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

Background 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. Methods Sections and protein blots from ganglionic and aganglionic colon were immunolabeled with MPZ-specific antibodies. Results 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 Conclusions 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.

Development of a Novel Double Antibody Sandwich ELISA for Quantitative Detection of Porcine Deltacoronavirus Antigen

Porcine deltacoronavirus (PDCoV) can cause diarrhea and dehydration in newborn piglets. Here, we developed a double antibody sandwich quantitative enzyme-linked immunosorbent assay (DAS-ELISA) for detection of PDCoV by using a specific monoclonal antibody against the PDCoV N protein and an anti-PDCoV rabbit polyclonal antibody. Using DAS-ELISA, the detection limit of recombinant PDCoV N protein and virus titer were approximately 0.5 ng/mL and 103.0 TCID50/mL, respectively. A total of 59 intestinal and 205 fecal samples were screened for the presence of PDCoV by using DAS-ELISA and reverse transcriptase real-time PCR (RT-qPCR). The coincidence rate of the DAS-ELISA and RT-qPCR was 89.8%. DAS-ELISA had a sensitivity of 80.8% and specificity of 95.6%. More importantly, the DAS-ELISA could detect the antigen of PDCoV inactivated virus, and the viral antigen concentrations remained unchanged in the inactivated virus. These results suggest that DAS-ELISA could be used for antigen detection of clinical samples and inactivated vaccines. It is a novel method for detecting PDCoV infections and evaluating the PDCoV vaccine.

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.

AKR1C3 Expression in T and B Acute Lymphoblastic Leukemia/Lymphoma for Clinical Use as a Biomarker

Objectives: To investigate aldo–keto reductase 1C3 (AKR1C3) expression in T and B acute lymphoblastic leukemia/lymphoma (ALL) patients.Methods: Three commercial antibodies were evaluated for AKR1C3 immunohistochemistry (IHC) staining performance: Polyclonal Thermofisher scientific (Clone#PA523667), rabbit monoclonal Abcam [EPR16726] (ab209899) and Sigma/Millipore anti-AKR1C3 antibody, mouse monoclonal, clone NP6.G6.A6, purified from hybridoma cell culture. Initial optimization was performed on cell line controls: HCT116 (negative control); genetically modified cell line HCT116 with AKR1C3 overexpression; Nalm and TF1 cell lines. Twenty normal bone marrows from archival B and T-ALL patient samples were subsequently examined. AKR1C3 expression levels in these samples were evaluated by immunohistochemistry, Protein Wes and quantitative RT-PCR.Results: Sigma/Millipore Anti-AKR1C3 antibody (mouse monoclonal, clone NP6.G6.A6) showed higher specificity compared to rabbit polyclonal antibody by immunohistochemistry. H-score was used to quantify percent of nuclear immunoreactivity for AKR1C3 with varying disease involvement. T-ALL samples had a higher H-score (172-190) compared to B-ALL cases (H-score, 30-160). The AKR1C3 expression in peripheral blood by Protein Wes and RT-qPCR showed concordance in relapsed/refractory and/or minimal residual T-ALL cases. Conclusions: Sigma/Millipore Anti-AKR1C3 antibody and mouse monoclonal, clone NP6.G6.A6 can be used to aid in AKR1C expression of T-ALL and in cases of relapsed/refractory and/or minimal residual disease.

Identification of Brachyspira spp. in the cecum of broiler chickens using histology and in situ diagnostic assays

The genus Brachyspira corresponds to the group of bacteria formerly classified into the genus Serpulina and includes several commensal and pathogenic intestinal spirochetes that affect pigs, poultry, and other animal species, including humans. In birds, some pathogenic species of this genus causes a condition known as avian intestinal spirochetosis, which remains underdiagnosed, thereby causing serious economic losses. Brachyspira is a fastidious organism that necessitates the employment of fast and efficient identification techniques. The aim of this study was to identify Brachyspira spp. using histology, immunohistochemistry (IHC) and fluorescence in situ hybridization (FISH) in formalin-fixed paraffin embedded (FFPE) tissue samples from the cecum of commercial poultry. Samples were collected from 129 birds aged between 35 and 45 days from commercial broiler farms. For evaluation, routine histology processing (H&E) and the histochemical technique, periodic acid–Schiff (PAS) were done. Additionally, FFPE tissue samples were evaluated for FISH and IHC. The histological lesions were analyzed and graded after H&E staining, and the goblet cells were counted and compared using PAS staining with the positive and negative samples obtained through FISH and IHC. For FISH, probes labeled with Brachyspira spp., B. pilosicoli, B. hyodysenteriae, and B. intermedia were used, whereas rabbit polyclonal antibody specific for Brachyspira spp. was used for IHC. Of 129 samples, 82 were positive with IHC and 86 were positive with FISH. The samples positive for the genus Brachyspira in the FISH technique were tested for B. pilosicoli, B. hyodysenteriae, and B. intermedia in which 56 were positive for B. pilosicoli, 75 for B. hyodysenteriae and 80 for B. intermedia. There was an increase in goblet cells in the samples positive for FISH and IHC. The techniques used were effective and gave corresponding results, thus serving as a fast and efficient tool for diagnosis.

An SPRi Biosensor for Determination of the Ovarian Cancer Marker HE4 in Human Plasma

Human epididymis protein 4 (HE4) is an ovarian cancer marker. Various cut-off values of the marker in blood are recommended, depending on the method used for its determination. An alternative biosensor for HE4 determination in blood plasma has been developed. It consists of rabbit polyclonal antibody against HE4, covalently attached to a gold chip via cysteamine linker. The biosensor is used with the non-fluidic array SPRi technique. The linear range of the analytical signal response was found to be 2–120 pM, and the biosensor can be used for the determination of the HE4 marker in the plasma of both healthy subjects and ovarian cancer patients after suitable dilution with a PBS buffer. Precision (6–10%) and recovery (101.8–103.5%) were found to be acceptable, and the LOD was equal to 2 pM. The biosensor was validated by the parallel determination of a series of plasma samples from ovarian cancer patients using the Elecsys HE4 test and the developed biosensor, with a good agreement of the results (a Pearson coefficient of 0.989). An example of the diagnostic application of the developed biosensor is given—the influence of ovarian tumor resection on the level of HE4 in blood serum.

Development and Optimization of Rabbit Polyclonal Antibodies for Cu/ZnSOD detection in Rice (Oryza sativa L.)

Superoxide dismutase (SOD) activity is an important measure of plant stress tolerance used in cultivar improvement. At present, we are unaware of any widely available immunological reagents for the detection of SOD in Oryza sativa (common Asian rice) or other plants. In this study, we used insilico B-cell epitope prediction tools to generate peptides which were immunized into rabbits to yield polyclonal antibodies against Cu/Zn SODs. Immunoblotting demonstrated that the antibody specifically recognized both native and denatured Cu/Zn SODs in rice. In addition, this antibody can confirm the expression tendency of endogenous OsCu/Zn SODs under heat stress by immunoblotting, and has a positive reaction in tomato leaf extracts, as well as human Hela cells. Chloroplast content of Cu/Zn SODs in rice can be identified by ELISA indirect competition method using this antibody. These results suggest that this Cu/Zn SOD rabbit polyclonal antibody may be a useful tool for elucidating the biological functions of Cu/Zn SODs in plants.

A Novel KRAS Antibody Highlights a Regulation Mechanism of Post-Translational Modifications of KRAS during Tumorigenesis

KRAS is a powerful oncogene responsible for the development of many cancers. Despite the great progress in understanding its function during the last decade, the study of KRAS expression, subcellular localization, and post-translational modifications remains technically challenging. Accordingly, many facets of KRAS biology are still unknown. Antibodies could be an effective and easy-to-use tool for in vitro and in vivo research on KRAS. Here, we generated a novel rabbit polyclonal antibody that allows immunolabeling of cells and tissues overexpressing KRAS. Cell transfection experiments with expression vectors for the members of the RAS family revealed a preferential specificity of this antibody for KRAS. In addition, KRAS was sensitively detected in a mouse tissue electroporated with an expression vector. Interestingly, our antibody was able to detect endogenous forms of unprenylated (immature) and prenylated (mature) KRAS in mouse organs. We found that KRAS prenylation was increased ex vivo and in vivo in a model of KRASG12D-driven tumorigenesis, which was concomitant with an induction of expression of essential KRAS prenylation enzymes. Therefore, our tool helped us to put the light on new regulations of KRAS activation during cancer initiation. The use of this tool by the RAS community could contribute to discovering novel aspects of KRAS biology.

The correlation between IL-10 expression and histopathological type in nasopharynx carcinoma patients

Background: Nasopharyngeal carcinoma (NPC) is a malignancy originated from nasopharyngeal epithelial cells. NPC therapy response could be predicted from histopathological type, but some patients with the same histopathological type, showed a different therapy response. Interleukin (IL)-10 expression is expected to be able to predict a better response of therapy in NPC patients. Purpose: To find out the correlation between IL-10 expression and histopathological type in NPC patients. Method: An analytic observational study with cross sectional approach towards 33 samples taken from the Oncology Polyclinic of Outpatient Unit of Otorhinolaryngology Head and Neck Surgery Department, Dr. Soetomo General Hospital. Formalin-fixed paraffin-embedded biopsy specimens were obtained. The IL-10 expression was studied with immunohistochemistry using rabbit polyclonal antibody Anti IL-10. Assessment of the staining used Allred scale. The Fisher exact test was utilized to determine the correlation of IL-10 expression and histopathological type of NPC, with p value = 0.05. Result: The result of IL-10 expression in NPC patients with histopathological WHO type I NPC obtained 1 sample (8.3%) with strong positive expression and 2 samples (9.5%) with weak positive expression. In patients with histopathological WHO type II NPC obtained 2 samples (16.7%) with strong positive expression and 12 samples (57.1%) with weak positive expression. In patients with histopathological WHO type III NPC obtained 9 samples (75%) with strong expression and 7 samples (33.3%) with weak positive expression. Conclusion: There was moderate positive correlation between IL-10 expression and histopathological type in NPC patients.Keywords: nasopharyngeal carcinoma, IL-10 expression, histopathological type ABSTRAK Latar belakang: Karsinoma nasofaring (KNF) adalah suatu keganasan yang berasal dari epitel nasofaring. Respon terapi KNF selama ini dapat dinilai berdasarkan tipe histopatologi, namun pada kenyataannya penderita KNF dengan tipe histopatologi sama dapat menunjukkan respon terapi berbeda. Pemeriksaan ekspresi interleukin (IL)-10 diharapkan dapat memberikan prediksi lebih baik mengenai respon terapi pada penderita KNF. Tujuan: Mengetahui hubungan antara ekspresi IL-10 dengan tipe histopatologi pada penderita KNF. Metode: Penelitian observasional analitik dengan pendekatan cross sectional pada 33 sampel yang diperoleh dari Poliklinik Onkologi Unit Rawat Jalan, Departemen THTBedah Kepala Leher, RSUD Dr Soetomo. Ekspresi IL-10 diperiksa dari blok parafin dengan teknik pemulasan imunohistokimia menggunakan rabbit polyclonal antibody Anti IL-10. Ekspresi IL-10 dinilai dengan menggunakan skala Allred. Uji Fisher exact digunakan untuk menentukan korelasi antara ekspresi IL-10 dan tipe histopatologi KNF, dengan p = 0,05. Hasil: Ekspresi IL-10 pada KNF WHO tipe I didapatkan ekspresi positif kuat 1 penderita (8,3%) dan ekspresi positif lemah 2 penderita (9,5%). Hasil ekspresi IL-10 pada KNF WHO tipe II didapatkan ekspresi positif kuat 2 penderita (16,7%) dan ekspresi positif lemah 12 penderita (57,1%). Hasil ekspresi IL-10 pada KNF WHO tipe III 9 penderita (75%) dengan ekspresi positif kuat dan 7 penderita (33,3%) dengan ekspresi positif lemah. Kesimpulan: Didapatkan korelasi positif sedang antara ekspresi IL-10 dan tipe histopatologi pada penderita KNF