Rabbit Polyclonal Antibody Research Articles

Molecular characterization and antibacterial immunity functional analysis of the antimicrobial peptide hepcidin from Coregonus ussuriensis berg

Antimicrobial peptides are immune system molecules existing in different organisms including mollusks, crustaceans and vertebrates. Hepcidins are a group of cysteine rich antimicrobial peptides, which plays an important role in fish response to a variety of pathogens. In this study, we cloned and identified Hepcidin from the Coregonus ussuriensis Berg, and its functions in vivo and in vitro was investigated. Our results showed that, CuHepc contains a 267 bp coding sequence (CDS) region that encodes 88 putative amino acids with a molecular weight of 9.77 kD. Hepcidin transcripts were most abundant in the liver of healthy C. ussuriensis Berg. The synthesized Hepcidin peptide exhibited a wide range of antibacterial activity against Gram-positive and Gram-negative bacteria in vitro, and the results of in vivo bacterial attack assays showed that the CuHepc gene was differentially up-regulated in the six tissues investigated after infection with Aeromonas hydrophila. To analyze the changes in protein levels in C. ussuriensis, we generated Hepc polyclonal antibodies in rabbits and verified that the protein expression was increased after bacterial infection with Western blot assay. MIC assay results showed a geometric mean value of 5.513 μM for CuHepc peptide. In the in vivo experiment, immune-related genes IL-10, NF-κB, TLR3 were up-regulated post-infection CuHepc peptide in liver and intestine. Finally, CuHepc peptide reduced the tissues microbial load compared to infection with Aeromonas hydrophila. The above results indicate that Hepc plays a role in the immune response of C. ussuriensis to exogenous disturbances, indicate that CuHepc might act a candidate for modulation of the innate immune system in C. ussuriensis.

GroEL Chaperonin-Based Assay for Early Diagnosis of Scrub Typhus.

A point-of-care diagnostic for early and rapid diagnosis of scrub typhus caused by Orientia tsutsugamushi is required for prompt and proper treatment of patients presenting with undifferentiated febrile illnesses. In this study, an immunochromatographic antigen detection test kit (ICT AgTK) that targets the highly conserved O. tsutsugamushi 60 kDa GroEL chaperonin (heat shock protein 60) was developed. E. coli-derived recombinant GroEL expressed from DNA coding for the consensus sequence of 32 GroEL gene sequences extracted from the GenBank database was used to immunize rabbits and mice. Rabbit polyclonal antibodies (pAb) were used for preparing a gold-pAb conjugate, and the rGroEL-specific mouse monoclonal antibody was used as the antigen detection reagent at the ICT test line. In-house validation revealed that the ICT AgTK gave 85, 100 and 95% diagnostic sensitivity, specificity and accuracy, respectively, compared to the combined clinical features and standard IFA when tested on 40 frozen serum samples. The test kits correctly identified 10 scrub typhus samples out of 15 fresh plasma/buffy coat samples of patients with febrile illnesses. For independent laboratory validation, the ICT AgTK was sent to one provincial hospital. The ICT AgTK utilized by the hospital medical technologist correctly identified six scrub typhus samples out of 20 serum samples of patients with fever, as confirmed by specific IgM/IgG detection by IFA. The ICT AgTK is easy to perform with rapid turn-around time. It has the potential to be used as an important tool for on-site and early scrub typhus diagnosis by allowing testing of freshly collected samples (serum, plasma or buffy coat), especially in resource-limited healthcare settings.

Development and performance evaluation of an In-House ELISA for the detection of group A rotavirus in diarrheal stool samples from children and domestic South American camelids

Group A rotavirus (RVA) is a prevalent pathogen causing acute gastroenteritis (AGE) in young children and animals. We developed an in-house ELISA (ROTA-GeFeK) for RVA detection, based on the expression of native recombinant VP6 protein in E. coli. To detect the RVA antigen, rabbit polyclonal IgG antibodies, produced against rVP6,were used as capture and detector antibodies in a sandwich ELISA. To validate the ROTA-GeFeK, 252 stool samples from children with AGE, were evaluated by conventional RT-PCR and commercial ELISA. Compared to RT-PCR, the ROTAGeFeK had a sensitivity of 88.2 % and a specificity of 94.4 %. Total detection rates with the ROTA-GeFeK, commercial ELISA and RT-PCR were 58 %, 58 % and 64 % respectively. The limit of detection was equal to 2.1 × 10 4 CCID 50 of the RVA strain RIX4414. No cross-reactivity with other enteric pathogens was observed. The RVApositive samples detected by the assay belonged to a diversity of G and [P] genotypes.This assay displayed reactivity and was proved to be useful for the detection of RVA in diarrheal samples of domestic South American Camelids. We suggest that the ROTAGeFeK can be used as an epidemiologic tool for rotavirus surveillance and for RVA detection in other animal species.

Morphogenetic and pathogenetic features of hypertrophic and keloid scars of the head and neck

Evaluate the morphogenetic and pathogenetic features of hypertrophic and keloid scars of the head and neck. The study included 286 patients, among them 176 (61.5%) patients with hypertrophic and 110 (38.5%) with keloid scars aged 18 to 65 years with a disease duration from 1 month to 2 years. Material for histological and immunohistochemical (IHC) studies of scar tissue was fixed in 10% buffered formalin. Serial paraffin sections were stained with H&E, according to Van Gieson and Weigert. IHC was performed using monoclonal mouse antibodies to collagen type I (clone 3G3, Santa Cruz, dilution 1:100), collagen type III (clone B-4, Santa Cruz, dilution 1:50), collagen type IV (clone COL-94, Santa Cruz, dilution 1:50), MMP-1 (clone 3B6, Santa Cruz, dilution 1:100), α-SMA1 (clone 1A4, Dako Agilent, dilution 1:100) and rabbit polyclonal anti-TGFβ antibodies (clone 3C11, Santa Cruz, 1:100 dilution). Pathogenetic, morphological and immunohistochemical differences in hypertrophic and keloid scars were established depending on their degree of maturity. In the formation of hypertrophic scars, the key factor in sclerotic processes is TGF-b on the background of low MMP1 activity. Keloid scars were distinguished not only by the accumulation of hard-to-degrade collagens, but also by the development of an osteoclast-like reaction with a high content of MMP1. Immature scar tissue was characterized by the presence of myofibroblastic α-SMA1 positive focus and center of inflammatory changes. The data obtained allow substantiating new approaches to the treatment of patients with hypertrophic and keloid scars.

Diagnostically misleading aberrant terminal deoxynucleotidyl transferase expression in germ cell tumors.

Terminal deoxynucleotidyl transferase (TdT) is aunique type of DNA polymerase predominantly expressed in precursor lymphoid cells and acute lymphoblastic leukemia. It participates in the junctional diversity of T-cell receptors and immunoglobulins. Recently, aberrant TdT expression was found in seminomas. Here, we evaluated the expression of TdT in our cohort of germ cell tumors (GCTs) with two anti-TdT antibody clones. We included 173 cases of testicular GCTs, 5 ovarian dysgerminomas, and one gonadoblastoma in the study. Tissue microarrays containing representative tumor samples were constructed and subsequently stained with anti-TdT monoclonal rabbit antibody EP266 (Dako) and TdT rabbit polyclonal antibody (Cell Marque). Expression was assessed with the H-score. No specific nuclear reaction was observed for the polyclonal anti-TdT antibody. The H-score values varied between the histological subtypes for the EP266 antibody. Positive nuclear staining was consistently seen in germ cell neoplasia in situ , seminoma, dysgerminoma, and embryonal carcinoma. Pure tumors had higher TdT H-scores than the mixed ones. Teratomas, yolk sac tumors, and choriocarcinomas were almost uniformly negative. Our study confirms that aberrant expression of TdT by testicular and ovarian GCTs exemplifies apotential diagnostic pitfall in histopathological diagnostics.

Expression of Poly(Adenosine Diphosphate-Ribose) Polymerase Protein in Breast Cancer.

The use of poly(adenosine diphosphate-ribose) polymerase (PARP) inhibitors for breast cancer (BC) therapy is the subject of debate, and there is an urgent need to understand much the expression and prognostic role of the PARP1 protein. In this study, we have compared the expression of PARP between BC and benign breast disease (BBD) patients and also analyzed the association of PARP expression with clinicopathological parameters in BC. The study consists of 30 patients with newly diagnosed operable BC who were planned for surgery without neoadjuvant chemotherapy and 15 patients of BBD as a control between 2019 and 2021. Immunohistochemical analyses were performed prospectively on tissue samples. Anti-human PARP1 rabbit polyclonal antibody gives strong nuclear positivity. Internal control was the adipose tissue and the BBD acted as the external control. PARP1 expression was evaluated using the multiplicative quickscore method. The mean age for BC patients was 51.30 ± 10.694 years (range: 25-75 years) while BBD was below 30 years. Overexpression of PARP was present in 25 (83.3%) and weak expression in 5 (16.7%) of BC patients compared to BBD, only 2 (13.3%) patients demonstrated an overexpression of PARP, and 13 (86.6%) patients showed weak expression which showed significant association (P < 0.001). In BC, nuclear PARP (nPARP) overexpression was seen in 22 (73.3%) patients and weak expression of nPARP in 8 (26.7%), whereas 5 (16.7%) patients showed cytoplasmic overexpression. On comparing expression of PARP with clinicopathological parameters, PARP overexpression was significantly associated with older population (age >50 years) (P = 0.002), postmenopausal women (P = 0.029), higher TNM stage (Stage II and III) (P = 0.014), higher histological grade (grade 2) (P = 0.043), and presence of lymphovascular invasion (P = 0.015). Enhanced PARP1 expression is closely correlated with positive estrogen receptor status (P = 0.001) and PR status (P = 0.001). Overall PARP and nPARP overexpression was significantly associated with ER- (P = 0.006 and P = 0.008) and PR-positive (P = 0.006 and P = 0.008) patients. The PARP and nPARP overexpression was significantly associated with nontriple-negative BC patients (P = 0.001 and P = 0.001). We have not come across any study in the literature to compare PARP expression in BC and BBD patients. On the basis of our observations, we concluded that PARP overexpression is a poor prognostic marker in BC.

An Endogenous miRNA-Initiated Hybridization Chain Reaction and Subsequent DNAzyme Activation for Cellular Theranostics

An Endogenous miRNA-Initiated Hybridization Chain Reaction and Subsequent DNAzyme Activation for Cellular Theranostics

Myelin Protein Zero Immunohistochemistry Is Not a Reliable Marker of Extrinsic Mucosal Innervation in Patients With Hirschsprung Disease.

Innervation of aganglionic rectum in Hirschsprung disease derives from extrinsic nerves which project from cell bodies located outside the bowel wall and markers that distinguish extrinsic from intrinsic innervation are diagnostically useful. Myelin protein zero (MPZ) is a putative marker of extrinsic glial cells which could distinguish mucosal innervation in aganglionic vs ganglionic colon. Sections and protein blots from ganglionic and aganglionic colon were immunolabeled with MPZ-specific antibodies. Immunolabeling of MPZ with a chicken polyclonal or mouse monoclonal antibody confirmed glial specificity and reliably labeled hypertrophic submucosal nerves in Hirschsprung disease. In contrast, a rabbit polyclonal antibody strongly labeled extrinsic and intrinsic nerves, including most mucosal branches. Immunoblots showed MPZ is expressed in mucosal glial cells, albeit at lower levels than in extrinsic nerves, and that the rabbit antibody is more sensitive that the other two probes. Unfortunately, none of these antibodies consistently distinguished mucosal innervation in aganglionic vs ganglionic rectum. The results suggest that (a) glial cell myelin protein zero expression is influenced more by location (mucosa vs submucosa) than the extrinsic vs intrinsic origin of the accompanied nerves and (b) myelin protein zero immunohistochemistry has limited value as a diagnostic adjunct for Hirschsprung disease.

Research Note: Development of a sandwich ELISA for determining plasma growth hormone concentrations in goose

Growth hormone (GH) is required for normal postnatal development in poultry; however, no immunoassay exists to assess its levels in geese plasma, hindering the study of endocrine regulation in this species. We developed a sandwich ELISA to determine the GH concentrations in the plasma of geese. Recombinant goose GH was produced using a eukaryotic expression system and purified for use as the reference standard in ELISA and the antigen for producing the polyclonal antibodies in rabbits. Rabbit anti-goose GH polyclonal antibody was used to coat the wells of the ELISA plate, and its biotinylated form served as the detection antibody. An avidin-conjugated horseradish peroxidase was used to bind the detection antibody and catalyze the chromogenic reaction of 3,3,5,5-tetramethylbenzidine and H2O2. A sigmoidal curve was fitted to the optical density and the log of the standard GH concentration using the four-parameter logistic model. The sensitivity of the assay was less than 0.156 ng/mL. The intra- and interassay coefficients of variation were less than 9 and 13%, respectively. The response curve of the serially diluted plasma samples from geese exhibited a good parallel relationship with that observed for the reference standards. The assay effectively detected differences in GH concentrations in plasma samples from geese at various physiological stages; thus, it will be useful for future study of their growth and metabolism.

Microscopy suggests that glutathione S‐transferase is stored in large granules of myeloid cells in bone marrow and sparse granulocytes of the regenerating tail of lizard

AbstractMicroscopy suggests that glutathione S‐transferase is stored in large granules of myeloid cells in bone marrow and sparse granulocytes of the regenerating tail of lizard. Acta Zoologica (Stockolm). Tail regeneration in lizards occurs after an immune‐privileged blastema and is formed where various immunosuppressive cells and biomolecules keep inflammation low. Molecules that limit inflammation may include glutathione and glutathione S‐transferase (GST). These molecules reduce reactive oxidative species (ROS) and detoxify extraneous molecules. In the present study, the presence of GST has been analysed in the early phases of tail regeneration in the lizard Podarcis muralis. Using a rabbit polyclonal antibody directed against epitopes present in GST of P. muralis, a light and electron microscopy immunohistochemical and Western blotting study has been done. The study detected proteins of 25 and 32 kDa in pale‐medium electron‐dense and large granules present in promyelocytes and myelocytes of likely basophilic or eosinophilic fate that is present in the bone marrow of tail vertebrae. These cells are still proliferating and give rise to basophilic or eosinophilic granulocytes that also migrate into the regenerating tail. The enzyme stored in granules of promyelocytes and myelocytes is also localized in the cytoplasm of granulocytes present in the regenerating blastema‐cone, basophilic or/and eosinophilic. It is suggested that these granulocytes in the blastema reduce ROS and detoxify potentially inflammatory metabolites, contributing with other molecules to limit inflammation and favour regeneration.

A novel ELISA test to detect soy in highly processed foods

Soy allergens detection is challenging when they are in trace amounts in foods, especially when foods are subjected to heat treatment, after which the target allergen maybe modified. Two ELISA formats, sandwich and indirect competitive, were developed using rabbit polyclonal antibodies against heat-denatured glycinin as the target protein. Sandwich format showed less cross-reactions and better sensitivity than the competitive format. The performance of both formats was tested using three model foods incurred with soy proteins. Results showed that the recovery decreased with the increase of heat treatment intensity applied to foods. Sandwich format could detect amounts of added soy isolate of 10 μg/g in sausage (pasteurized), 100 μg/g in bread (baked) and 1000 μg/g in pâté (sterilized); whereas competitive format only detected 2500 μg/g in sausage and 10000 μg/g in bread, and no glycinin was detected in pâté samples. A prototype of the sandwich ELISA was in-house validated giving acceptable results of precision and robustness. Recovery of model foods spiked with heat-denatured glycinin after processing ranged between 88 % and 149 %. In a survey of retail foods, the sandwich ELISA could differentiate most of the commercial foods with soy or derivatives declared or not in the ingredient list and could detect soy in highly processed foods.

Construction of Genomic Library and Screening of Edwardsiella tarda Immunogenic Proteins for Their Protective Efficacy Against Edwardsiellosis.

Edwardsiella tarda is a severe aquaculture pathogen that can infect many hosts including humans, animals, and fish. Timely diagnosis and treatment are crucial for the control of edwardsiellosis in the aqua industry. By using rabbit polyclonal antibody, an expression gene library of virulent Edwardsiella tarda strain ED-BDU 1 isolated in south India was constructed and screened. The identified immune expressive proteins were characterized, and the corresponding coding sequences were cloned, expressed, and the purified recombinant proteins were used as antigens. The identified immunoreactive proteins namely HflC, HflK, and YhcI were studied for their immune protective potential in vivo by challenge experiments. The protective efficacy of HflC, HflK, and YhcI showed that the clearance of Edwardsiella from the host with ~ 60% survivability. Further, the immunoreactive proteins induce a strong immune response upon infection and elicit the significant production of IL-10, IFN-γ, Th1, and Th2 mediated mRNA expression and were therefore effective in vaccine production for edwardsiellosis.

Immunohistochemical Evaluation of Adaptor Protein FAM159B Expression in Normal and Neoplastic Human Tissues.

FAM159B is a so-called adaptor protein. These proteins are essential components in numerous cell signalling pathways. However, little is known regarding FAM159B expression in normal and neoplastic human tissues. The commercially available rabbit polyclonal anti-human FAM159B antibody HPA011778 was initially characterised for its specificity using Western blot analyses and immunocytochemistry and then applied to a large series of formalin-fixed, paraffin-embedded normal and neoplastic human tissue samples. Confirmation of FAM159B’s predicted size and antibody specificity was achieved in BON-1 cells, a neuroendocrine tumour cell line endogenously expressing FAM159B, using targeted siRNA. Immunocytochemical experiments additionally revealed cytoplasmic expression of the adaptor protein. Immunohistochemical staining detected FAM159B expression in neuronal and neuroendocrine tissues such as the cortex, the trigeminal ganglia, dorsal root and intestinal ganglia, the pancreatic islets and the neuroendocrine cells of the bronchopulmonary and gastrointestinal tract, but also in the syncytiotrophoblasts of the placenta. FAM159B was also expressed in many of the 28 tumour entities investigated, with high levels in medullary and anaplastic thyroid carcinomas, parathyroid adenomas, lung and ovarian carcinomas, lymphomas and neuroendocrine tumours of different origins. The antibody HPA011778 can act as a useful tool for basic research and identifying FAM159B expression in tissue samples.

Anti-HNA-3a Alloantibodies Can Cause Differential Endothelial Damage through a Direct Neutrophil Activation Pathway

INTRODUCTIONThe human neutrophil antigen (HNA) 3a/b is associated with a single amino acid substitution (R 154Q polymorphism) on the first extracellular loop of choline transporter-like protein 2 (CTL2). Antibodies against HNA-3a have been associated with severe and fatal transfusion-related acute lung injury (TRALI). Epitope mapping suggests the existence of two types of HNA-3a antibodies. Type I antibodies only require first extracellular loop of the CTL2 for expression of the antigen, type II antibodies require at least the first 3 extracellular loops of CTL2 in a native configuration (i.e. expressed on a cell membrane). This study aimed to evaluate the activity of type I and type II anti-HNA-3a antibodies in an in vitro TRALI model.STUDY DESIGN & METHODS: The granulocyte agglutination test (GAT) and granulocyte immunofluorescence test (GIFT) were used to confirm anti-HNA-3a activity in two donor sera (Q49 and Q50). Flow cytometry was used to confirm antibody binding to freshly isolated human or mouse neutrophils. A rabbit polyclonal antibody against an epitope in the third extracellular loop of CTL2's was coated onto the wells of an ELISA plate and neutrophil HNA-3a antigen was captured and sera tested. For the TRALI model, human lung microvascular endothelial cells (HLMVECs) were grown to confluence before being treated with lipopolysaccharide (LPS). Freshly isolated neutrophils from HNA-3aa homozygote donors were then added with 10% Q49 or Q50. Microscopic counting of Trypan blue staining was used to measure rate HLMVEC death. Significance was determined using a one-way ANOVA (p<0.05), followed by Dunnett's post-hoc test. RESULTS AND DISCUSSIONGAT and GIFT confirmed that both Q49 and Q50 contained an anti-HNA-3a antibody, and that neither contained antibodies against any other HNA or HLA molecules. In flow cytometry, both Q49 and Q50 bound to human neutrophils, but only Q49 bound to mouse neutrophils. In the ELISA, signal was detected only for Q49. These data provided evidence that Q49 was a type I antibody and Q50 was a type II antibody. Within the TRALI model, HLVMEC death was observed with both Q49 and Q50 only in the presence of LPS and neutrophils (Table 1). Previously reported HLMVEC permeability in the absence of neutrophils (Bayat et al. Arterioscler Thromb Vasc Biol. 2013) was not observed as in this study the outcome measured was HLMVEC death. In the present study, the effect for Q49 (type I) was significantly larger than for Q50 (type II; Table 1; p-value <0.0001). CONCLUSIONSIn this model, LPS and neutrophils were required for anti-HNA-3a mediated HLMVEC death thought to reflect events involved in the initiation of TRALI. The observation that type I antibodies elicited a stronger response provides a direction for further research. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Expression of the recombinant C-terminal of the S1 domain and N-terminal of the S2 domain of the spike protein of porcine epidemic diarrhea virus.

Background and Aim:Porcine epidemic diarrhea virus (PEDV) causes severe diarrhea in suckling piglets, leading to severe economic losses in the swine industry. Commercial vaccines have limited effectiveness against different genogroups of PEDV and the shedding of virus. The C-terminal of the S1 domain and the N-terminal of the S2 domain (S1-2) protein of the spike (S) protein have four neutralizing epitopes. However, research on the expression of the S1-2 segment of the S gene has been limited. In this study, we expressed a recombinant S1-2 protein of the S protein of the PEDV Thai isolate and characterized the immunological properties of the recombinant S1-2 protein.Materials and Methods:The S1-2 segment of the S gene of the PEDV Thai isolate (G2b) was amplified, cloned into the pBAD202/D-TOPO® vector (Invitrogen, Carlsbad, CA, USA), and expressed in Escherichia coli. The optimum concentration of arabinose and the optimum induction time for the expression of the recombinant S1-2 protein were determined using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The immunogenic reactivity of the recombinant S1-2 protein was determined using Western blot analysis with rabbit polyclonal antibodies against the SM98 strain of PEDV (G1a).Results:The recombinant S1-2 segment of the S gene of the PEDV Thai isolate protein was cloned and the recombinant S1-2 protein was successfully expressed. The optimum concentration of arabinose and the optimum induction time for the induction of the recombinant S1-2 protein were 0.2% and 8 h, respectively. The recombinant S1-2 protein reacted specifically with both rabbit anti-histidine polyclonal antibodies and rabbit anti-PEDV polyclonal antibodies.Conclusion:The recombinant S1-2 protein reacted with rabbit anti-PEDV polyclonal antibodies induced by the different PEDV genogroup. Therefore, the recombinant S1-2 protein may be a useful tool for the development of a diagnostic test for PEDV or for a vaccine against PEDV.

A case of TFE-3-positive non-neoplastic pseudodecidualized endometrium presenting as a cervical mass

Abstract Introduction/Objective TFE-3 gene encodes a transcription factor that promotes the expression of genes involved in cell growth and proliferation. Its overactivation can result in oncogenic activity. Although TFE-3 seems to be almost universally expressed in normal tissues, this expression should be at very low levels and strong nuclear expression of TFE-3 is seen almost exclusively in tumors containing or lacking the TFE-3 gene fusion. These include renal cell carcinoma, alveolar soft part sarcomas, epithelioid hemangioendotheliomas, PEComas, granular cell tumour, solid pseudopapillary neoplasm of the pancreas, and ovarian sclerosing stromal tumors. It must be emphasized that only nuclear expression of TFE-3 is of diagnostic value, as non-specific cytoplasmic staining is common. Methods/Case Report A 30-year-old woman with pelvic pain, heavy vaginal bleeding and ureteral stricture on oral contraceptive pill was found to have a cervical mass on exam. Cervical biopsy showed fragments of benign squamous epithelium and polypoid endometrial tissue with atrophic glandular component, stromal pseudodecidualization and abundant mixed inflammation. The stroma was positive for CD10, and negative for P16, desmin, cytokeratin ae1/ae3, CD34, calretinin. There was patchy moderate to strong nuclear staining for TFE-3 (Anti-TFE-3 rabbit monoclonal primary antibody, Cell MarqueTM). No evidence of a neoplastic process was seen, and the overall findings fit with either prolapsed endometrial tissue or endometriosis. TFE-3 by FISH showed no rearrangement of the TFE-3 gene region, ruling out alveolar soft part sarcoma. Results (if a Case Study enter NA) NA Conclusion The Human Protein Atlas, a program mapping all the human proteins in cells and tissues, shows that endometrial stromal and glandular cells can have moderate TFE-3 nuclear expression, using Anti-TFE-3 rabbit polyclonal antibody (Prestige Antibodies ®). In our case, focal strong expression was seen using a monoclonal antibody. In the pathology literature this finding has not been previously reported. Pathologists should be aware of the possibility of strong nuclear expression of TFE-3 in non-neoplastic endometrium to avoid potential misdiagnosis.

Altered Development of Mesencephalic Dopaminergic Neurons in SIDS: New Insights into Understanding Sudden Infant Death Pathogenesis.

Sudden infant death syndrome (SIDS) is defined as the unexpected sudden death of an infant under 1 year of age that remains unexplained after a thorough case investigation. The SIDS pathogenesis is still unknown; however, abnormalities in brain centers that control breathing and arousal from sleep, including dramatic changes in neurotransmitter levels, have been supposed in these deaths. This is the first study focusing on mesencephalic dopaminergic neurons, so far extensively studied only in animals and human neurological diseases, in SIDS. Dopaminergic structures in midbrain sections of a large series of sudden infant deaths (36 SIDS and 26 controls) were identified using polyclonal rabbit antibodies against tyrosine hydroxylase, the rate-limiting enzyme in catecholamine biosynthesis, and the dopamine transporter, a membrane protein specifically expressed in dopaminergic cells. Dopamine-immunolabeled neurons were observed concentrated in two specific structures: the pars compacta of the substantia nigra and in the subnucleus medialis of the periaqueductal gray matter. Anatomical and functional degenerations of dopaminergic neurons in these regions were observed in most SIDS cases but never in controls. These results indicate that dopamine depletion, which is already known to be linked especially to Parkinson’s disease, is strongly involved even in SIDS pathogenesis.

Rapid Detection of Orthopoxviruses

The aim of the study was to develop a sensitive and fast immunochemical test for the detection of orthopoxviruses (OPXV) in the “point of care” format.Materials and methods. The analyses were performed in cultured crude and purifed preparations of vaccinia virus, cowpoxvirus, rabbitpoxvirus and ectromelia virus, as well as in the blood and tissue suspensions of infected mice and rabbits. OPXV-antigen was detected by one-stage and two-stage protocols of dot-immunoassay based on flat protein arrays using rabbit polyclonal antibodies as capture and detection reagents.Results and discussion. The results show that the detection limit of OPXV is inversely related to the degree of their purifcation. The one-stage (rapid) protocol is specifc and allows detecting OPXV in crude culture samples of the virus and in clinical samples in the range of 104–103 PFU/ml within 39 minutes. Rapid dot-immunoassay can be applied to detect or exclude the presence of a viral threat in samples and can be useful in various aspects of biosafety provision. The simplicity of the one-stage protocol, the possibility to visually account the results and easy interpretation of the results allow the rapid test to be used in the “point of care” format.

A paper-based ELISA for rapid sensitive determination of anaphylaxis-related MRGPRX2 in human peripheral blood

Mas-related G-protein-coupled receptor X2 (MRGPRX2) has recently been reported to be associated with anaphylaxis. Detection of MRGPRX2 levels in human peripheral blood might serve as a powerful tool for predicting the predisposition of patients to anaphylactic reactions. For rapid measurement of MRGPRX2, we established a paper-based double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) using mouse monoclonal antibody and horseradish peroxidase (HRP)-labelled rabbit polyclonal antibody as capture antibody and detection antibody, respectively. We avoided chemical functionalization of the cellulose paper by introducing bovine serum albumin (BSA) to provide COOH and NH2 groups for covalent immobilization of the capture antibody. Through amide condensation, a two-layer immobilization strategy was applied with BSA-BSA and BSA-capture antibody networks as the first and second layers, respectively. This strategy improved the quantity, activity and stability of the immobilized antibody. We then established a paper-based ELISA to detect MRGPRX2 in human peripheral blood. Our method is less laborious, easier to implement, and more cost-effective than conventional ELISA, while offering similar sensitivity, specificity, and accuracy. Therefore, it could serve as an innovative clinical point-of-care diagnostic tool, especially in areas that lack advanced clinical equipment.

The role of interleukin-17 (IL-17) in the pathogenesis of discoid lupus erythematosus and lichen planopilaris: is immunohistochemistry for IL-17 a promising way to differentiate these entities?

Interleukin-17 (IL-17) is a pro-inflammatory mediator which its excess secretion by immune cells is found in many chronic autoimmune diseases. This survey is aimed to compare the amount and pattern of distribution of IL-17 positive cells in lichen planopilaris (LPP) and discoid lupus erythematosus (DLE) which are of the most frequent causes of primary cicatricial alopecia (CA). Biopsies of lesional scalp skin from adult LPP (n = 30), DLE patients (n = 19), and control scalp skin (n = 18) were analyzed by immunohistochemistry (IHC) method using rabbit polyclonal antibody against IL-17. Cells stained positive for IL-17 and their pattern of distribution were assessed and compared between these groups. The mean of IL-17 positive cell counts per high power field (HPF) in both LPP and DLE groups was significantly higher in comparison with the control group (both p < .0001). The LPP group had higher values of IL-17 positive cell counts per HPF (47.56 ± 13.37) compared to the DLE group (22.21 ± 11.06) (p < .0001). More than 30 IL-17 positive cell counts in IHC have a sensitivity of 90% and a specificity of 78% for differentiating LPP from DLE. Our findings suggest that IL-17 is effective in the pathogenesis of DLE and LPP and its role is more prominent in LPP. IL-17 positive cells in IHC can distinguish DLE from LPP with satisfactory sensitivity and specificity.