Increased expression of in situ IL-31RA and circulating CXCL8 and CCL2 in pemphigus herpetiformis suggests participation of the IL-31 family in the pathogenesis of the disease.

BACKGROUND: Pemphigus herpetiformis (PH) is a rare clinical subtype of pemphigus with the presence of urticarial plaques, severe pruritus, rare acantholysis and eosinophilic spongiosis. OBJECTIVES: The aim of this study was to investigate the influence of IL-31 and pro-inflammatory cytokines/chemokines in the pathogenesis of PH. METHODS: Twenty-five patients with PH and three groups: pemphigus foliaceus (PF = 14), pemphigus vulgaris (PV = 15) and healthy controls (HC = 20) were selected for this study. The groups were analysed by immunohistochemistry utilizing IL-31, IL-31RA, IL-4, IL-17 and TNF-α antibodies. Serum levels of IL-4, IL-13, TNF, CXCL8, CCL5 and CCL2 were evaluated by cytometric bead array. RESULTS: Analysis of IL-31 family of PH patients revealed the following findings: (i) Enhanced in situ expression of IL-31 in PH samples, compared to PF and to PV (epidermis); (ii) Cutaneous IL-31RA expression in PH samples was higher than in PF, PV and HC groups (epidermis and dermis); (iii) PF patients that evolved to PH showed significant increased IL-31RA epidermal expression during the PH phase. Profile of pro-inflammatory cytokines (IL-4, IL-17 and TNF-α) in PH patients' skin exhibited: (i) Enhanced IL-4 expression, when compared to patients with PF (epidermis and dermis) and with PV (epidermis); (ii) Augmented IL-17 expression than PF and PV patients (epidermis); (iii) Augmented expression of TNF-α when compared to PF at the epidermal level. Evaluation of circulating cytokines and chemokines showed higher levels of CXCL8 and CCL2 in PH sera compared to HC group. CONCLUSIONS: IL-31 and IL-31RA, cytokines related to pruritus, and pro-inflammatory chemokines (CXCL8 and CCL2) seem to exert a role in the pathogenesis of PH. These findings support future studies to clarify the role of IL-31 pathway as a potential therapeutic target for patients with PH.

Exploring the in situ expression of vascular endothelial growth factor and endoglin in pemphigus foliaceus variants and pemphigus vulgaris.

BACKGROUND: Erythroderma is a severe manifestation of pemphigus foliaceus (PF), a blistering disease mediated by IgG autoantibodies against desmoglein 1. Increasing evidence supports the contribution of angiogenic mediators in the pathogenesis of erythroderma. OBJECTIVE: To evaluate the in situ expression of vascular endothelial growth factor (VEGF) and endoglin in patients with PF with erythroderma. METHODS: Formalin-fixed paraffin-embedded skin samples obtained from patients with erythrodermic PF (n = 19; 12 patients with endemic PF), non-erythrodermic PF (n = 17), pemphigus vulgaris (PV; n = 10), psoriasis (n = 10) and healthy individuals (HI; n = 10) were processed in an automated immunohistochemistry platform utilizing anti-VEGF and anti-endoglin as primary antibodies. Reactivity was evaluated both manually (0 = negative; 1+ = mild; 2+ = intense) and through an automated microvessel analysis algorithm. RESULTS: Vascular endothelial growth factor expression in erythrodermic PF was higher than in non-erythrodermic PF (P = 0.034) and in HI (P = 0.004), and similar to psoriasis (P = 0.667) and PV (P = 0.667). In non-erythrodermic PF, VEGF positivity was similar to HI (P = 0.247), and lower than psoriasis (P = 0.049) and PV (P = 0.049). Both erythrodermic and non-erythrodermic PF presented similar endoglin expression (P = 0.700). In addition, endoglin positivity during erythrodermic PF was similar to psoriasis (P = 0.133) and lower than PV (P = 0.0009). Increased expression of in situVEGF suggests that healing processes are triggered in response to tissue damage led by autoantibodies in PF, especially during erythroderma. Reduced endoglin positivity suggests that an unbalanced angiogenesis may occur during erythrodermic PF. Further studies may help to confirm if the regulation of VEGF and endoglin expression in patients with PF can contribute to control the healing process and enable disease remission. CONCLUSION: Overexpression of VEGF in erythrodermic PF as well as in PV and psoriasis points out a dysregulated repair process in severe forms of these diseases and suggests VEGF and endoglin could act as prognostic markers and future therapeutic targets to enable proper healing in PF.

Increased serum levels of vascular endothelial growth factor in pemphigus foliaceus patients with erythroderma.

BACKGROUND: Erythroderma is a clinical skin syndrome shared by patients with cutaneous disorders of distinct aetiologies as a result of the combined actions of chemokines, adhesion molecules, and cytokines, such as vascular endothelial growth factor (VEGF). OBJECTIVE: To evaluate the profile of serum levels of VEGF and soluble vascular endothelial growth factor receptor 1 (sVEGFR-1) in pemphigus foliaceus (PF) patients with erythroderma. METHODS: We conducted a retrospective study, which included (i) a chart review of all PF patients from the Autoimmune Blistering Clinic, University of Sao Paulo, Brazil, from January 1991 to December 2014, together with an evaluation of demographic variables, hospitalization duration and complications and (ii) analysis of the circulating VEGF and sVEGFR-1 levels in PF patients with erythroderma by ELISA. The controls included patients with pemphigus vulgaris or psoriasis. RESULTS: We observed higher serum VEGF levels in PF patients during erythroderma than during the non-erythrodermic phase. PF patients showed increased serum levels of sVEGFR-1 during the erythrodermic phase in comparison to controls. Interestingly, the sVEGFR-1 and antidesmoglein-1 levels were positively correlated during the non-erythrodermic period. CONCLUSION: Erythroderma, which represents one clinical form of PF, implies more severe outcomes. The circulating levels of VEGF, a potent endothelial activator, are increased in PF patients with erythroderma; this result suggests the contribution of the blood vessel endothelium to the pathogenesis of this clinical syndrome. Interestingly, our findings showed a positive correlation between the sVEGFR-1 and antidesmoglein-1 antibody levels, indicating a suppressive response to VEGF augmentation during the erythrodermic phase of PF.

Drug reaction with eosinophilia and systemic symptoms/drug-induced hypersensitivity syndrome: clinical features of 27 patients.

BACKGROUND: Drug reaction with eosinophilia and systemic symptoms (DRESS) [also called drug-induced hypersensitivity syndrome (DIHS)] includes severe reactions to drugs that need to be promptly recognized by physicians. AIM: To explore heterogeneity in the clinical presentation of DRESS/DIHS at a large academic hospital in Latin America, using the criteria defined by the European Registry of Severe Cutaneous Adverse Reactions (RegiSCAR) scoring system. METHODS: A retrospective medical record review of 60 patients with diagnostic suspicion of DRESS/DIHS admitted to our hospital between July 2008 and April 2012 was performed, including demographic data, clinical features, laboratory findings and treatment. RESULTS: Of the 60 patients, 27 fulfilled the criteria for DRESS/DIHS. Maculopapular exanthema (85.1%), fever (96.2%) and hepatic involvement (85.1%) were the most common features. Anticonvulsants were the most common causal drugs (77.7%); Phenytoin was the most common individual drug (44.4%), followed by carbamazepine (29.6%). All patients were treated initially with prednisone 1 mg/kg/day. Mortality rate was 4%. CONCLUSION: The major findings of this study (to our knowledge the largest collection of data on DRESS/DIHS in Latin America) include a positive statistical association between presence of atypical lymphocytes and higher levels of alanine aminotransferase (P < 0.001) and reinforce the importance of anticonvulsants in the pathogenesis of this severe reaction.

Clinical and immunological profile of umbilical involvement in pemphigus vulgaris and pemphigus foliaceus.

BACKGROUND: Pemphigus vulgaris (PV) and pemphigus foliaceus (PF) are autoimmune vesicobullous disorders with IgG autoantibodies directed against desmoglein (Dsg)1 and 3, which lead to intraepidermal acantholysis. AIM: To characterize the clinical and immunological profile of patients with PF or PV with umbilical involvement. METHODS: In total, 10 patients (7 women, 3 men; age range 24-70 years, disease duration 3-16 years) diagnosed with either PV (n = 5) or mucocutaneous PF (n = 5) were assessed according to their clinical features, histopathology and immunological findings [direct and indirect immunofluorescence (DIF and IIF) and ELISA with recombinant Dsg1 and Dsg3]. RESULTS: Erythema, erosions, crusts and vegetating skin lesions were the main clinical features of the umbilical region. DIF of the umbilical region gave positive results for intercellular epidermal IgG and C3 deposits in eight patients and for IgG alone in the other two. Indirect immunofluorescence with IgG conjugate showing the typical pemphigus pattern was positive in all 10 patients, with titres varying from 1 : 160 to 1 : 2560. ELISA with recombinant Dsg1 gave scores of 24-266 in PF and 0-270 in PV. Reactivity to recombinant Dsg3 was positive in all five patients with PV (ELISA 22-98) and was negative in all PF sera. CONCLUSIONS: All 10 patients with pemphigus with umbilical presentation had the clinical and immunopathological features of either PF or PV. This peculiar presentation, not yet completely elucidated, has rarely been reported in the literature. A possible explanation for this unique presentation may be the presence of either novel epitopes or an association with embryonic or scar tissue located in the umbilical-cord region.

Laryngeal involvement in pemphigus vulgaris: a proposed classification.

OBJECTIVE: (1) To investigate the incidence of laryngeal involvement in a large series of patients with pemphigus vulgaris, using endoscopic examination, (2) to describe the lesions, and (3) to establish a classification of laryngeal involvement in pemphigus vulgaris based on the location of the lesions. STUDY DESIGN: Prospective study. METHODS: A total of 40 sequentially treated pemphigus vulgaris patients, diagnosed using clinical, histological and immunofluorescence criteria, were evaluated for laryngeal manifestations using endoscopic examination. The results were used to establish a graded classification of laryngeal involvement according to the location of the lesions. RESULTS: Active laryngeal lesions (ulcers or blisters) were found in 16 patients (40 per cent). Of these, 37.5 per cent were classified as grade I, 20 per cent as grade II, 20 per cent as grade III and 17.5 per cent as grade IV. CONCLUSION: Laryngeal involvement is common in pemphigus vulgaris and must be considered at the point of diagnosis. Grade I lesions are the most frequent.

Clinical and immunopathological evaluation of epidermolysis bullosa acquisita.

BACKGROUND: Epidermolysis bullosa acquisita (EBA) is a subepidermal blistering disease with IgG antibodies against collagen VII. The disease is heterogeneous and can lead to significant morbidity. AIM: To characterize the clinical and laboratory profile of patients with EBA from Sao Paulo, Brazil. METHODS: In total, 12 patients (mean age 24 years) were analysed for cutaneous and mucosal involvement, laboratory data and response to treatment. RESULTS: Mucosal involvement occurred in 11 of the 12 patients (eyes in 4/12, nose in 4/9, pharynx-larynx in 5/9 and oesophagus in 4/10; 3 patients did not undergo nasopharyngeal examination and 2 paediatric patients did not undergo endoscopy). Using direct immunofluorescence, different patterns of deposits were found at the basement membrane zone: IgG (12/12), IgA (6/12), IgM (4/12), C3 (11/12). Indirect immunofluorescence (IIF) was positive in 6 of 12 patients, and IIF on salt-split skin detected dermal deposition in 10 of 12 patients. Antinuclear antibodies were found in 3 of 12 patients, but none of them fulfilled the criteria for systemic lupus erythematosus. After treatment, total remission was achieved in three patients and partial remission in five (three were maintained on minimal treatment, one on the full treatment and one was able to come off treatment). Two patients were lost to follow-up and the remaining two had disease flares. Complications were mainly mucosal (oesophageal stenosis, laryngeal synechia, symblephara and trichiasis). CONCLUSIONS: Mucosal involvement in EBA is a determining factor for disease morbidity. Complete evaluation of the patient, focusing on both cutaneous and extracutaneous sites is essential, as EBA may evolve to refractory disease, severely compromising its outcome.

Changes in the autoimmune blistering response: a clinical and immunopathological shift from pemphigus foliaceus to bullous pemphigoid.

We describe a 64-year-old Brazilian man who developed bullous pemphigoid (BP) 12 years after pemphigus foliaceus (PF) was diagnosed. On his first presentation in 1992, histological examination revealed intraepidermal blistering and acantholysis at the granular layer, direct immunofluorescence (DIF) demonstrated intercellular deposits of C3 in the epidermis, and indirect immunofluorescence showed the presence of IgG antibodies against the intercellular spaces. In 2004, laboratory findings revealed a subepidermal blister with neutrophils and eosinophils (by histology), DIF demonstrated deposition of IgG and C3 along the basement membrane zone, salt-split skin showed IgG deposition in the epidermal side of the blister, and immunoblotting showed reactivity against BP180. The occurrence of two autoimmune blistering conditions in the same patient is a rare event, and may suggest an intermolecular epitope-spreading phenomenon.

Calmodulin antagonists inhibit T-type Ca(2+) currents in mouse spermatogenic cells and the zona pellucida-induced sperm acrosome reaction.

The sperm acrosome reaction (AR) is a regulated exocytotic process required for gamete fusion. It depends on an increase in [Ca(2+)](i) mediated by Ca(2+) channels. Although calmodulin (CaM) has been reported to regulate several events during the AR, it is not known whether it modulates sperm Ca(2+) channels. In the present study we analyzed the effects of CaM antagonists W7 and trifluoroperazine on voltage-dependent T-type Ca(2+) currents in mouse spermatogenic cells and on the zona pellucida-induced AR in sperm. We found that these CaM antagonists decreased T-currents in a concentration-dependent manner with IC(50) values of approximately 10 and approximately 12 microM, respectively. W7 altered the channels' voltage dependence of activation and slowed both activation and inactivation kinetics. It also induced inactivation at voltages at which T-channels are not activated, suggesting a promotion of inactivation from the closed state. Consistent with this, W7 inhibited the ZP-induced [Ca(2+)](i) transients in capacitated sperm. Likewise, W7 and TFP inhibited the AR with an IC(50) of approximately 10 microM. In contrast, inhibitors of CaM-dependent kinase II and protein kinase A, as well as a CaM-activated phosphatase, had no effect either on T-currents in spermatogenic cells or on the sperm AR. Together these results suggest a functional interaction between CaM and the sperm T-type Ca(2+) channel. They are also consistent with the involvement of T-channels in the AR.

Immunopathologic characterization of the tissue response in endemic pemphigus foliaceus (fogo selvagem).

BACKGROUND: The research on endemic pemphigus foliaceus (fogo selvagem) has mainly focused on the humoral immune response, but little attention has been given to the function of cell-mediated immune response and the nature of the cellular elements of the tissue reaction in the lesions of fogo selvagem. OBJECTIVE: The purpose of this study was the immunophenotype characterization of the inflammatory cells as well as the expression of adhesion molecules and HLA-DR in the perilesional and lesional skin of fogo selvagem. METHODS: Twenty biopsy specimens of lesional and perilesional skin were analyzed by immunohistochemical techniques. The panel of monoclonal antibodies consisted of CD8, CD4, CD1a, HLA-DR, IL-2R, LFA-1, ICAM-1, and PAN-B. RESULTS: The semiquantitative analysis of the cell population revealed a predominance of CD4 T lymphocytes in the tissue response of perilesional and lesional skin. The population of epidermal Langerhans cells was decreased in lesional skin when compared with the perilesional skin, whereas CD1a(+) dermal dendritic cells predominated in lesional skin. Keratinocyte expression of ICAM-1 and HLA-DR was negative in both lesional and perilesional skin. CONCLUSION: The overall results suggest the participation of the cell-mediated immunity in endemic pemphigus foliaceus (fogo selvagem). The lack of keratinocyte ICAM-1 expression may be related to the pattern of cytokines secreted by the CD4(+) T cells of the tissue reaction in fogo selvagem.

Giant nevus lipomatosus cutaneus superficials: case report and review of the literature.

Nevus lipomatosus superficialis is a rare disorder characterized by a nevoid fatty growth within the papillary and reticular dermis. Lesions more often occur in the sacral, abdominal, or pelvic regions. A 36-year-old Brazilian female with giant nevus lipomatosus is reported. Our case seems to be the biggest reported in the literature.

Localisation of inositol trisphosphate and ryanodine receptors during mouse spermatogenesis: possible functional implications.

During spermatogenesis the activity of intracellular Ca(2+)-release channels is likely to play an important role in different specific cellular functions. Accordingly, messenger RNAs for the three inositol 1,4,5-trisphosphate receptor (IP3R) subtypes were found to be present throughout spermatogenesis. Immunocytochemical analysis revealed distinct distribution patterns of the mature IP3Rs during sperm differentiation. At early stages, IP3Rs are distributed throughout the cytoplasm, and as differentiation proceeds they become selectively localised to the Golgi complex. Consistently, spermatogonia underwent large intracellular Ca2+ release in response to thapsigargin (TG), while smaller responses were detected in late spermatocytes and spermatids. The distribution of IP3Rs and the larger Ca(2+)-release responses found in spermatogonia, suggest that IP3Rs may be involved in cell proliferation at this stage. This notion is supported by our observations in a spermatogenic cell line that depletion of intracellular Ca2+ pools using TG inhibits cell division, and that incubation with an IP3R-I antisense oligonucleotide completely inhibited proliferation. Furthermore, the three genes encoding ryanodine receptor proteins (RyRs) are expressed at all stages of spermatogenesis. However, immunocytochemical studies with specific antibodies against each of the RyR subtypes detected types 1 and 3 in spermatogenic cells and only type 3 in mature sperm. In contrast to IP3Rs, RyRs remain scattered in the cytoplasm throughout differentiation. Functional responses to caffeine and ryanodine were absent in spermatogenic cells and in mature sperm. These findings suggest that IP3Rs have significantly more important roles in spermatogenesis than RyRs, and that one of these roles is crucial for cell proliferation.

T-type Ca2+ channels and alpha1E expression in spermatogenic cells, and their possible relevance to the sperm acrosome reaction.

There is pharmacological evidence that Ca2+ channels play an essential role in triggering the mammalian sperm acrosome reaction, an exocytotic process required for sperm to fertilize the egg. Spermatozoa are small terminally differentiated cells that are difficult to study by conventional electrophysiological techniques. To identify the members of the voltage-dependent Ca2+ channel family possibly present in sperm, we have looked for the expression of the alpha 1A, alpha 1B, alpha 1C, alpha 1D and alpha 1E genes in mouse testis and in purified spermatogenic cell populations with RT-PCR. Our results indicate that all 5 genes are expressed in mouse testis, and in contrast only alpha 1E, and to a minor extent alpha 1A, are expressed in spermatogenic cells. In agreement with these findings, only T-type Ca2+ channels sensitive to the dihydropyridine nifedipine were observed in patch-clamp recordings of pachytene spermatocytes. These results suggest that low-threshold Ca2+ channels are the dihydropyridine-sensitive channels involved in the sperm acrosome reaction.

A cAMP regulated K+-selective channel from the sea urchin sperm plasma membrane.

Ion channels are deeply involved in sperm physiology. In sea urchin sperm cyclic nucleotide levels increase during quimotaxis and in the acrosome reaction (AR). Although cyclic nucleotides are second messengers known to directly or indirectly modulate ion channels, it is not clear how they modulate sperm responses to the egg outer layer. Here, we describe a cAMP regulated K+-selective channel from sea urchin sperm plasma membranes fused into planar bilayers that may have a role during sea urchin sperm quimotaxis and/or the AR. Its single channel conductance in 100 mM KCl is 103 pS. In bi-ionic experiments, the channel displayed a K+/Na+ permeability ratio (PK+/PNa+) of approximately 5. Thus, in sea water its reversal potential would be approximately -13 mV and channel opening would depolarize spermatozoa. The channel has low open probability (Po = 0.8 +/- 0.2% at 0 mV applied voltage) and weak voltage dependence. Channel activity is reversibly up-regulated by cAMP in the cis bilayer side, but not by cGMP. This modulation followed a single Langmuir isotherm with an apparent kd of 200 microM. At this concentration the channel open probability at 0 mV increased up to 11- fold. TEA+ blocked the channel only from the trans side. Also Ba2+ in trans blocked the channel in a voltage-dependent manner.

Cellular immunity in paracoccidioidomycosis.

Paracoccidioidomycosis was studied in 62 patients from Brazil in the 10 year period between 1978 and 1988. In 46 patients included in a first group, the disease was active and in 16 patients included in a second group, the disease was cured. The study was conducted according to both the clinical form of the disease and the response to paracoccidioidin in both groups. In the first group, 10 patients presented the acute form, 12 presented the chronic unifocal form and 24 had the chronic multifocal form of the disease. As to the response to paracoccidioidin, in the first group 16 patients were negative and 30 were positive; in the second group, 11 were positive and 5 were negative. An immunological study was performed in all patients using in vivo methods such as skin tests and sensitization to DNCB and in vitro techniques such as total lymphocyte counts, T and B cell counts, leukocyte migration inhibition test, chemotaxis of total leukocytes and mononuclear leukocyte phagocytosis.