Indirect Immunoperoxidase Research Articles

Isolation and identification of Bovine coronavirus (BCoV) from diarrheatic calves in Dakahlia Governorate, Egypt

In the current study, a total of 107 fecal samples were collected from diarrhetic calves from 1 day to 3 weeks of age from different localities in Dakahlia governorate, Egypt (Gamasa, Belkas, Sherbin, Nabaroh, Talkha, Almanzala, Algamalia, and Aga) during the period from year 2015 to year 2018. These calves were suffering from watery diarrhea, dehydration, weakness and recumbency. All samples were taken from calves which were born from vaccinated cows. The fecal samples from 107 cases were screened for the presence of BCoV by using Ag-ELISA and RT-PCR. From ELISA and PCR positive identified samples, we visualized the Coronavirus particles when negatively stained by transmission electron microscopy. From ELISA and PCR positive identified samples, virus isolation was done via MDBK tissue culture, six passages were carried out followed by identification of BCoV in harvested tissue culture via DFA test and indirect immunoperoxidase technique. Furthermore, nucleotide sequence analysis of amplified N gene of the identified BCoV nucleic acid was done and compared with published reference sequences on GeneBank. The results of ELISA and RT-PCR revealed that: out of 107 tested fecal samples, 4 samples showed positive results (3.7%) and 103 samples showed negative results (96.2%). The viral particles showed pleomorphism with characteristic radial projections forming a corona, by transmission electron microscopy. All tested samples gave positive results with DFAT as yellowish green coloration was detected in stained MDBK cells which increased gradually with increased passages. All tested samples gave positive results with IP as brownish coloration was detected in stained MDBK cells which increased gradually with increased the period of incubation. further studies is required for better understanding the genetic diversity among BCoV circulating in Egyptian farms based on full genome sequencing.

Conventional and Molecular Detection of Avipoxviruses from Chickens, Pigeons and Turkeys

In the present study, a total of 90 cutaneous lesions samples were collected from chickens, pigeons, and turkeys farms in Dakahlia Governorate, Egypt during summer 2016. These farms suspected to be infected with Avipoxviruses (APVs).Thirty pooled samples were created (10 from chickens, 10 from pigeons and 10 from turkeys). Hyperimmune serum was prepared against standard fowlpox virus in adult white New Zealand rabbits. APV were identified in the collected samples using agar gel precipitation test (AGPT), indirect immunoperoxidase, and polymerase chain reaction (PCR) based on 4b gene of APVs. The results revealed that out of 30 tested samples there were 16 samples (53.3%) tested positive via AGPT including, 6 chicken samples (60%) , 5 pigeon samples (50%) and 5 turkey samples (50%). while using indirect immunoperoxidase, positive results were detected in 23 samples (76.7%) including, 8 chicken samples (80%), 8 pigeon samples (80%) and 7 turkey samples (70%).The 4b gene of APVs was detected using PCR in all tested samples (100%). In conclusion, Indirect immunoperoxidase is superior over AGPT in APVs detection in collected samples from chickens, pigeons and turkeys. PCR could efficiently used in molecular diagnosis of the virus.

Isolation and identification of Bovine coronavirus (BCoV) from diarrheatic calves in Dakahlia Governorate, Egypt

In the current study, a total of 107 fecal samples were collected from diarrhetic calves from 1 day to 3 weeks of age from different localities in Dakahlia governorate, Egypt (Gamasa, Belkas, Sherbin, Nabaroh, Talkha, Almanzala, Algamalia, and Aga) during the period from year 2015 to year 2018. These calves were suffering from watery diarrhea, dehydration, weakness and recumbency. All samples were taken from calves which were born from vaccinated cows. The fecal samples from 107 cases were screened for the presence of BCoV by using Ag-ELISA and RT-PCR. from ELISA and PCR positive identified samples, we visualized the Coronavirus particles when negatively stained by transmission electron microscopy. from ELISA and PCR positive identified samples, virus isolation was done via MDBK tissue culture, six passages were carried out followed by identification of BCoV in harvested tissue culture via DFA test and indirect immunoperoxidase technique. Furthermore, nucleotide sequence analysis of amplified N gene of the identified BCoV nucleic acid was done and compared with published reference sequences on GeneBank. The results of ELISA and RT-PCR revealed that: out of 107 tested fecal samples, 4 samples showed positive results (3.7%) and 103 samples showed negative results (96.2%). The viral particles showed pleomorphism with CHARacteristic radial projections forming a corona, by transmission electron microscopy. All tested samples gave positive results with DFAT as yellowish green coloration was detected in stained MDBK cells which increased gradually with increased passages. All tested samples gave positive results with IP as brownish coloration was detected in stained MDBK cells which increased gradually with increased the period of incubation. further studies is required for better understanding the genetic diversity among BCoV circulating in Egyptian farms based on full genome sequencing.

Immunohistochemistry and morphometric analysis of pelviureteric junction complexes in children with hydronephrosis.

The study aimed to analyze the musculature and innervations of pelviureteric junction (PUJ) complex and upper ureter by morphometry and immunohistochemistry in surgically resected specimens of PUJ obstruction and compares the findings with normal controls. Specimens of the PUJ were obtained from twenty patients with hydronephrosis due to PUJ obstruction undergoing surgical treatment in this unit from 2013 to 2015. In the control group, seven PUJ complexes were taken from patients who had nephrectomy due to trauma and Wilms tumor. The sections were stained with H and E stain and elastic von Gieson stain. For immunohistochemistry, the tissue sections were immunostained by an indirect immunoperoxidase method for S-100 and neuron-specific enolase. Histopathologically, the predominant arrangement of muscle fibers was layered, i.e., a thick circular layer surrounded by two longitudinal layers on either side in PUJ of obstruction patients. S-100 immunoreactivity in the muscle layer showed dense neural innervation in PUJ complex of obstructed patients. Circular enhanced musculature may cause a sphincter-like activity with holding up of urine. The higher density of neuronal expression in PUJ might be responsible for causing constant contraction and intrinsic obstruction.

435. Serological Cross-Reactivity Between Rickettsia japonica and Orientia tsutsugamushi, and Among Orientia tsutsugamushi Serotypes

BackgroundThe rickettsial diseases Japanese spotted fever (JSF) and scrub typhus (ST) are caused by Rickettsia japonica and Orientia tsutsugamushi, respectively. The diseases share clinical symptoms, such as fever, rash, and eschar. However, there are no systematical investigations of the serological cross-reactivity between R. japonica and O. tsutsugamushi. Also, it has still been unclear the serological cross-reactivity among O. tsutsugamushi serotypes.MethodsWe analyzed 1,406 cases tested by indirect immunoperoxidase assay (IP) using seven rickettsial antigens—one R. japonica and six O. tsutsugamushi serotypes (Kato, Karp, Gilliam, Irie/Kawasaki, Hirano/Kuroki, and Shimokoshi)—between 2003 and 2016 at two reference centers in Japan. Patient sera were 2-fold diluted from 1:40 to 1:40,960, and the titer was expressed as the reciprocal values. We defined the serology diagnosis as positive when a ≥4-fold increase in the IP IgM/IgG titer against R. japonica or O. tsutsugamushi was observed in the paired samples or if the IP IgM titer was ≥320.ResultsOf the 1,406 cases, 154 JSF and 138 ST cases were diagnosed by paired samples, and 13 JSF and 52 ST cases were diagnosed by a single sample. Figure 1a shows the serology results of 154 JSF cases—6 cases showed an IgG titer of ≥40 against O. tsutsugamushi without any significant elevation and four cases showed a non-significant IgM elevation of <320, which had none/few cross-reactions with other O. tsutsugamushi serotypes. Figure 1b shows the serology results of 138 ST cases, none showing any rise in the IgM/IgG titer against R. japonica except for one with an IgG titer of 80 only in the acute phase sample. We observed a massive degree of cross-reactivity between O. tsutsugamushi serotypes. When the correlation was further analyzed, cross-reactivity was significant among Karp, Hirano/Kuroki, and Kato types and between Gilliam and Irie/Kawasaki types in IgM. In contrast, the Shimokoshi type was less cross-reactive than the others (Figure 2).ConclusionThere is no serological cross-reaction or no recall reaction between R. japonica and O. tsutsugamushi. The cross-reactivity among O. tsutsugamushi vary. Disclosures All authors: No reported disclosures.

Keratinocytes derived from embryonic stem cells induce wound healing in mice

ABSTRACTThe skin plays an important role in defending the body against the environment. Treatments for burns and skin injuries that use autologous or allogenic skin grafts derived from adult or embryonic stem cells are promising. Embryonic stem cells are candidates for regenerative and reparative medicine. We investigated the utility of keratinocyte-like cells, which are differentiated from mouse embryonic stem cells, for wound healing using a mouse surgical wound model. Mice were allocated to the following groups: experimental, in which dressing and differentiated cells were applied after the surgical wound was created; control, in which only the surgical wound was created; sham, in which only the dressing was applied after the surgical wound was created; and untreated animal controls with healthy skin. Biopsies were taken from each group on days 3, 5 and 7 after cell transfer. Samples were fixed in formalin, then stained with Masson’s trichrome and primary antibodies to interleukin-8 (IL-8), fibroblast growth factor-2 (FGF-2), monocyte chemoattractant protein-1 (MCP-1), collagen-1 and epidermal growth factor (EGF) using the indirect immunoperoxidase technique for light microscopy. Wound healing was faster in the experimental group compared to the sham and control groups. The experimental group exhibited increased expression of IL-8, FGF-2 and MCP-1 during early stages of wound healing (inflammation) and collagen-1 and EGF expression during late stages of wound healing (proliferation and remodeling). Keratinocytes derived from embryonic stem cells improved wound healing and influenced the wound healing stages.

Comparative diagnosis of Mycobacterium avium subspecies paratuberculosis in the tissues of clinical and subclinical sheep of paratuberculosis endemic farm

Paratuberculosis (Johne’s disease) is the chronic infectious granulomatous enteritis of ruminants caused by Mycobacterium avium subspecies paratuberculosis. Due to the lack of definite diagnostic tests, the early detection of the disease is difficult. The present study was undertaken to assess comparative efficacy of diagnostic methods routinely used in the identification and confirmation of Mycobacterium avium subspecies paratuberculosis (MAP) in the tissue samples of naturally infected clinical and subclinical sheep. The ileum and mesenteric lymph node (MLN) tissues were collected from 20 paratuberculous sheep from organized farm of Rajasthan. These animals were further classified as paucibacillary (PB) (n = 8) or multibacillary (MB) (n = 12) on the basis of histopathological findings and mycobacterial loads. The ileum and MLN sections from PB and MB and uninfected control groups were tested by indirect immunoperoxidase technique (IPT), Ziehl Neelsen’s (ZN) method, bacterial culture, IS900 PCR and 251 gene PCR. In PB sheep, IPT and ZN were positive in 87.5 and 50% of intestinal sections and 75 and 37.5% of MLN sections, respectively. In MB sheep, IPT and ZN had equal sensitivity. The overall sensitivity of IPT was found to be superior (95%) than ZN method (80%) in demonstration of acid-fast bacteria in intestinal tissues. On the bacterial cultural examination of the intestinal and MLN tissues using Herrold’s egg yolk medium, MAP was isolated in eight (66.6%) MB and in four (50%) PB sheep. Among 20 sheep, IS900 PCR detected 13 (65.0%) and 251 gene PCR detected 16 (80%) sheep as positive for MAP. In the PB sheep, the culture, ZN test and PCR sensitivity was found low in comparison to MB sheep. The sensitivity of IPT in PB sheep was better than other tests in detecting the MAP infection, which underlines its utility in confirmation of subclinical and clinical cases of paratuberculosis in sheep. The sensitivity of 251 gene PCR assay was found better than IS900 gene PCR and has potential to be used as screening test for clinical and subclinical paratuberculosis in sheep flocks.

Periostin expression in neoplastic and non-neoplastic diseases of bone and joint

BackgroundPeriostin is a matricellular protein that is expressed in bone and joint tissues. To determine the expression of periostin in primary bone tumours and to assess whether it plays a role in tumour progression, we carried out immunohistochemistry and ELISA for periostin in a range of neoplastic and non-neoplastic bone and joint lesions.Methods140 formalin-fixed paraffin-embedded sections of bone tumours and tumour-like lesions were stained by an indirect immunoperoxidase technique with a polyclonal anti-periostin antibody. Periostin expression was also assessed in rheumatoid arthritis (RA) and non-inflammatory osteoarthritis (OA) synovium and synovial fluid immunohistochemistry and ELISA respectively.ResultsPeriostin was most strongly expressed in osteoid/woven bone of neoplastic and non-neoplastic bone-forming lesions, including osteoblastoma, osteosarcoma, fibrous dysplasia, osteofibrous dysplasia, fracture callus and myositis ossificans, and mineralised chondroid matrix/woven bone in chondroblastoma and clear cell chondrosarcoma. Reactive host bone at the edge of growing tumours, particularly in areas of increased vascularity and fibrosis, also stained strongly for periostin. Vascular elements in RA synovium strongly expressed periostin, and synovial fluid levels of periostin were higher in RA than OA.ConclusionsIn keeping with its known role in modulating the synthesis of collagen and other extracellular matrix proteins in bone, strong periostin expression was noted in benign and malignant lesions forming an osteoid or osteoid-like matrix. Periostin was also noted in other bone tumours and was found in areas of reactive bone and increased vascularity at the edge of growing tumours, consistent with its involvement in tissue remodelling and angiogenesis associated with tumour progression.

Congenital Zika Syndrome and Extra-Central Nervous System Detection of Zika Virus in a Pre-term Newborn in Mexico

BackgroundDuring pregnancy, the Zika virus (ZIKV) replicates in the placenta and central nervous system (CNS) of infected fetuses; nevertheless, the ability of ZIKV to replicate in other fetal tissues has not been extensively characterized.MethodsWe researched whether dissemination of congenitally-acquired ZIKV outside the CNS exists by searching for the accumulation of the viral envelope protein, ZIKV ribonucleic acid (RNA), and infectious viral particles in different organs of a deceased newborn with Congenital Zika Syndrome. A real-time qualitative polymerase chain reaction (qPCR) was used to detect ZIKV RNA in the brain, thymus, lungs, kidneys, adrenal glands, spleen, liver, and small intestine. The same tissues were analyzed by indirect immunofluorescence and immunoperoxidase assays using the monoclonal antibody 4G2 to detect ZIKV envelope antigens. Isolation of infectious ZIKV in a cell culture was carried out using brain and kidney samples.ResultsA postmortem, virological analysis of multiple organs, such as the kidneys (epithelial cells in the renal tubules), lungs (bronchial epithelia), thymus (epithelial cells inside the Hassall’s corpuscles), and brain (neurons, ependymal cells, and macrophages) revealed the presence of ZIKV RNA and envelope antigens. Other tissues of the deceased newborn tested positive by qPCR for Epstein-Barr virus and human herpesvirus 6, including the brain cortex (Epstein-Barr) and the thymus, kidneys, and adrenal glands (human herpesvirus 6). The kidneys were identified as a significant niche for viral replication, given that infectious particles were successfully isolated from renal tissues.ConclusionsOur findings demonstrate the ability of congenitally-acquired ZIKV to produce disseminated infections and the viral tropism towards epithelial cells.

Persistent BVD virus infections in offspring from imported heifers.

The aim of this study was to investigate the possible risk of bovine viral diarrhea virus transport from imported live animals. For this purpose, two different groups of animals were sampled in this study. Group 1 consisted of pregnant heifers; group 2 consisted of male beef cattle imported during 2011-2012 and 2015, respectively. Blood samples were tested for pestivirus antigen using a commercial BVDV antigen ELISA. All the pregnant heifers were negative, but 9 out of 412 offspring and 5 of the 332 male cattle were BVDV antigen positive. Virus isolation and also investigation by RT-PCR were carried out by using 14 ELISA-positive samples. At the end of three blind passages, eight non-cytopathogenic isolates were obtained by indirect immunoperoxidase monolayer assay, which were also RT-PCR positive using panpesti-virus primers. After discriminative RT-PCR, all the isolates that were identified as BVDV-1 and 5'UTR-based analysis demonstrated the existence of BVDV-1b (n = 4), BVDV-1f (n = 2), BVDV-1l (n = 1), and BVDV-1r (n = 1) subgenotypes. There was no BVDV subgroup that is newly introduced into the country. However, detection of persistent infection in calves born from imported animals demonstrates the risk of BVDV virus introduction by imported animals into the receiving country. Viral strains from persistently infected animals were characterized as BVDV-1b, which is predominant subgroup in the country where animals are imported. These results highlight a possible problem for the areas where a BVDV control program is currently ongoing. Additionally, sequences obtained in this study also showed that there are two distinct branches identified in BVDV-1l.

Serological Cross-Reactivity among Orientia tsutsugamushi Serotypes but Not with Rickettsia japonica in Japan.

The rickettsial diseases Japanese spotted fever (JSF) and scrub typhus (ST) are caused by Rickettsia japonica and Orientia tsutsugamushi, respectively. The diseases share clinical symptoms, such as fever, rash, and eschar. However, there are no systematical investigations of the serological cross-reactivity between R. japonica and O. tsutsugamushi. Also, the serological cross-reactivity among O. tsutsugamushi serotypes is still unclear. We analyzed 1406 cases tested by indirect immunoperoxidase assay using seven rickettsial antigens—one R. japonica and six O. tsutsugamushi serotypes—between 2003 and 2016 at two reference centers in Japan. Of these, 167 JSF and 190 ST cases were serologically diagnosed. None of the ST cases had a significant increase in IgM titers against R. japonica. Six JSF cases showed IgG titers of ≥40 against O. tsutsugamushi, but no IgG titer showed a significant elevation in the convalescent phase sample. We observed a substantial degree of cross-reactivity between O. tsutsugamushi serotypes. Cross-reactivity was significant among Karp, Hirano/Kuroki, and Kato types and between Gilliam and Irie/Kawasaki types in IgM, while the Shimokoshi type was less cross-reactive than the others. In conclusion, there is no serological cross-reaction between R. japonica and O. tsutsugamushi. The cross-reactivity among O. tsutsugamushi varies depending on serotypes.

Procaine and saline have similar effects on articular cartilage and synovium in rat knee

BackgroundIntra-articular local anaesthetics are widely used for providing postoperative analgesia and decreasing the need for opioids. Procaine has proven positive effects in carpal tunnel syndrome and chondromalacia patella. However, the effect of procaine on articular cartilage has not yet been studied. The aim of this study was to evaluate the effects of intra-articular procaine injection on the articular cartilage and the synovium.MethodsTwenty adult Sprague-Dawley rats were enrolled in the study. After providing anaesthesia and aseptic conditions, 0.25 ml of 10% procaine was injected to the right knee joint, and 0.25 ml of normal saline (as control group) was injected to the left knee joint. Knee joint samples were obtained from four rats in each group after appropriate euthanasia on days 1, 2, 7, 14 and 21. The histological sections of the articular and periarticular regions and the synovium were evaluated by two histologists, and inflammatory changes were graded according to a five-point scale in a blinded manner. The apoptosis of chondrocytes was determined by the caspase-3 indirect immunoperoxidase method.ResultsThere were no significant differences in inflammation between procaine and saline groups at any of the time intervals. Slight inflammatory infiltration due to injection was seen in both groups on the 1st day. Haemorrhage was observed in both groups at days 1 and 2, and the difference between groups was not found to be significant. No significant difference was detected in the percentage of apoptotic chondrocytes between groups at any of the time intervals.ConclusionsInjection of procaine seems safe to use intra-articularly based on this in vivo study on rat knee cartilage. However, further studies investigating both the analgesic and histopathological effects of procaine on damaged articular cartilage and synovium models are needed.

Keratin 17 is a Negative Prognostic Biomarker for Endometrial Carcinoma

BACKGROUNDKeratin 17 (K17) is a negative prognostic biomarker in cervical and ovarian cancers but has not previously been evaluated as a prognostic biomarker in endometrial adenocarcinoma. The association of K17 with decreased patient survival may be explained in part by the recent discovery that K17, unique among all keratins, drives tumor aggression by serving as a nuclear shuttle of p27, leading to cell cycle progression. Analysis of gene expression data from The Cancer Genome Atlas (TCGA) demonstrated that high K17 mRNA expression is prognostic for decreased overall survival in patients with endometrial carcinoma (n=228), prompting to test in the current study the hypothesis that immunohistochemical (IHC) staining for K17 correlates with survival of patients with endometrial cancer.METHODSCases of endometrial carcinoma were selected from Stony Brook Medicine Cancer Registry and UMass Memorial Cancer Registry. Histologic subtypes included endometrioid carcinoma (n=81), serous carcinoma (n=44), clear cell carcinoma (n=37), and malignant mixed Müllerian tumor (MMMT) (n=48). K17 was detected in sections of formalin‐fixed, paraffin‐embedded tissue blocks by indirect immunoperoxidase methods. Staining was scored based on the proportion of tumor cells that showed strong staining for K17 (PathSQ Score). Cases with “high‐K17” were defined as those with PathSQ score ≥10% and “low‐K17” cases were those with PathSQ &lt;10% of tumor cells. Survival analyses were determined using the Kaplan‐Meier method and Mantel‐Haenszel method.RESULTS AND DISCUSSIONHigh‐K17 was detected in 31/210 cases of endometrial carcinoma, including 18/81 endometrioid carcinomas, 5/37 clear cell carcinomas, 6/44 serous carcinomas, and 17/48 MMMTs. Staining was localized to the malignant epithelial component in each case and was not detected in benign stromal cells, myometrium, or sarcomatous elements of MMMTs. Overall, High‐K17 correlated with decreased patient survival (HR: 4.8, n=210, p=0.0002). This trend was also found in clear cell carcinoma, serous carcinoma, and MMMT subtypes. K17 expression was also independently prognostic for both cases with localized (HR: 6.8, p=0.0009) and metastatic disease (HR: 2.4, p=0.0625), as well as for high‐grade tumors (HR: 2.3, p=0.0142), across all histologic subtypes of endometrial carcinoma.Prognostic biomarkers for endometrial adenocarcinoma have the potential to identify patients that are most likely to benefit from aggressive surgical intervention, including complete staging at the time of primary resection. The results of this study support the hypothesis that K17 is a powerful negative prognostic biomarker for not only high‐grade endometrioid adenocarcinomas, but also for other high‐grade histologic types of uterine carcinoma and MMMT, including cases limited to the uterus and those with advanced stage disease. Further studies are indicated to address the potential role of K17 as prognostic biomarker, to inform patient treatment decisions.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Gross and Pathomorphological Alterations of Infectious Bursal Disease Virus (IBDV) Infection and Its Localization by Indirect Immunoperoxidase in Broilers

The present research was undertaken to study the gross and pathomorphological alterations due to infectious bursal disease virus infection in broilers. A total of 29 outbreaks were recorded from Srinagar and adjoining districts during the study period. Out of the 29 recorded outbreaks, 22 (75.86%) were clinical outbreaks and the rest 7 (24.13%) were subclinical outbreaks. The average size of the affected flocks ranged between 2500 to 6000 birds. The affected birds were mostly between 17 and 35 days of age. Among the affected flocks morbidity varied from 30% to 60%, however mortality varied from 9 % to 25%. On postmortem examination the carcasses were found to be emaciated, dehydrated with darkened discoloration and presence of petechial and ecchymotic hemorrhages in thigh and breast muscles. Histopathological alterations mostly comprised of depletion of B- lymphocytes with RE cell hyperplasia. Indirect immunoperoxidase staining of the tissues revealed viral antigen in the form of coarsely brown deposits.

Identification of Bulge Stem Cells in Mouse and Human Hair Follicles

The skin contains various populaions of stem cells, but its characterization has been hampered by lack of markers and unclear location. The hair follicle has a niche for stem cells called a “bulge” which acts as a reservoir of multipotent stem cells. In the study reported here, an immunohistochemical and immunofluorescence analysis was performed on mouse and human tissues in order to determine the possible presence of stem cells of hair follicle through cytokeratin 15 (CK15), CD34, and CD200 markers identified as crucial to the stem cells and to identify the bulge region. Mouse (n = 7) and human (n = 7) skin samples were used. The expression of proteins was determined by the indirect immunoperoxidase technique and a secondary antibody bound to a fluorochrome. The specificity of staining was evaluated by negative controls. The results revealed that the stem cells associated with CD34 and CD200 antibodies were differentially expressed in the interfollicular epidermis, sebaceous glands, and bulge region, indicating that, in mice, CD34 and, in humans, CD200 are more specific than CK15 in detecting bulge cells. It also suggests that CD34 is specific for mouse bulge cells, while CD200 might have specificity for progenitor cells and partially differentiated cells in humans.

Keratin 17 Is a Prognostic Biomarker in Endocervical Glandular Neoplasia.

Previous work in our laboratory identified keratin 17 (K17) as a specific and sensitive biomarker for high-grade squamous intraepithelial lesions and cervical squamous cell carcinoma (SCC). K17, however, has not been previously evaluated in endocervical glandular neoplasia. Based on the similar pathogenesis of squamous and glandular lesions of the cervix, we hypothesized that K17 overexpression could also be a diagnostic and/or prognostic biomarker for endocervical neoplasia. Cases of endocervical adenocarcinoma (n = 90), adenocarcinoma in situ (AIS) (n = 32), benign glandular lesions (n = 36), and normal endocervical mucosa (n = 5) were selected from Stony Brook Medicine and the University of Massachusetts from 2002 to 2013. Immunohistochemical staining for K17 was performed by an indirect immunoperoxidase method and was scored based on the proportion of cells that showed strong (2+) staining. K17 was highly expressed in 21 (65.6%) of 32 AIS and in 75 (83.0%) of 90 adenocarcinoma cases. In adenocarcinomas, K17 staining was detected in a mean of 33.9% of malignant cells. Staining tended to be strongest at the periphery of pseudoglandular groups and at the invasive front of tumors. K17 was not detected in the epithelial cells of benign glandular lesions, but groups of cuboidal cells, residing beneath the epithelial layer of benign glands, were frequently positive for K17, especially in cases of microglandular hyperplasia. High levels of K17 expression were significantly associated with decreased patient survival. K17 is highly expressed in most cases of both invasive adenocarcinoma and in AIS and is a powerful, negative prognostic marker for patient survival.

Detection of pantothenic acid-immunoreactive neurons in the rat lateral septal nucleus by a newly developed antibody.

The available immunohistochemical techniques have documented restricted distribution of vitamins in the mammalian brain. The aim of the study was to develop a highly specific antiserum directed against pantothenic acid to explore the presence of this vitamin in the mammalian brain. According to ELISA tests, the anti-pantothenic acid antiserum used showed a good affinity (10-8 M) and specificity. The antiserum was raised in rabbits. Using an indirect immunoperoxidase technique, the mapping of pantothenic acid-immunoreactive structures was carried out in the rat brain. Pantothenic acid-immunoreactive perikarya were exclusively found in the intermediate part of the lateral septal nucleus. These cells were generally small, round, fusiform or pyramidal and showed 2-3 long (50-100 μm) immunoreactive dendrites. Any immunoreactive axons containing pantothenic acid were detected. The very restricted anatomical distribution of the pantothenic acid suggests that this vitamin could be involved in some specific neurophysiological mechanisms.

Role of LIGHT in the pathogenesis of joint destruction in rheumatoid arthritis.

AIMTo characterise the role of substitutes for receptor-activator nuclear factor kappa-B ligand (RANKL) in rheumatoid arthritis (RA) joint destruction.METHODSSynovial fluid (SF) macrophages isolated from the knee joint of RA patients were incubated with 25 ng/mL macrophage-colony stimulating factor (M-CSF) and 50 ng/mL LIGHT (lymphotoxin-like, exhibits inducible expression and competes with herpes simplex virus glycoprotein D for herpes virus entry mediator, a receptor expressed by T lymphocytes) in the presence and absence of 25 ng/mL RANKL and 100 ng/mL osteoprotegerin (OPG) on glass coverslips and dentine slices. Osteoclastogenesis was assessed by the formation of multinucleated cells (MNCs) expressing tartrate-resistant acid phosphatase (TRAP) on coverslips and the extent of lacunar resorption pit formation on dentine slices. The concentration of LIGHT in RA and osteoarthritis (OA) synovial fluid was measured by an enzyme-linked immunosorbent assay (ELISA) and the expression of LIGHT in RA and OA synovium was determined by immunohistochemistry using an indirect immunoperoxidase technique.RESULTSIn cultures of RA SF macrophages treated with LIGHT and M-CSF, there was significant formation of TRAP + MNCs on coverslips and extensive lacunar resorption pit formation on dentine slices. SF-macrophage-osteoclast differentiation was not inhibited by the addition of OPG, a decoy receptor for RANKL. Resorption pits were smaller and less confluent than in RANKL-treated cultures but the overall percentage area of the dentine slice resorbed was comparable in LIGHT- and RANKL-treated cultures. LIGHT significantly stimulated RANKL-induced lacunar resorption compared with RA SF macrophages treated with either RANKL or LIGHT alone. LIGHT was strongly expressed by synovial lining cells, subintimal macrophages and endothelial cells in RA synovium and the concentration of LIGHT was much higher in RA compared with OA SF.CONCLUSIONLIGHT is highly expressed in RA synovium and SF, stimulates RANKL-independent/dependent osteoclastogenesis from SF macrophages and may contribute to marginal erosion formation.

The brain after ischemic stroke: a clinical/histological study

To analyze the histological features of changes in the brain tissue after ischemic stroke. Brain tissue samples obtained in autopsy from 9 people died as a result of a left middle cerebral artery ischemic stroke from 3 to 7 days after admission were studied. Tissue samples were taken from 3 areas of the brain: 1) contiguous to the tissue necrotic damage site zone, 2) 5-10 cm distant from the previous one, 3) the contralateral hemisphere zone symmetrical to damage site. Samples were Nissl and hematoxylin-eosin stained. Detection of p53 protein, NSE, GFAP was performed by indirect immunoperoxidase immunohistochemistry. A decrease in the total number of neurons and glial elements, their spatial redistribution, change in cell structure and their functional activity was found. The changes of the artery wall and impaired regional blood flow were detected. The more intense NSE reactivity; p53-positive neurons, reduced neuron-astrocytes distance were identified in zones 2 and 3. Ischemic stroke is accompanied by severe histological changes. These changes with varying degrees of severity occur both in the areas adjacent to necrotic core, and in remote areas, and it is a substrate for neuroplasticity.

Co-existence of bovine viral diarrhea and border disease viruses in a sheep flock suffering from abortus and diarrhea

Pestivirus infections have a huge economic impact on livestock production. In 2014, an aborted fetus from a sheep flock suffering from abortus and diarrhea was submitted for virological diagnosis. Due to the positive result of the sample for pestivirus and continuing clinical symptoms in the flock, all of the animals were sampled individually. Blood samples for serum and peripheral blood mononuclear cells were collected from 93 animals (5 rams, 26 lambs, and 62 sheep). During sampling 1 ocular and 4 rectal swab samples were obtained from lambs that had a clinical eye problem and diarrhea, respectively. Additionally 5 aborted fetuses were submitted after the initial sampling. Thirteen of the 93 blood samples tested positive for pestivirus by antigen-detection ELISA. Propagation of noncytopathogenic virus was detected in blood samples from 6 lambs and in 1 aborted fetus sample by using the indirect immunoperoxidase monolayer assay. Pestivirus RNA was detected in 10 of 13 samples by RT-PCR employing pan-pestivirus primers. Border disease virus (BDV) RNA was identified with PBD1/PBD2 specific primers in all 10 samples that tested positive for pan-pestivirus primers. Differentiating RT-PCR further identified BVDV-1 sequences in 3 of the 10 samples. The Sequenced BDV strain (KY-57) was located in the cluster of BDV-7 (Aydin-like) while the BVDV strain was close close to BVDV-1c. The results of this study highlight the possibility of dual infection in sheep with BDV and BVDV-1.