Serration pattern analysis as a practical adjunct tool for categorization of subepidermal autoimmune blistering diseases

Background: Serration pattern analysis helps in the classification of subepidermal autoimmune blistering disorders; more precisely, it helps to differentiate epidermolysis bullosa acquisita from other subepidermal autoimmune blistering disorders. Most of the published reports of this tool have come from a single center. Objectives: The objectives of the study were to study the utility of serration pattern analysis in classifying subepidermal autoimmune blistering disorders. Methods: Seventy five cases of subepidermal autoimmune blistering disorders were enrolled in this prospective study. A three millimeter punch biopsy was taken from the perilesional skin or mucosa for direct immunofluorescence; indirect immunofluorescence was carried out using salt-split skin. Subclassification of subepidermal autoimmune blistering disorders was done based on direct immunofluorescence, indirect immunofluorescence on salt-split skin, indirect immunofluorescence using knockout skin and serration pattern analysis findings. Results: Indirect immunofluorescence was positive in 68 cases; 14 cases showed a dermal staining pattern while the rest showed either an epidermal or a combined pattern. All patients with epidermal or combined staining patterns showed “n” serrated pattern on direct immunofluorescence. Nine patients with dermal staining on indirect immunofluorescence also revealed an “n” serration pattern on direct immunofluorescence indicating the diagnosis of anti-p200 pemphigoid, and the rest showed a “u” serrated pattern. Three patients with negative indirect immunofluorescence showed “u” serration on direct immunofluorescence while the rest showed “n” serration. Limitations: ELISA and immunoblotting could not be performed due to resource constraints. Conclusion: Based on indirect immunofluorescence and serration pattern analysis, classification of the majority of patients with subepidermal autoimmune blistering disorders was possible in our study. Pattern recognition is a cost-effective tool and can be easily learnt. It is recommended to be practiced in all laboratories where facilities for advanced immunological diagnosis are unavailable.

Development of a Multi-Epitope Recombinant Protein for the Diagnosis of Human Visceral Leishmaniasis

Background: Iran is one of the endemic areas of Mediterranean Visceral Leishmaniasis, a disease caused by Leishmania infantum. In this work, we examined whether Proteína quimérica 10 (PQ10) recombinant protein is suitable for immunological diagnosis of human visceral leishmaniasis. Methods: The study was carried out in Tarbiat Modares University during 2016- 2018. The coding sequence of PQ10 recombinant protein was sub-cloned in pET28 expression vector and was commercially synthesized by GENERAY Biotechnology, China. Sequencing with proper primers was done, the expression, optimization of expression and protein purification were performed, and the purified recombinant protein was confirmed by western blot. The efficacy of PQ10 for serodiagnosis was evaluated with 50 positive and 50 negative serum samples, which confirmed by the direct agglutination test and collected from individuals living in the visceral leishmaniasis endemic areas of Iran. ELISA was performed with the PQ10 recombinant protein. Results: The 95% CI sensitivity of ELISA that was evaluated with sera from naturally infected individuals was 84%. The 95% CI specificity value of the ELISA determined with sera from healthy individuals (50 serum samples) and from individuals with other infectious diseases was 82%. The 95% CI positive predictive value (PPV) and negative predictive value (NPV) were exterminated 82.35% and 83.67%, respectively. Conclusion: We have used a recombinant synthetic protein to improve serodiagnosis of human visceral leishmaniasis. PQ10 could be useful for diagnosis of asymptomatic cases, as well as in the early phase of infections.

Immunological detection of human and camel cystic echinococcosis using different antigens of hydatid cyst fluid, protoscoleces, and germinal layers

Background and Aim: Cystic echinococcosis (CE)/hydatidosis is one of the most prevalent neglected zoonotic diseases. It is initially asymptomatic and does not produce any clinical signs until the cyst becomes enlarged, causing localized pressure on internal organs and tissues. Therefore, the detection of Echinococcus granulosus antibodies is highly essential. This study evaluated the antigens of hydatid cyst fluid, protoscoleces, and germinal layers for efficient immunological diagnosis of CE in humans and camels. Materials and Methods: Hydatid cyst fluid (FLc), protoscoleces (Psc), and the germinal layer (GLc) antigens were prepared from camel-lung hydatid cysts. In the same way, hydatid cyst fluid (FLh) and protoscoleces (Psh) antigens from human-liver cyst aspirate were produced. The comparative immunodiagnostic efficacy of the prepared antigens was verified using indirect enzyme-linked immunosorbent assay (ELISA), SDS-PAGE, and immunoblotting. Results: ELISA proves that FLc and GLc antigens were higher than FLh and Psh antigens. This shows that binding reactivity in naturally infected human sera, camel sera, and Psc is the most potent, exhibiting 100% sensitivity with 78.26% and 76.47% specificity in camel and human sera, respectively. The CE prevalence using diagnostic Psc was 54.79% and 61.32% in tested human and camel sera, respectively. The electrophoretic profiles of all shared antigens showed similarities at 52, 41, and 22 kDa. Immunoblotting demonstrated common immune-reactive bands in all antigen types at 52 and 41 kDa against positive human and camel sera. Conclusion: This immunological study introduces camel hydatid cyst Psc as a potent diagnostic antigen and new immune-reactive fractions of 52 and 41 kDa for diagnosing hydatidosis in humans and camels.

Immunologic Responses against SARS-CoV-2

Coronavirus disease 2019 (COVID-19) emerged in Wuhan, China, in December 2019 and quickly spread worldwide becoming a global health problem unprecedented. The infection is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that is characterized as a RNA virus with an envelope derived from host cell with glycoprotein spikes, appearing like a crown-like external structure under electron microscope. Due to the aggressive spread profile of SARS-CoV-2, the scientific community is under pressure to generate knowledge about the morphology of the virus and the immune response against SARS-CoV-2, in order to generate useful information for the development of vaccines and methods of immunological diagnosis. Previous knowledge about other coronaviruses, such as SARS-CoV-1 and MERS-CoV, were the pillars for understanding the immune response of SARS-CoV-2. Until now, we know that the anti-SARS-CoV-2 immune response in the host involves mechanisms related to innate immunity, activation of CD4+ and CD8+ T cells and production of antibodies (IgA, IgG and IgM) against the virus. In spite of being a new pathogen, the literature on SARS-CoV-2 has increased dramatically in the past few months, especially in the immunology field. Here, we review the literature on SARS-CoV-2 immunology, focusing on the innate and adaptative immune responses.

Development of a nanobody tagged with streptavidin-binding peptide and its application in a Luminex fluoroimmunoassay for alpha fetal protein in serum

A nanobody/streptavidin-binding peptide fusion protein was developed and proved to be a very promising immunological diagnosis reagent for disease-related biomarkers.