Indirect Immunofluorescence Studies Research Articles

Radiation-Induced Localized Bullous Pemphigoid.

Radiation-Induced Localized Bullous Pemphigoid.

Mucous membrane pemphigoid - a report of four cases

Introduction. Mucous membrane pemphigoid (MMP) is a rare autoimmune, chronic inflammatory disease that affects mucous membranes, most commonly the eyes and mouth, with or without skin involvement. It is a complex disease with several complications, including scarring, especially on conjunctival mucosa, that can lead to visual loss. Case report. We report four patients (two men and two women) with MMP. In all patients, the disease started between seventy and eighty years of age. The diagnosis was confirmed based on clinical appearance, histology, direct and indirect immunofluorescence studies, indirect split skin technique, and enzyme-linked immunosorbent assay (ELISA) test. The majority of lesions were on the gums and buccal mucosa; one patient had laryngeal involvement and a lesion on the umbilicus. No ocular involvement and no malignancy were detected. Direct immunofluorescence tests revealed continuous linear IgG deposition in the basal membrane zone in two patients, and they were treated with oral nicotinamide and tetracycline hydrochloride. In two patients, we detected IgG along with IgA linear deposition; they received treatment with methylprednisolone. Complete remission was achieved in all patients. Conclusion. Early diagnosis and an adequate therapeutic approach are necessary for the MMP treatment in long-term disease control and reduction of disease-related complications.

Utility of Direct Immunofluorescence Using Buccal Mucosal Biopsies in Those with Suspected Isolated Ocular Mucous Membrane Pemphigoid

To determine the rate of positivity of immunofluorescence studies in buccal biopsies in patients with cicatrizing conjunctivitis undergoing workup for ocular mucous membrane pemphigoid (MMP)/ocular cicatricial pemphigoid (OCP). Retrospective cohort review. Forty-one patients with cicatrizing conjunctivitis undergoing workup for OCP. A retrospective chart review of direct immunofluorescence (DIF) studies in buccal mucosal biopsies was performed. The primary outcome measure was the rate of positivity of direct and indirect immunofluorescence studies on buccal mucosal biopsies. Twenty-two patients (54%) had a positive buccal mucosal biopsy; 64% of patients (14/22) demonstrated+DIF on initial biopsy and an additional 36% of patients (8/22) on the second biopsy. Eighteen patients underwent conjunctival biopsy. In the 6 patients with a negative conjunctival biopsy, 4 (67%) had a positive buccal biopsy. Buccal mucosal immunofluorescence studies may be positive in patients with OCP even in the absence of extraocular disease. Buccal mucosal biopsy may be considered as an alternative to or attempted before conjunctival biopsy for the diagnosis of OCP, particularly in patients in whom conjunctival biopsy may be difficult or imminently visually threatening.

Bipolar Patients and Bullous Pemphigoid after Risperidone Long-Acting Injectable: A Case Report and a Review of the Literature.

Neuropsychiatric disorders are found to be associated with bullous pemphigoid (BP), an autoimmune subepidermal blistering disease. Antipsychotics have emerged as possible inducing factors of BP. However, large sample studies concerning BP associated with antipsychotics, as well as with specific mental disorders, are still lacking. Our review retrieved a few clinical studies and case reports on the topic, producing controversial results. We report for the first time a bipolar patient case presenting BP following five-month therapy with risperidone long-acting injectable (LAI). We hypothesize that the dermatological event is associated with the medication administered. The issue emerged during psychiatric consultation and was confirmed by histological examination, direct and indirect immunofluorescence studies, plus positive plasma and cutaneous BP180 and BP230 IgG. Neurodegeneration or neuroinflammation might represent a primary process leading to a cross-reactive immune response between neural and cutaneous antigens and contributing to self-tolerance failure. Furthermore, the time sequence of the shared biological mechanisms leading to clinical manifestations of the neuropsychiatric disorder and BP remains undefined. BP comorbid with bipolar disorder might occasionally represent a serious health risk and affect patients’ physical and psychosocial quality of life. Thus, clinicians treating psychiatric patients should consider BP as a possible adverse effect of psychotropic medications.

Inhibitions of human parainfluenza virus type 2 replication by ribavirin and mycophenolate mofetil are restored by guanosine and S-(4-nitrobenzyl)-6-thioinosine.

The antiviral activities of a nucleoside analog antiviral drug (ribavirin) and a non-nucleoside drug (mycophenolate mofetil) against human parainfluenza virus type 2 (hPIV-2) were investigated, and the restoration of the inhibition by guanosine and S-(4-nitrobenzyl)-6-thioinosine (NBTI: equilibrative nucleoside transporter 1 inhibitor) were also investigated. Ribavirin (RBV) and mycophenolate mofetil (MMF) inhibited cell fusion induced by hPIV-2. Both RBV and MMF considerably reduced the number of viruses released from the cells. Virus genome synthesis was inhibited by RBV and MMF as determined by polymerase chain reaction (PCR) and real time PCR. mRNA syntheses were also reduced. An indirect immunofluorescence study showed that RBV and MMF largely inhibited viral protein syntheses. Using a recombinant green fluorescence protein (GFP)-expressing hPIV-2 without matrix protein (rhPIV-2ΔMGFP), it was found that virus entry into the cells and multinucleated giant cell formation were almost completely blocked by RBV and MMF. RBV and MMF did not disrupt actin microfilaments or microtubules. Both guanosine and NBTI completely or partially reversed the inhibition by RBV and MMF in the viral replication, syntheses of genome RNA, mRNA and protein, and multinucleated giant cell formation. NBTI caused a little damage in actin microfilaments, but had no effect on microtubules. Both RBV and MMF inhibited the replication of hPIV-2, mainly by inhibiting viral genome RNA, mRNA and protein syntheses. The inhibition was almost completely recovered by guanosine. These results indicate that the major mechanism of the inhibition is the depletion of intracellular GTP pools.

A Monoclonal Antibody to M-Phase Phosphoprotein 1/Kinesin-Like Protein KIF20B.

Kinesin-like protein KIF20B, originally named M-phase phosphoprotein 1 (MPP1), is a plus-end-directed kinesin-related protein that exhibits in vitro microtubule-binding and -bundling properties as well as microtubule-stimulated ATPase activity. It has been characterized as a slow molecular motor that moves toward the plus-end of microtubules. Human autoantibodies directed against KIF20B have been described in up to 25% of patients with idiopathic ataxia and less commonly in other neuropathies and autoinflammatory conditions. One of the limitations of research into the structure and function of KIF20B has been a reliable monoclonal antibody that can be used in a variety of applications. To establish a reference standard for anti-KIF20B immunoassays and facilitate studies on the role of KIF20B in developmental cell biology, we developed an IgG1 monoclonal antibody, 10C7, which reacts with the cognate KIF20B protein in Western immunoblots and in addressable laser bead immunoassays. In HEp2 cells, leptomeningeal pericytes, and transfected HEK293T cells, indirect immunofluorescence studies showed that reactivity was mainly localized to a proportion of interphase nuclei, but during metaphase, it was redistributed throughout the cytoplasm and perichromatin mass. Later in telophase/anaphase, KIF20B was localized to the stem body and midzone of the midbody. 10C7 also showed remarkable staining of a subset of cells in the cerebellum, ovary, and testis tissues. KIF20B was shown to have extensive coiled-coil domains. The monoclonal antibody, 10C7, will be of value to diagnostic laboratory scientists interested in having a reliable reference standard for anti-KIF20B immunoassays as well as cell, molecular, and developmental biology researchers.

Proteomic Analysis of Histones H2A/H2B and Variant Hv1 in Tetrahymena thermophila Reveals an Ancient Network of Chaperones.

Epigenetic information, which can be passed on independently of the DNA sequence, is stored in part in the form of histone posttranslational modifications and specific histone variants. Although complexes necessary for deposition have been identified for canonical and variant histones, information regarding the chromatin assembly pathways outside of the Opisthokonts remains limited. Tetrahymena thermophila, a ciliated protozoan, is particularly suitable to study and unravel the chromatin regulatory layers due to its unique physical separation of chromatin states in the form of two distinct nuclei present within the same cell. Using a functional proteomics pipeline, we carried out affinity purification followed by mass spectrometry of endogenously tagged T. thermophila histones H2A, H2B and variant Hv1.We identified a set of interacting proteins shared among the three analyzed histones that includes the FACT-complex, as well as H2A- or Hv1-specific chaperones. We find that putative subunits of T. thermophila versions of SWR- and INO80-complexes, as well as transcription-related histone chaperone Spt6Tt specifically copurify with Hv1. We also identified importin β6 and the T. thermophila ortholog of nucleoplasmin 1 (cNpl1Tt) as H2A–H2B interacting partners. Our results further implicate Poly [ADP-ribose] polymerases in histone metabolism. Molecular evolutionary analysis, reciprocal affinity purification coupled to mass spectrometry experiments, and indirect immunofluorescence studies using endogenously tagged Spt16Tt (FACT-complex subunit), cNpl1Tt, and PARP6Tt underscore the validity of our approach and offer mechanistic insights. Our results reveal a highly conserved regulatory network for H2A (Hv1)–H2B concerning their nuclear import and assembly into chromatin.

Immunohistochemistry for Immunoglobulin G4 in the Diagnosis of Pemphigus.

Introduction:Pemphigus comprises of a group of autoimmune bullous disorders with intraepithelial lesions involving the skin and mucous membranes. Pemphigus is characterized histologically by an intraepidermal blister and immunopathologically by the finding of in vivo bound immunoglobulin G (IgG) antibodies against desmosomal adhesion proteins on the surface of keratinocytes. Indirect immunofluorescence for IgG is considered as a gold standard method for diagnosis of this group of bullous disorders on the condition that fresh frozen tissue is accessible.Aim:We designed a new diagnostic method by immunohistochemistry (IHC) for IgG4 on paraffin sections instead of fresh frozen tissue and evaluated sensitivity, specificity, and positive and negative predictive values of this method.Materials and Methods:We searched our pathologic archive of pemphigus of 35 patients, including 29 cases of pemphigus vulgaris (PV) and 6 patients with pemphigus foliaceus (PF). In all cases, the diagnosis was confirmed by indirect immunofluorescence studies. Thirty-five specimens served as controls, including 31 specimens of safe margins of basal cell carcinoma and 4 specimens of normal skin. Sections with condensed and continuous immunoreactivity localized to the intercellular junctions of keratinocytes were considered as positive.Results:Sensitivity of IgG4 was estimated to be 72.4% in PV group and 83.3% in PF group. The overall sensitivity and specificity of IgG4 IHC for diagnosis of pemphigus were 74.2% and 82.8%, respectively, in PV and PF groups. Furthermore, positive predictive value and negative predictive value were 81.2% and 76.3%, respectively.Conclusion:Immunohistochemical labeling for IgG4 on paraffin-embedded tissue provides a sensitive and specific test for diagnosing pemphigus in a situation when fresh frozen tissue is unavailable.

Detection of IgE autoantibodies in mucous membrane pemphigoid and their association with disease severity.

Mucous membrane pemphigoid (MMP) is an autoimmune disease characterized by scarring lesions at mucosal sites. Although the pathogenic role of specific IgG and/or IgA has been already demonstrated and the detection of these immunoglobulins is a criterion in the diagnosis of MMP, little is known about IgE role in this disease. Therefore, the main purpose of this study was to assess the presence of circulating and tissue bound IgE in patients with MMP and their possible correlations with clinical presentation and disease course. We conducted a retrospective study on 29 patients affected by MMP, recruited from a single center. Direct and indirect immunofluorescence studies were assessed to analyze the presence of specific IgE directed against the basal membrane zone. For each patient, fluorescence data were compared to clinical features. Linear deposits of C3, IgG and IgA were present in 86.2%, 62% and 37.9% of cases respectively, while IgE linear deposits were detected in 17 out of 29 patients (58.6%) including one case with isolated IgE positivity. Circulating IgE and IgA anti-BMZ were present in 7 (24.1%) and 5 (17.2%) patients, respectively. Both the presence of circulating IgA and of tissue bound IgE deposits correlated with disease activity index (P<0.014). Our results demonstrated the presence of IgE autoantibodies in MMP, particularly in more severe cases. Thus, IgE detection may represent an additional useful diagnostic tool in this disease.

Clinical and Immunological Study of 30 Cases With Both IgG and IgA Anti-Keratinocyte Cell Surface Autoantibodies Toward the Definition of Intercellular IgG/IgA Dermatosis.

Several sporadic cases, in which direct and indirect immunofluorescence studies simultaneously detected IgG and IgA autoantibodies to keratinocyte cell surfaces, have been reported mainly under the name of IgG/IgA pemphigus. However, there have been no systematic studies for this condition. In this study, we collected 30 cases of this condition from our cohort of more than 5,000 autoimmune bullous disease cases, which were consulted for our diagnostic methods from other institutes, and summarized their clinical and immunological findings. Clinically, there was no male–female prevalence, mean age of disease onset was 55.6 years, and mean duration before this condition was suspected was 18 months. The patients showed clinically bullous and pustular skin lesions preferentially on the trunk and extremities, and histopathologically intraepidermal pustules and blisters with infiltration of neutrophils and eosinophils. Immunologically, ELISAs frequently detected IgG and IgA autoantibodies to both desmogleins and desmocollins. From the characteristic clinical, histopathological, and immunological features, which are considerably different from those in classical IgG types of pemphigus, we propose this disease as a new disease entity with preferential name of intercellular IgG/IgA dermatosis (IGAD). This was the largest study of IGAD to date.

Identification of Glucose-Regulated Protein 78 (GRP78) as a Receptor in BHK-21 Cells for Duck Tembusu Virus Infection.

Since 2010, outbreak and spread of tembusu virus (TMUV) caused huge losses to the breeding industry of waterfowl in several provinces of China. In this study, we identify the glucose-regulated protein 78 (GRP78) as a receptor in BHK-21 cells for duck TMUV infection. Using cell membrane from BHK-21 cells, a TMUV-binding protein of approximately 70 kDa was observed by viral overlay protein binding assay (VOPBA). LC-MS/MS analysis and co-immunoprecipitation identified GRP78 as a protein interacting with TMUV. Antibody against GRP78 inhibited the binding of TMUV to the cell surface of BHK-21 cells. Indirect immunofluorescence studies showed the colocalization of GRP78 with TMUV in virus-infected BHK-21 cells. We found that GRP78 over-expression increased TMUV infection, whereas GRP78 knockdown by using a specific small interfering RNA inhibited TMUV infection in BHK-21 cells. Taken together, our results indicate that GRP78 is a novel host factor involved in TMUV entry.

Regulation of CNKSR2 protein stability by the HECT E3 ubiquitin ligase Smurf2, and its role in breast cancer progression

BackgroundSmurf2 E3 ubiquitin ligase physically associates with and regulate the stability of distinct cellular protein substrates. The multi-functional scaffold protein Connector enhancer of kinase suppressor of ras 2 (CNKSR2) plays a key role in regulating cell proliferation, and differentiation through multiple receptor tyrosine kinase pathways. The aim of this study was to investigate whether the interaction between Smurf2 and CNKSR2 has any significant role in the post transcriptional regulation of CNKSR2 expression in breast cancer. Methods: Here we demonstrate a novel interaction of CNKSR2 with Smurf2 by co-immunoprecipitation, indirect immunofluorescence studies, and surface plasmon resonance (SPR) analysis, which can ubiquitinate, but stabilize CNKSR2 by protecting it from proteasome mediated degradation.ResultsCNKSR2 protein levels were significantly increased upon forced overexpression of Smurf2, indicating the role of Smurf2 in regulating the stability of CNKSR2. Conversely, Smurf2 knockdown resulted in a marked decrease in the protein level expression of CNKSR2 by facilitating enhanced polyubiquitination and proteasomal degradation and reduced the proliferation and clonogenic survival of MDA-MB-231 breast cancer cell lines. Tissue microarray data from 84 patients with various stages of mammary carcinoma, including (in order of increasing malignant potential) normal, usual hyperplasia, fibrocystic changes, fibroadenoma, carcinoma-in-situ, and invasive ductal carcinoma showed a statistically significant association between Smurf2 and CNKSR2 expression, which is also well correlated with the ER, PR, and HER2 status of the tissue samples. A comparatively high expression of Smurf2 and CNKSR2 was observed when the expression of ER and PR was low, and HER2 was high. Consistently, both Smurf2 and CNKSR2 showed an integrated expression in MCF10 breast progression model cell lines.ConclusionsAltogether, our findings reveal that Smurf2 is a novel positive regulator of CNKSR2 and suggest that Smurf2-CNKSR2 interaction may serve as a common strategy to control proliferation of human breast cancer cells by modulating CNKSR2 protein stability.

Anti-neutrophil cytoplasmic antibodies and their clinical significance

Anti-neutrophil cytoplasmic antibodies (ANCA) are a group of autoantibodies that cause systemic vascular inflammation by binding to target antigens of neutrophils. These autoantibodies can be found in serum from patients with systemic small-vessel vasculitis and they are considered as a biomarker for ANCA-associated vasculitis (AAV). A conventional screening test to detect ANCA in the serum is indirect immunofluorescence study, and subsequently confirmed by enzyme-linked immunosorbent assay. A positive staining of ANCA can be classified into three main categories based on the staining patterns: cytoplasmic, perinuclear, and atypical. Patients with granulomatosis with polyangiitis (GPA) mostly have a positive cytoplasmic staining pattern (c-ANCA) whilst a perinuclear pattern (p-ANCA) is more common in microscopic polyangiitis (MPA) and eosinophilic granulomatosis with polyangiitis (EGPA) patients. Atypical pattern (a-ANCA) is rarely seen in patients with systemic small-vessel vasculitis but it can be found in other conditions. Here, techniques for ANCA detection, ANCA staining patterns and their clinical significances are reviewed.

An Immunofluorescence Study About Staining Pattern Variability of Sarcoglycans in Rat’s Cerebral and Cerebellar Cortex

Title: An Immunofluorescence Study About Staining Pattern Variability of Sarcoglycans in Rat’s Cerebral and Cerebellar Cortex. Background: Sarcoglycans are transmembrane glycoproteins which connect extracellular matrix components to cytoskeleton. This protein system has been long studied in muscle but there are few data about its localization in non-muscular tissues. Methods and Findings: In the present report, we have conducted an indirect immunofluorescence study on normal rat’s cerebral and cerebellar cortex. Our results show that in these districts each sarcoglycan is expressed by a “spot-like” staining pattern, with spots of 0.5-2 μm average diameter, extending mainly around the soma of neurons and glial cells. In cerebral cortex, although all sarcoglycans are present, a staining pattern variability for each sarcoglycan, in the different cerebral cortex areas, exists. Instead, the pattern distribution level of sarcoglycans in cerebellar cortex doesn’t change. We also performed a statistical analysis which confirms the immunofluorescence results. Conclusions: Then, the presence of a sarcoglycans variability in cerebral cortex, where it is known the existence of several synaptic network, and the absence of a sarcoglycans variability in cerebellar cortex, where the same synaptic networks are repeated unchanged, suggest that in brain sarcoglycans may be associated with synaptic networks. Moreover, the distribution of sarcoglycans, mainly in post-synaptic regions of the neurons, suggests a role of these proteins in cellular signalling, regulating membrane receptors assembly. We also support that sarcoglycans in glial cells could be associated with the regulation of the mechanism in the brain-blood-barrier.

Induction of Sirt1 and PGC1α Signalling Pathway in Cardiac Mitochondrial Biogenesis by Aspirin

Title: An Immunofluorescence Study About Staining Pattern Variability of Sarcoglycans in Rat’s Cerebral and Cerebellar Cortex. Background: Sarcoglycans are transmembrane glycoproteins which connect extracellular matrix components to cytoskeleton. This protein system has been long studied in muscle but there are few data about its localization in non-muscular tissues. Methods and Findings: In the present report, we have conducted an indirect immunofluorescence study on normal rat’s cerebral and cerebellar cortex. Our results show that in these districts each sarcoglycan is expressed by a “spot-like” staining pattern, with spots of 0.5-2 μm average diameter, extending mainly around the soma of neurons and glial cells. In cerebral cortex, although all sarcoglycans are present, a staining pattern variability for each sarcoglycan, in the different cerebral cortex areas, exists. Instead, the pattern distribution level of sarcoglycans in cerebellar cortex doesn’t change. We also performed a statistical analysis which confirms the immunofluorescence results. Conclusions: Then, the presence of a sarcoglycans variability in cerebral cortex, where it is known the existence of several synaptic network, and the absence of a sarcoglycans variability in cerebellar cortex, where the same synaptic networks are repeated unchanged, suggest that in brain sarcoglycans may be associated with synaptic networks. Moreover, the distribution of sarcoglycans, mainly in post-synaptic regions of the neurons, suggests a role of these proteins in cellular signalling, regulating membrane receptors assembly. We also support that sarcoglycans in glial cells could be associated with the regulation of the mechanism in the brain-blood-barrier.

Cardiac rhythm and pacemaking abnormalities in patients affected by endemic pemphigus in Colombia may be the result of deposition of autoantibodies, complement, fibrinogen, and other molecules

We previously showed that one-third of patients affected by endemic pemphigus foliaceus in El Bagre, Colombia (El Bagre-EPF), display autoreactivity to the heart. The purpose of this study was to investigate rhythm disturbances with the presence of autoantibodies and correlate them with ECG changes in these patients. We performed a study comparing 30 patients and 30 controls from the endemic area, matched by demographics, including age, sex, weight, work activities, and comorbidities. ECG as well as direct and indirect immunofluorescence, immunohistochemistry, and confocal microscopic studies focusing on cardiac node abnormalities were performed. Autopsies of 7 patients also were reviewed. The main ECG abnormalities seen in the El Bagre-EPF patients were sinus bradycardia (in one-half), followed by left bundle branch block, left posterior fascicular block, and left anterior fascicular block compared with the controls. One-third of the patients displayed polyclonal autoantibodies against the sinoatrial and/or AV nodes and the His bundle correlating with rhythm anomalies and delays in the cardiac conduction system (P <.01). The patient antibodies colocalized with commercial antibodies to desmoplakins I and II, p0071, armadillo repeat gene deleted in velo-cardio-facial syndrome (ARVCF), and myocardium-enriched zonula occludens-1-associated protein (MYZAP; Progen Biotechnik) (P <.01). One-third of the patients affected by El Bagre-EPF have rhythm abnormalities that slow the conduction of impulses in cardiac nodes and the cardiac conduction system. These abnormalities likely occur as a result of deposition of autoantibodies, complement, and other inflammatory molecules. We show for the first time that MYZAP is present in cardiac nodes.

Species-specific and differential expression of BSP-5 and other BSP variants in normozoospermic and asthenozoospermic buffalo (Bubalus bubalis) and cattle (Bos taurus) seminal plasma

Binder of sperm-5 (BSP-5) is one of the fertility-associated proteins of cattle seminal plasma. Binding of sperm to the oviductal epithelium is mediated by BSP group of proteins. However, it is not clear, whether this protein is also involved in sperm motility. In the present study, attempts were made to characterize BSP-5 protein in both normozoospermic (NS) and asthenozoospermic (AS) Murrah buffalo (n = 18; Bubalus bubalis), Holstein Friesian (n = 8, Bos taurus) and Jersey cattle (n = 8; Bos taurus) bull seminal plasma and also study its expression pattern in these species. 1-D Western blot demonstrated three major BSP-5 immunoreactive protein bands (24.2 kDa, 20.5 kDa, and 12.3 kDa) in buffalo seminal plasma. Of these, the intensities of 24.2 and 20.5 kDa protein bands reduced significantly (P ≤ 0.05) in seminal plasma of AS group compared to that of NS group. On the contrary, the expression of 12.3 kDa protein band did not vary significantly between the groups. In Holstein Friesian seminal plasma, at least six BSP-5 immunoreactive protein bands (25.1, 23.6, 19.5, 13.8, 13.1 and 12.3 kDa) could be detected. Of these, the intensities of 23.6, 13.8/13.1 and 12.3 kDa protein bands decreased (P = 0.058, 0.111, 0.053) in AS group bulls compared to NS bulls. Holstein Friesian bull seminal plasma demonstrated a BSP-5 immunoreactive duplex protein band of 13.8/13.1 kDa, which was not evident in buffalo seminal plasma. In 2-D Western blot, a train of five BSP-5 immunoreactive duplex protein spots (Mr 21.0–27.6 kDa, pI of ∼3.9–5.1) was detected. Mass spectrometry of one of the representative duplex spot confirmed that these were BSP-5 and BSP-3 proteins, respectively. Indirect immunofluorescence studies showed that BSP-5 is primarily localized to the mid-piece/mitochondrial region of buffalo spermatozoa. To conclude, the findings of the present study could establish the significance and association of BSP-5 proteins in sperm motility and how their level differ in semen from two different clinical groups of buffalo bull (NS vs. AS). Further, the study also demonstrated that the expression pattern of BSP-5 and other BSP variants in seminal plasma of bulls is species-specific.

358 Treg dysfunction leads to spontaneous production of autoantibodies to bullous pemphigoid antigens in mice

Bullous pemphigoid (BP) is the most common autoimmune blistering disease caused by autoantibodies to hemidesmosomal proteins; type XVII collagen (COL17 or BP180) and BP230. Regulatory T cells (Tregs) are crucial for peripheral immune tolerance and maintaining cutaneous immune homeostasis. However, it remains largely unknown whether failure of peripheral immune tolerance has an influence on the production of autoantibodies to the epidermal components. To address this, we investigated scurfy mice, which lack functional Tregs due to the mutation in Foxp3, a master transcriptional factor of Tregs. Utilizing direct and indirect immunofluorescence (IF) studies, we found that scurfy mice developed autoantibodies to the dermal-epidermal junction of the skin. The production of IgG autoantibodies, which had been class-switched from IgM, started within 12 days after birth. The autoantibodies reacted to the epidermal side of artificial blisters induced by 1M NaCl by indirect IF. From these findings, we focused on BP antigens. Immunoblotting using recombinant proteins of murine BP230 and COL17 demonstrated the presence of autoantibodies to those proteins in most scurfy sera. However, autoantibodies in scurfy sera have no reactivity to NC14A domain of murine COL17, corresponding to NC16A domain of human COL17, a domain responsible for subepidermal blister formation. This result may explain the fact that blister formation is not observed in scurfy mice. Furthermore, by using a CD4+ T cell-transferred model we revealed that CD4+ T cells mediate the production of those autoantibodies in scurfy mice. These results suggested that Tregs contribute to maintain immune tolerance to COL17 and BP230 at a steady state in mice.

Glycyrrhizin inhibits human parainfluenza virus type 2 replication by the inhibition of genome RNA, mRNA and protein syntheses.

The effect of glycyrrhizin on the replication of human parainfluenza virus type 2 (hPIV-2) was examined. Cell fusion induced by hPIV-2 was inhibited by glycyrrhizin, and glycyrrhizin reduced the number of viruses released from the cells. Glycyrrhizin did not change cell morphology at 1 day of culture, but caused some damage at 4 days, as determined by the effect on actin microfilaments. However, it affected the cell viability at 1 day: about 20% of the cells were not alive by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay at 1 day of culture. Real-time polymerase chain reaction (PCR) and PCR showed that virus genome synthesis was largely inhibited. mRNA synthesis was also inhibited by glycyrrhizin. Viral protein synthesis was largely inhibited as observed by an indirect immunofluorescence study. Multinucleated giant cell formation was studied using a recombinant green fluorescence protein (GFP)-expressing hPIV-2 without matrix protein (rhPIV-2ΔMGFP). A few single cells with fluorescence were observed, but the formation of giant cells was completely blocked. Taken together, it was shown that viral genome, mRNA and protein syntheses, including F and HN proteins, were inhibited by glycyrrhizin, and consequently multinucleated giant cell formation was not observed and the infectious virus was not detected in the culture medium.

Ocular Onset of Paraneoplastic Pemphigus Presenting as Hyperemic Conjunctivitis and Massive Bilateral Eyelid Ulceration: A Case Report and Literature Review

ABSTRACTIntroduction: Paraneoplastic clinical signs are characterized by a large and heterogeneous variety of manifestations due to several possible underlying neoplasms. Paraneoplastic pemphigus (PNP) is a particular paraneoplastic variety that usually primarily affects the dermic and/or oral mucosa and is characterized by a high rate of mortality (90%). Therefore, it is important to recognize its possible signs early. This report describes a case of ocular paraneoplastic pemphigus (PNP) presenting with recalcitrant eyelid ulceration and hyperemic conjunctivitis caused by an undiagnosed prostate cancer.Methods: A 77-year-old man was admitted to our department because of recalcitrant hyperemic conjunctivitis in both eyes, complicated with large ulceration of both upper eyelids in spite of topical therapy. After 3 weeks, oral mucositis and bullous dermatitis on the chest and arms developed.Results: Complete slit lamp ocular study, conjunctival swabs, routine hematologic tests, serum neoplasm markers, indirect immunofluorescence study, immunoblotting, and oral mucose biopsy with direct immunofluorescence were performed under the hypothesis of a paraneoplastic sign. Total body computed tomography scan and ultrasound-guided needle prostate biopsy completed the diagnostic process and confirmed the diagnosis of prostate PNP. Complete remission of ocular clinical signs was achieved by treatment of the prostate malignancy with systemic immunosuppressive therapy and chemotherapy.