheral blood mononuclear cells (PBMCs), the humanized antibody versions induced a predominantly anti-inflammatory cytokine profile, in contrast with the pro-inflammatory profile induced by OKT3. Neither OKT3 nor the humanized versions induced the expression of IL-4, IL-2 or TGF-beta. Both humanized antibodies induced significantly lower production of IFN-gamma and IL-5 and slightly higher production of IL-10 than OKT3. This immunomodulatory profile was most evident by the 80-fold higher ratio of IL-10/IFN-gamma production in PBMCs cultured in the presence of the humanized antibodies, compared to those stimulated with CKT3. Furthermore, these humanized anti-CD3 antibodies induced a late FOXP3 gene expression while OKT3 led to a more transient expression of FOXP3. Taken our results, we suggest that these humanized anti-CD3 antibodies may promote the development of T cells with immunoregulatory activity. (C) 2009 Elsevier B.V. All rights reserved.

Evaluation of RET polymorphisms in a six-generation family with G533C RET mutation: specific RET variants may modulate age at onset and clinical presentation

P>Context We previously described a six-generation family with G533C RET mutation and medullary thyroid carcinoma, in the largest family reported do date. Of particular interest, phenotype variability regarding the age of onset and clinical presentation of the disease, was observed. Objective We evaluate whether single SNPs within RET oncogene or haplotype comprising the RET variants (defined by Haploview) could predispose to early development of MTC in this family and influence the clinical manifestation. Design Eight SNPs were selected based on their previous association with the clinical course of hereditary or sporadic MTC, in particular promoting an early onset of disease. The variants were initially tested in 77 G533C-carriers and 100 controls using either PCR-direct sequencing or PCR-RFLP. Association between a SNP or haplotype and age at diagnosis or presence of lymph node metastasis was tested in 34 G533C-carries with MTC. Different bioinformatic tools were used to evaluate the potential effects on RNA splicing. Results An association was found between IVS1-126G > T and age at diagnosis. The variant [IVS8 +82A > G; 85-86 insC] was associated with the presence of lymph node metastases at diagnosis. In silico analysis suggested that this variant may induce abnormal splicing. This in silico analysis predicted that the [IVS8 +82A > G; 85-86 insC] could alter the splicing by disrupting and/or creating exonic splicing enhancer motifs. Conclusions We here identified two RET variants that were associated with phenotype variability in G533C-carriers, which highlights the fact that the modifier effect of a variant might depend on the type of mutation.

The frequency of anti-beta(2)-glycoprotein I antibodies is low and these antibodies are associated with pulmonary hypertension in mixed connective tissue disease

The objective of this study is to evaluate the prevalence of antiphospholipid antibodies, mainly anti-beta(2)-glycoprotein I (anti-beta(2)-GPI), and their possible clinical and laboratory relevance in mixed connective tissue disease (MCTD). This study included 39 consecutive patients with MCTD (Kasukawa`s criteria) from January, 2005, to March, 2007, and compared them with 21 age- and sex-matched healthy controls. IgG and IgM anticardiolipin (aCL) and anti-beta(2)-GPI were measured by ELISA. Lupus anticoagulant (LA) was detected by functional coagulation tests. Medium to high titres of aCL and anti-beta(2)-GPI antibodies were found in sera from four (10.2%) MCTD patients. One of these patients was found to be positive for IgM aCL, IgM anti-beta(2)-GPI and LA antibodies simultaneously. Additionally, this patient had a previous history of foetal loss in the second trimester and new-onset pulmonary arterial hypertension (PAH). The other three patients had none of the manifestations of antiphospholipid syndrome (APS) or PAH. The mean value of IgG anti-beta(2)-GPI was higher among those MCTD patients with PAH than in the group without PAH (34.2 +/- 46.8 vs 12.3 +/- 9.1, P = 0.018). None of the controls were positive for antiphospholipid antibodies. High to moderate titres of anti-beta(2)-GPI as well as APS were rare in MCTD, and these antibodies may be correlated with the development of PAH in these patients. Lupus (2009) 18, 618-621.

A Duplex Allele-Specific Amplification PCR to Detect SMN1 Deletion

Spinal muscular atrophy (SMA), the leading genetic cause of death in childhood, is an autosomal recessive neuromuscular disorder characterized by progressive muscle weakness, associated with deletions of the survival motor neuron (SMN) gene identified and mapped to chromosome 5q13. SMN is present in two highly homologous copies (SMN1 and SMN2). In the general population, normal individuals (noncarriers) have at least one telomeric (SMN1) copy, and 5% of them have no copies of SMN2. Approximately 95% of SMA patients carry homologous deletions of SMN1 exon(s) 7 (and 8). SMN1 and SMN2 exons 7 and 8 differ only by 1 bp each, and SMA diagnosis might be performed by single-strand conformational polymorphism, PCR amplification followed by restriction fragment length polymorphism (RFLP), multiple ligation-dependent probe amplification, or realtime PCR of SMNs exons 7 and 8. We developed a simpler and cost-effective method to detect SMN1 exon 7 deletion based on allele-specific amplification PCR.

Autoimmune bullous dermatoses

Autoimmune bullous dermatoses are diseases in which blisters and vesicles are the primary and fundamental types of skin lesion. Their classification is based on the location of the blister; intraepidermal and subepidermal. Patients produce autoantibodies against self-specific structures of the skin detectable by immunofluorescence techniques, immunoblotting and ELISA. Recent advances in molecular and cellular biology have brought to knowledge these self-antigens, against which patients are sensitized, and which are found in epidermis or in the dermo-epidermal junction. These are low incidence, but high morbidity diseases that may be fatal. The aim of this article is to review and describe the progress of four autoimmune vesiculobullous disorders; endemic pemphigus foliaccous (wild fire), pemphigus vulgaris, bullous pemphigoid and dermatitis herpetiformis.

HLA-DR in Brazilian patients with polyarteritis nodosa (PAN) and microscopic polyangiitis (MPA)

The aim of this study was to evaluate the frequency and clinical associations of HLA-DR alleles in Brazilian Caucasian patients with polyarteritis nodosa (PAN) or microscopic polyangiitis (MPA). We evaluated 29 Caucasian patients with vasculitis classified as PAN or MPA according to the American College of Rheumatology (ACR) 1990 Criteria, Chapel Hill Consensus Conference (CHCC) nomenclature for vasculitis and EULAR recommendations for conducting clinical studies in systemic vasculitis. HLA-DR alleles were typed using polymerase chain reaction-amplified DNA, hybridized with sequence-specific low resolution primers. DNA obtained from 59 Caucasian healthy blood donors were used as control. In order to evaluate if a specific HLA may have influence on the clinical profile of those diseases, we also divided the patients according to Birmingham vasculitis score (BVAS) and Five-Factors Score (FFS) at the time of diagnosis. Increased frequency of HLA-DRB1*16 (p = 0.023) and DRB4*01 (p = 0.048) was found in patients with higher disease activity at the time of diagnosis (BVAS >= 22). Patients with less severe disease (FFS = 0) had a higher frequency of HLA-DRB1*03 (p = 0.011). Patients with gastrointestinal tract involvement had significantly increased frequency of HLA-DRB1*11 or B1*12 (p = 0.046), B1*13 (p = 0.021) and B3 (p = 0.008). In contrast, patients with renal disease, had higher frequency of DRB1*15 or DRB1*16 (p = 0.035) and B5 (p = 0.035). In the subgroup of patients with MPA, increased frequency of HLA-DRB1*15 was found in patients with BVAS >= 22 (p = 0.038) and FFS >= 1 (p = 0.039) suggesting that this allele is associated with more aggressive disease. Antineutrophil cytoplasmic antibodies (ANCA) negative MPA patients had significantly increased frequency of HLA-DRB1*11 or DRB1*12 when compared to ANCA positive patients (p = 0.023). Our results suggest that HLA-DR alleles may influence PAN and MPA clinical expression and outcome and that in MPA they participate in the mechanisms involved in the development to ANCA.

Western blotting method (TESAcruzi) as a supplemental test for confirming the presence of anti-Trypanosoma cruzi antibodies in finger prick blood samples from children aged 0-5 years in Brazil

Some Latin American countries have plans for total control and/or eradication of Chagas disease by the main vector (Triatoma infestans) and by blood transfusion. To achieve this, patients with Chagas disease must be identified. A Western blotting test, TESAcruzi, is described as a supplemental test for diagnosis of Chagas disease using samples collected from children <5 years living in different states of Brazil. Blood samples collected by finger prick on filter paper were sent to the test laboratory by a central laboratory to confirm results obtained previously. Ten percent of negative samples, all doubtful and all positive samples were received. Commercial reagents, IgG indirect immunofluorescence, enzyme immunoassay, and a recently introduced TESAcruzi test were used. From 8788 samples, 163 (1.85%) were reactive by IgG-ELISA and 312 (3.55%) by IgG IIF. From these, 77 (0.87%) were reactive in the TESAcruzi test. The results had high clinical value to identify those truly infected. (C) 2010 Elsevier B.V. All rights reserved.

Immunization with pVAXhsp65 Decreases Inflammation and Modulates Immune Response in Experimental Encephalomyelitis

Background: A DNA vaccine (pVAXhsp65) containing the gene of a heat-shock protein (hsp65) from Mycobacterium leprae showed high immunogenicity and protective efficacy against tuberculosis in BALB/c mice. A possible deleterious effect related to autoimmunity needed to be tested because hsp65 is highly homologous to the correspondent mammalian protein. In this investigation we tested the effect of a previous immunization with DNAhsp65 in the development of experimental autoimmune encephalomyelitis (EAE), a rat model of multiple sclerosis. Methods: Female Lewis rats were immunized with 3 pVAXhsp65 doses by intramuscular route. Fifteen days after the last DNA dose the animals were evaluated for specific immunity or submitted to induction of EAE. Animals were evaluated daily for weight loss and clinical score, and euthanized during the recovery phase to assess the immune response and inflammatory infiltration at the central nervous system. Results: Immunization with pVAXhsp65 induced a specific immune response characterized by production of IgG(2b) anti-hsp65 antibodies and IFN-gamma secretion. Previous immunization with pVAXhsp65 did not change EAE clinical manifestations (weight and clinical score). However, the vaccine clearly decreased brain and lumbar spinal cord inflammation. In addition, it downmodulated IFN-gamma and IL-10 production by peripheral lymphoid organs. Conclusion: Our data demonstrated that this vaccine does not trigger a deleterious effect on EAE development and also points to a potential protective effect. Copyright (C) 2010 S. Karger AG, Basel

Representational Difference Analysis (RDA) reveals differential expression of conserved as well as novel genes during caste-specific development of the honey bee (Apis mellifera L.) ovary

In highly eusocial insects, such as the honey bee, Apis mellifera, the reproductive bias has become embedded in morphological caste differences. These are most expressively denoted in ovary size, with adult queens having large ovaries consisting of 150-200 ovarioles each, while workers typically have only 1-20 ovarioles per ovary. This morphological differentiation is a result of hormonal signals triggered by the diet change in the third larval instar, which eventually generate caste-specific gene expression patterns. To reveal these we produced differential gene expression libraries by Representational Difference Analysis (RDA) for queen and worker ovaries in a developmental stage when cell death is a prominent feature in the ovarioles of workers, whereas all ovarioles are maintained and extend in length in queens. In the queen library, 48% of the gene set represented homologs of known Drosophila genes, whereas in the worker ovary, the largest set (59%) were ESTs evidencing novel genes, not even computationally predicted in the honey bee genome. Differential expression was confirmed by quantitative RT-PCR for a selected gene set, denoting major differences for two queen and two worker library genes. These included two unpredicted genes located in chromosome 11 (Group11.35 and Group11.31, respectively) possibly representing long non-coding RNAs. Being candidates as modulators of ovary development, their expression and functional analysis should be a focal point for future studies. (C) 2011 Elsevier Ltd. All rights reserved.

NOVEL 65 kDa RNA-BINDING PROTEIN IN SQUID PRESYNAPTIC TERMINALS

A polyclonal antibody (C4), raised against the head domain of chicken myosin Va, reacted strongly towards a 65 kDa polypeptide (p65) on Western blots of extracts from squid optic lobes but did not recognize the heavy chain of squid myosin V. This peptide was not recognized by other myosin Va antibodies, nor by an antibody specific for squid myosin V. In an attempt to identify it, p65 was purified from optic lobes of Loligo plei by cationic exchange and reverse phase chromatography. Several peptide sequences were obtained by mass spectroscopy from p65 cut from sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) gels. BLAST analysis and partial matching with expressed sequence tags (ESTs) from a Loligo pealei data bank indicated that p65 contains consensus signatures for the heterogeneous nuclear ribonucleoprotein (hnRNP) A/B family of RNA-binding proteins. Centrifugation of post mitochondrial extracts from optic lobes on sucrose gradients after treatment with RNase gave biochemical evidence that p65 associates with cytoplasmic RNP complexes in an RNA-dependent manner. Immunohistochemistry and immunofluorescence studies using the C4 antibody showed partial co-labeling with an antibody against squid synaptotagmin in bands within the outer plexiform layer of the optic lobes and at the presynaptic zone of the stellate ganglion. Also, punctate labeling by the C4 antibody was observed within isolated optic lobe synaptosomes. The data indicate that p65 is a novel RNA-binding protein located to the presynaptic terminal within squid neurons and may have a role in synaptic localization of RNA and its translation or processing. (C) 2010 IBRO. Published by Elsevier Ltd. All rights reserved.

Reliability and validity of the Brazilian-Portuguese version of the Burns Specific Pain Anxiety Scale (BSPAS)

Background: Pain and anxiety are a common problem in all recovery phases after a burn. The Burns Specific Pain Anxiety Scale (BSPAS) was proposed to assess anxiety in burn patients related to painful procedures. Objectives: To assess internal consistency, discriminative construct validity, dimensionality and convergent construct validity of the Brazilian-Portuguese version of the Burns Specific Pain Anxiety Scale. Design: In this cross-sectional study, the original version of the BSPAS, adapted into Brazilian Portuguese, was tested for internal consistency (Cronbach`s Alpha), discriminative validity (related to total body surface area burned and sex), dimensionality (through factor analysis), and convergent construct validity (applying the Visual Analogue Scale for pain and State-Anxiety-STAI) in a group of 91 adult burn patients. Results: The adapted version of the BSPAS displayed a moderate and positive correlation with pain assessments: immediately before baths and dressings (r = 0.32; p < 0.001), immediately after baths and dressings (r = 0.31; p < 0.001) and during the relaxation period (r= 0.31; p < 0.001) and with anxiety assessments (r = 0.34; p < 0.001). No statistically significant differences were observed when comparing the mean of the adapted version of the BSPAS scores with sex (p = 0.194) and total body surface area burned (p = 0.162) (discriminative validity). The principal components analysis applied to our sample seems to confirm anxiety as one single domain of the Brazilian-Portuguese version of the BSPAS. Cronbach`s Alpha showed high internal consistency of the adapted version of the scale (0.90). Conclusion: The Brazilian-Portuguese version of the BSPAS 9-items has shown statically acceptable levels of reliability and validity for pain-related anxiety evaluation in burn patients. This scale can be used to assess nursing interventions aimed at decreasing pain and anxiety related to the performance of painful procedures. (c) 2010 Elsevier Ltd. All rights reserved.