Immunoperoxidase Technique Research Articles

Distribution of calcium-binding proteins immunoreactivity in the bottlenose dolphin entorhinal cortex.

The entorhinal cortex has been shown to be involved in high-level cognitive functions in terrestrial mammals. It can be divided into two main areas: the lateral entorhinal area (LEA) and the medial entorhinal area (MEA). Understanding of its structural organization in cetaceans is particularly important given the extensive evidence for their cognitive abilities. The present study describes the cytoarchitectural and immunohistochemical properties of the entorhinal cortex of the bottlenose dolphin (Tursiops truncatus, Montagu, 1821), perhaps the most studied cetacean species and a paradigm for dolphins and other small cetaceans. Four bottlenose dolphins' entorhinal cortices were processed. To obtain a precise overview of the organization of the entorhinal cortex we used thionin staining to study its laminar and regional organization, and immunoperoxidase technique to investigate the immunohistochemical distribution of three most commonly used calcium-binding proteins (CBPs), calbindin D-28k (CB), calretinin (CR) and parvalbumin (PV). Entorhinal cortex layers thickness were measured, morphological and morphometric analysis for each layer were conducted and statistically compared. Six layers in both the LEA and MEA were identified. The main difference between the LEA and the MEA is observed in layers II and III: the neurons in layer II of the LEA were denser and larger than the neurons in layer II of MEA. In addition, a relatively cell-free zone between layers II and III in LEA, but not in MEA, was observed. The immunohistochemical distribution of the three CBPs, CB, CR and PV were distinct in each layer. The immunostaining pattern of CR, on one side, and CB/PV, on the other side, appeared to be distributed in a complementary manner. PV and CB immunostaining was particularly evident in layers II and III, whereas CR immunoreactive neurons were distributed throughout all layers, especially in layers V and VI. Immunoreactivity was expressed by neurons belonging to different morphological classes: All CBPs were expressed in non-pyramidal neurons, but CB and CR were also found in pyramidal neurons. The morphological characteristics of pyramidal and non-pyramidal neurons in the dolphin entorhinal cortex are similar to those described in the entorhinal cortex of other species, including primates and rodents. Interestingly, in primates, rodents, and dolphins, most of the CBP-containing neurons are found in the superficial layers, but the large CR-ir neurons are also abundant in the deep layers. Layers II and III of the entorhinal cortex contain neurons that give rise to the perforant pathway, which conveys most of the cortical information to the hippocampal formation. From the hippocampal formation, reciprocal projections are directed back to the deep layer of the entorhinal cortex, which distributes the information to the neocortex and subcortical area. Our data reveal that in the dolphin entorhinal cortex, the three major CBPs label morphologically heterogeneous groups of neurons that may be involved in the information flow between entorhinal input and output pathways.

Cell Death Mechanism of Organometallic Ruthenium(II) and Iridium(III) Arene Complexes on HepG2 and Vero Cells.

Due to side effects and toxicity associated with platinum-derived metal-based drugs, extensive research has been conducted on ruthenium (Ru) complexes. We aim to synthesize a highly oil soluble Ru(II)-p-cymene complex (Ru1) with an aliphatic chain group, a bimetallic Ru(II)-p-cymene complex (Ru2) with N,S,S triple-coordination and a bimetallic Ir(III)-pentamethylcyclopentadienyl complex (Ir1) with S,S double-coordination. Subsequently, we investigate the effects of these complexes on Vero and HepG2 cell lines, focusing on cell death mechanisms. Characterization of the complexes is performed through nuclear magnetic resonance spectroscopy (1H and 13C NMR) and Fourier-transform infrared spectroscopy. The effective doses are determined using the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide) (MTT) assay, applying different doses of the complexes to the two cell lines for 24 and 48 h, respectively. Immunoreactivities of Bax, Bcl2, caspase-3, RIP3, and RIPK1 are analyzed using the indirect immunoperoxidase technique. Notably, all the complexes (Ru1, Ru2, and Ir1) exhibit distinct cell death mechanisms, showing greater effectiveness than cisplatin. This study reveals the diverse mechanisms of action of Ru and Ir complexes based on different ligands. To the best of our knowledge, this study represents the first investigation of a novel RAED-type complex (Ru1) and unexpected bimetallic complexes (Ru2 and Ir1).

OVER SERÖZ KARSİNOMUNDA VE BORDERLİNE SERÖZ TÜMÖRDE İMMÜNHİSTOKİMYASAL PINCH/LIMS-1 ANTİKOR EKSPRESYONUNUN KLİNİKOPATOLOJİK DEĞERİ

Objective
 Particularly interesting cys-his rich protein (PINCH/
 LIMS-1), a protein implicated in cell adhesion, is
 assumed to oversee the development and invasion of
 cancer cells in tumors and tumor-associated stroma.
 This study aimed to assess PINCH-1 expression in
 serous borderline tumor (SBT) and serous carcinoma
 (SC) in the tumor and peritumoral stroma and
 scrutinize any associations between its expression
 and various clinical and pathological parameters.
 Material and Method
 In this study, the expression of the PINCH-1 antibody
 was analyzed in 21 cases of SBT and 89 cases of
 SC using the indirect immunoperoxidase technique
 with streptavidin/HRP-biotin. The staining pattern of
 PINCH-1 in the tumor and peritumoral stroma was
 evaluated using a semiquantitative scoring method.
 The staining procedure used in the study allowed for
 the accurate identification of PINCH-1 expression,
 and the data obtained through the semiquantitative
 scoring method provided a reliable of assessing
 the degree and intensity of PINCH-1 staining. Thus,
 the correlation between PINCH-1 expression and
 various pathologic factors such as patient age, tumor
 size, FIGO stage, intra-abdominal washing cytology,
 capsule invasion, tumor location in the ovary, tumor
 grade, and cancer antigen 125 (CA125) levels at the
 time of diagnosis was examined.
 Results
 The study found that PINCH-1 was more prevalent
 in cases of SC than in SBT cases. The tumors in SC
 cases had stronger staining than those in SBT cases
 (p

Boron-Doped Carbon Nanodots as a Theranostic Agent for Colon Cancer Stem Cells.

Carbon nanodots have drawn a great deal of attention due to their green and expedient opportunities in biological and chemical sciences. Their high fluorescence capabilities and low toxicity for living cells and tissues make them excellent imaging agents. In addition, they have a fluorimetric response against inorganic and organic species. Boron-doped carbon nanodots (B-CDs) with high fluorescence yield were produced from phenylboronic acid and glutamine as boron and carbon sources, respectively, by a hydrothermal method. First, the effects of the temperature on their fluorescence yield and the structural characteristics of B-CDs were investigated. Second, their cytotoxicity and cell death and proliferation behaviors were examined. The cytotoxicity was evaluated by the MTT assay. The cellular properties were evaluated with the distribution of caspase 3, Ki67, lamin B1, P16, and cytochrome c after the indirect immunoperoxidase technique. After the MTT assay, 1:1 dilution of all applicants for 24 h was used in the study. After immunohistochemical analyses, the application of B-CDs synthesized at 230 °C did not change control cell (Vero) proliferation, and also apoptosis was not triggered. Colo 320 CD133+ and CD133- cell-triggered apoptosis and cellular senescence were found to be synthesis temperature dependent. In addition, Colo 320 CD133- cells were affected relatively more than CD133+ cells from B-CDs. While B-CDs did not affect the control cells, the colon cancer stem cells (Colo 320 CD133+) were affected in a time-dependent manner. Therefore, the use of the synthesized B-CD product may be an alternative method for controlling or eliminating cancer stem cells in the tumor tissue.

Иммунные реакции при лимфоме Ходжкина

HL is characterized by significantly enlarged lymph nodes and the presence of rare Hodgkin and Reed-Sternberg cells. Pathogenesis is not fully understood. The increase in the disease risk can be associated with immunosuppression, HIV, parenchymal organ transplantation, autoimmune disorders, etc. The possibility of differentiating pathogenetic and protective immune responses associated with this disease will help understand the causes of the disease and the treatment prognosis. The study was aimed to determine the features of immune responses in HL depending on the disease duration and the circulating lymphocyte counts. A total of 134 patients with HL were assessed. The cytogram and phagocytosis were assessed in blood smears stained by the Wright-Giemsa procedure. The expression of lymphocyte markers in lymphocytes was determined using the indirect immunoperoxidase technique and flow cytometry. Serum levels of cytokines, immunoglobulins, autoantibodies and circulating immune complexes were assessed by enzyme immunoassay. Comparative analysis of the immune responses depending on peripheral blood leukocyte counts is provided. It has been found that prolonged HL course is associated with the decrease in the functionally active T cell counts, progressive neutropenia and monocytopenia, along with the increased activity of the reaginic reactions and autosensitization. In individuals with lymphocytopenia, mainly small lymphocytes die, the 3-fold decrease in the counts of such lymphocytes is observed; lymphocytopenia is associated with the deficiency of circulating T cells, both mature and immature, the concentrations of which decrease by 2.5–3 times, while B cell counts show no dramatic changes. The disease progression is associated with reduction of the lymphocyte homeostasis control by granulocytes and monocytes, along with progressive neutropenia and monocytopenia.

Clinicopathological Challenges in Tumors of the Nasal Cavity and Paranasal Sinuses: Our Experience.

Background and objectiveNasal and paranasal lesions are one of the most common otorhinolaryngological presentations encountered in clinical practice. Common presenting symptoms of these lesion range from nasal blockades, facial swellings, pain, nasal discharge, and epistaxis to orbital and ear symptoms. Diagnosis can be tricky as these symptoms are common in inflammatory conditions and tumors. The aim of our study was to observe the epidemiology and clinical pathological findings in patients with nasal and paranasal masses presenting to our institute and discuss the challenges in proper diagnosis and management due to similar presentations, and the role of histopathological examination (HPE) and immunohistochemistry (IHC) in overcoming these challenges.MethodsThe IPD records of 396 patients were taken up for the study. All the specimens were sent in 10% neutral buffered formalin for examination as biopsy for diagnosis or after surgical excision. After adequate fixation, the biopsy specimen was submitted for routine processing, followed by paraffin embedding, and stained with hematoxylin and eosin (H&E). Special stains like periodic acid-Schiff (PAS) and Ziehl-Neelsen (ZN) stains for acid-fast bacilli (AFB) were used as required. IHC was performed in the required samples. IHC markers were performed on representative paraffin-embedded sections according to the streptavidin-biotin immunoperoxidase technique as needed. The findings were noted, and histology was correlated with clinical presentations and investigations, tabulated, and statistically analyzed using SPSS Statistics (IBM, Armonk, NY).ResultsOf note, 67.92%% were non-neoplastic lesions whereas 18.18% came out to be benign neoplasms and 13.88% were malignant lesions on HPE. Nasal obstruction was the most common presenting symptom (73.23% of patients) followed by nasal mass (64.14% of cases). Inflammatory sinonasal polyps were the most prominent cases in our study, accounting for 41.16% of all lesions; 18.68% fungal RS (mucormycosis) were seen in our study and 4.54% were cases of juvenile nasopharyngeal angiofibroma (JNA). The majority of benign neoplasms encountered were Schneiderian papilloma or inverted papilloma (06.81%). Eighteen (4.54%) cases of squamous cell carcinomas (SCC) were seen in our study and 2.77% (n=11) cases were of adenoid cystic carcinoma. Of 18 cases of SCC, moderately differentiated SCC carcinoma accounted for 10 cases followed by poorly differentiated SCC (5/18) and nonkeratinizing SCC (3/18). IHC for p40 was performed in all the cases of nonkeratinizing SCC, which showed strong and diffuse nuclear positivity.ConclusionThe nasal cavity is the site of the most varied presentation of tumors in the upper respiratory tract. Mass in the nose and paranasal sinus (PNS) form a heterogeneous group of lesions with varied histopathological features. The proximity of the area to the eyes and brain warrants early definitive diagnosis so that the lesion is treated before it can involve important and vital centers. Even though malignant nasal tumors have a very low incidence, they cause a lot of morbidity due to their long course and frequent local recurrences. Nasal tumors tend to become polypoidal. Epithelioid papilloma of the nasal cavity often resembles a nasal polyp. Clinical diagnosis can be challenging due to similar presentations and appearances, and hence histological examination is a vital tool for the timely diagnosis of such patients.

IMMUNOEXPRESSION OF STEM CELL MARKERS IN DEVELOPMENTAL ODONTOGENIC CYSTS

<h3>Objectives</h3> Developmental odontogenic cysts (DOCs) originate from remnants of the odontogenesis, rich in multipotent stem cells (SC). The aim was to evaluate the immunoexpression of multipotent SC markers in DOCs and to compare aggressive and indolent cysts. <h3>Study Design</h3> Immunohistochemistry using the immunoperoxidase technique was performed on 42 DOCs using monoclonal antibodies for CD44 and CD90. The sample consisted of 20 odontogenic keratocysts (OKs), 7 dentigerous cysts (DCs), 7 glandular odontogenic cysts (GOCs), 4 calcifying odontogenic cysts (COC), 2 adult gingival cysts, 1 lateral periodontal cyst, 1 botryoid odontogenic cyst, and 1 orthokeratinized odontogenic cyst. <h3>Results</h3> DOCs revealed immunopositivity in epithelium and capsule for CD44 and CD90. CD44 immunoexpression in epithelium and capsule was superior in OK, followed by DC and GOC (<i>P</i> = .000). For CD90, only the capsule showed greater expression in OK, followed by calcifying odontogenic cyst and DC (<i>P</i> = .000). Aggressive cysts (OK and GOC), when compared to indolent cysts, showed superior positivity for CD44 in the epithelium (<i>P</i> = .007) and for CD90 in the capsule (<i>P</i> = .006). <h3>Conclusions</h3> Multipotent SC can participate in the histogenesis and pathogenesis of DOCs. This study also suggests that multipotent SC can be associated with the distinct biological behaviors of these lesions.

Comparative Study of Cytokeratin Immunostaining of Parotid Gland Parenchyma in Normal, Diabetic, and Excretory Duct Ligation of Mongrel Dogs.

The present study aimed to give a glimpse of the normal distribution of intermediate filaments within the parotid gland parenchyma of mongrel dogs and to reveal the pathological changes that may occur as a result of the effects of diabetes mellitus or atrophy of the gland caused by the ligation of the excretory duct to discover whether there is a similarity in these pathological behaviors. Twelve healthy mongrel dogs were used in the experiment and were divided into three groups: group I (the control group), group II (dogs with alloxan-induced diabetes), and group III (dogs with the right-side duct-ligated parotid gland). The dogs were sacrificed 45 days after the parotid excretory duct were tied. The right parotid gland of all groups was dissected and prepared for histological and immunohistochemical expression of cytokeratin 17 assay. Histological findings confirmed that the parotid gland parenchyma of the diabetic group had glandular atrophy characterized by the loss of gland structure, degenerated acini, and dilatation of the duct system. Moreover, there is a predominance of the fibrous component with the presence of fat cells within the gland compartments. On the contrary, the excretory duct-ligated group undergoes severe glandular atrophy of the previous character with the presence of duct-like structure as well as extravasation and vasodilatation. Immunohistochemical expression of cytokeratin 17 in control parotid using an immunoperoxidase technique showed that cytokeratin expression varies from negative to mild in all ducts and some serous acinar cells. The gland parenchyma of the diabetic group showed mild to strong cytokeratin expression of duct cells more concentrated in the apical part with moderate to strong expression of diffuse type in some serous acini. The intensity of cytokeratin 17 in gland compartments of the excretory duct-ligated group revealed a variation in expression that ranged from negative to strong diffuse staining throughout the gland. The severity and prevalence of cytokeratin 17 in our results are predictive of the pathological influence of both diabetes mellitus and duct ligation on the cytokeratin intracellular filaments of the salivary gland parenchyma in a different way that interferes with saliva production and/or secretion leading to xerostomia.

Topical Administration of Carvacrol Improves Healing in Nasal Septal Perforation: An Experimental Animal Study.

Clinicians are exploring alternative treatments for nasal septum perforation since surgical treatment is challenging. The effects of topical carvacrol on the healing of nasal septal perforation were investigated in an animal model. Twenty-one male New Zealand rabbits were randomly divided into three equal groups. A 5-mm circular biopsy punch was used to perforate the nasal septum behind the columella. For 14 days, bilateral gelatin sponges impregnated with carvacrol in olive oil in group 1, and only olive oil in group 2 were placed. Perforation only was performed in the control group. Animals were then sacrificed, and their nasal septums were removed. The closure of the perforation was measured, and samples were examined histopathologically. MMP-9 reactivity was evaluated using the immunoperoxidase technique. Histopathologic parameters were scored as 0 = none, 1 = mild, 2 = moderate, and 3 = strong. The septum perforation closure in the carvacrol group was statistically significant compared with the other groups (p < 0.001). Cartilage regeneration, connective tissue density, and MMP-9 immunoreactivity were significantly higher in the carvacrol group (p = 0.020, p = 0.009, and p = 0.008, respectively). Topically administered carvacrol enhances wound healing in rabbit nasal septum perforation. It accelerated perforation closure by increasing cartilage regeneration, connective tissue, and MMP-9 expression.

ALDH1 expression and potential clinical implications in chronic inflammatory periapical lesions.

Aldehyde dehydrogenase 1 (ALDH-1) is a marker of stem cells in a variety of diseases, but its role in individuals with chronic inflammatory periapical lesions remains unknown. The aim of this study was to investigate the presence of cells with a stem cell profile based on the immunoexpression of ALDH-1 in periapical granulomas (PGs) and radicular cysts (RCs). A total of 51 cases of periapical lesions (25 PGs and 26 RCs) were subjected to immunohistochemical study. The anti-ALDH-1 antibody was applied using the immunoperoxidase technique. An immunoexpression score (intensity vs. percentage of cells) was used, with the cases being classified as low expression (score: 0 to 4) and high expression (score: 6 to 9). The Chi-square test was used with a 5% level of significance. Immunoexpression of ALDH-1 was detected in all cases of PGs and RCs. In PG cases, the expression was diffuse in connective tissue cells, with most cases exhibiting high expression (n = 18; 69.2%), while in RC cases the expression revealed focal distribution in cells of the capsule and epithelial cells of the cystic lining, with most cases classified as low expression (n = 18; 72%). Significant differences in the expression scores of ALDH-1 were observed in PGs (p = 0.003). The variable expression of ALDH-1 suggests the presence of cells with stem cell profiles in PGs and RCs. These findings suggest that periapical tissues infiltrated by chronic inflammation can recruit important cells for the repair or evolution of periapical lesions.

Indirect Immunoperoxidase Test (IPT) for Detection of Antibodies Against African Swine Fever Virus (ASFV) on African Green Monkey Cell Lines (Vero, MS).

The immunoperoxidase technique (IPT) is an immunocytochemistry technique on fixed infected cells to determine the antibody-antigen complex formation through the action of the peroxidase enzyme. In this procedure, African green monkey established cell lines, such as Vero or MS, are infected with African swine fever virus (ASFV)-adapted isolates to these cell cultures. The infected cells are fixed and then used as antigens to determine the presence of the specific antibodies against ASFV in samples of swine origin. IPT has many advantages for the serological diagnosis of ASF. Firstly, IPT has a higher sensitivity than ELISA or immunoblot assay (IB) for the detection of antibodies against ASFV. Secondly, it has greater versatility to analyze any type of clinical sample of porcine origin and to conduct research studies on the epidemiological situation, through the presence of antibody titers in exudates from organs and tissues. Thirdly, the preparation of IPT plates with cells infected with ASFV is simple, although laborious, that can be stored for a long time and allow the analysis of a large number of samples.

Successful Therapy of a Patient with Prospected Autoimmune Encephalitis and Negative Laboratory Results-Confirmation of Diagnosis by Immunocytochemistry

Autoimmune encephalitis (AIE) presents with a broad spectrum of neurological and psychiatric symptoms and can cause persistent brain damage. However, the absence of pathognomonic findings in CT and MRI screening, and the fact that relevant antigens for laboratory detection of autoantibodies mostly are unknown, makes its diagnosis still a challenging task. In the present report the latter problem is overcome by the immunocytochemical detection of autoantibodies using brain sections, which in fact present nearly all neuronal antigens. Furthermore, we used a highly optimized immunoperoxidase technique, which allowed morphological high-resolution analyses of the immunoproducts. The 82-old female patient presented with unsteady gait pattern, ataxia, myoclonus, apraxia, as well as an unclear language. She suffered from panic attacks, cognitive and mnestic deterioration as well as delusions. She was alert but disoriented. Despite of normal MRI and EEG results and normal CSF laboratory data we prospectively diagnosed autoimmune encephalitis. After five days high dose corticosteroid therapy all symptoms disappeared completely. The autoimmune pathogenesis subsequently was verified unequivocally, when the patient´s CSF strongly stained neuronal and glial cell bodies in brain sections. This report should be taken as proof of principle that immunocytochemistry with CSFs of neurological patients provides the possibility to recognize a considerable number morphological details at high resolution. This will provide the possibility to form groups of patients showing similar distributions of immunoreactivities. Such groups may represent distinct nosological entities and finally may allow for elaborating selective treatments for patients with distinct types of autoimmune inflammation of the CNS.

Techniques of blood sampling for detection of African swine fever virus in wild boar and domestic pigs in the field conditions

It is thought that due to the high virulence of the African swine fever virus its circulation in the Russian Federation is accompanied by a low seroprevalence. However taking into account a long-term ASF unfavourable situation, the introduction of the virus into the wild boar population, and the occurrence of attenuated viral variants, the significance of serological testing aimed at the detection of viral antibodies is increasing. To collect field samples of biological material from animals for molecular genetic, virological, and serological tests, filter paper, as well as swabs, can be used. The specificity and sensitivity of enzyme-linked immunosorbent assay when testing blood absorbed by filter paper are worse than those shown when testing sera, but they allow effective detection of African swine fever virus antibodies. It was demonstrated that blood absorbed on filter paper can be used for the immunoblot analysis, but the optimum performance could be achieved when the immunoperoxidase technique in combination with samples, taken by swabs was used. When comparing results of enzyme-linked immunosorbent assay performed on sera collected from domestic pigs (infected with ASFV isolates Antonovo 07/14 and Sobinka 07/15), and blood from ear veins absorbed on filter paper the sensitivity was 88.9%, specificity – 90.6%. However, the use of the immunoperoxidase technique for testing blood from swabs showed 100% coincidence with ELISA, while testing of sera with immunoperoxidase technique was superior to ELISA in sensitivity. This means blood sampling using swabs may be recommended for tests after proper validation. This technique can be especially useful for collecting data about infected wild boars because effective eradication strategies are impossible without such data.

Histologic and physiologic analysis of the relationship between the dorsal nerve of the penis and the corpus cavernosum on a rat model. A complementary pathway on the innervation of penile erection?

The dorsal nerve of the penis (DNP) is the terminal branch of the pudendal nerve which is responsible for the somatic innervation of the penis. This study aims to outline any direct role of the DNP in the hemodynamics of erection histologically and physiologically. Fifteen Wistar albino rats were sorted into the electrical activity (n = 6), intracavernous pressure (n = 4), and control (n = 5) groups. The dorsal nerve was electrostimulated and the simultaneous changes in intracavernous pressure and smooth muscle activity were recorded. Penile tissues were collected, fixed, and sectioned, the slides were stained with either hematoxylin-eosin for morphological evaluation or using the indirect immunoperoxidase technique to analyze the distributions of eNOS, iNOS, and nNOS. During electrostimulation, there was a simultaneous statistically significant decrease in the electrical activity inside the corpora in electromyography and an increase in intracavernous pressure. eNOS and iNOS immunoreactivities were higher in the study group than in the control group. nNOS immunoreactivity was moderate in both study and control groups. Some fibers in the dorsal nerve of penis continue into the corpora cavernosa through the tunica albuginea and have an active, direct role in the hemodynamic process of erection, which may be complementary to the main route of innervation.

An Autopsy Case of Idiopathic Rhabdomyolysis in 1979: Immunoperoxidase Detection of Myoglobin Casts in Formalin-Fixed, Paraffin-Embedded Sections of the Kidney

In 1979, the author in his younger days experienced an autobiographical case of idiopathic rhabdomyolysis. The heme casts in formalin-fixed, paraffin-embedded sections of the kidney were immunoreactive for myoglobin. In these days, the immunoperoxidase technique had been utilized as a research seed by using paraformaldehyde-fixed frozen sections. The precious experience prompted the young author of his younger days to apply the immunoperoxidase method to diagnostic pathology using formalin-fixed paraffin-embedded sections. A brief history of early development of chromogenic immunostaining in diagnostic pathology in Japan is summarized.

Bone marrow trephine immunohistochemistry is useful in characterizing malignancy-associated myelonecrosis: A retrospective observational study.

We aim to describe the utility of immunohistochemistry (IHC) in characterizing malignancy-associated myelonecrosis (MN) on bone marrow trephine biopsies (BMBx) as a part of initial workup. Patten and intensity of antigenic immunoexpression in necrotic tumor cells on BMBx were evaluated in a series of cases using standardized avidin-biotin-complex immunoperoxidase technique after heat-induced epitope retrieval and compared the same with viable tumor cells wherever available. Fifteen out of 2494 (0.6%) cases (median age: 28years; range: 4 to 66years) had evidence of MN (extensive in eight, moderate in five, and focal in two) secondary to hematological (N=9) and solid (N=6) malignancies. Five (33.3%) had pancytopenia, and eight (53.3%) had difficult and/or hemodiluted aspirate. Antigenic expression for CD10, CD79a, CD3, CD7, and CD20 was retained by necrotic leukemic blasts or lymphoma cells; CD34, TdT, and PAX5 showed heterogeneous expression; and a weak Golgi zone (dot like) CD30 positivity was noted in Reed-Sternberg (RS) or RS-like giant cells. Necrotic epithelial metastases retained pancytokeratin in all and showed variable positivity for prostate-specific antigen, carcinoembryonic antigen, CK20, ER, PR, and GATA3. Necrotic neuroblastomas (N=2) retained positivity for synaptophysin and chromogranin, whereas retained nuclear positivity for NKX2.2 in necrotic Ewing family of tumor (N=1) aided in early diagnosis. Myelonecrosis may retain tumor antigenicity, and immunohistochemistry using selected panel of antibodies should be tried in such challenging cases for an early presumptive diagnosis and further decision making.

Tumor Associated Macrophages and Topoisomerase II Alpha As Prognostic Factors in Advanced Stage Hodgkin's Lymphoma

Background : Classic Hodgkin's lymphoma (cHL) is associated with a high cure rate, but approximately 20% of patients are refractory to initial treatment and 10-30% of patients will relapse after achieving complete remission (CR) , the search of new prognostic factors to identify these high risk patients remains challenging in daily practice, the aim of this study is to evaluate CD68 positive Tumor associated macrophages(TAM) and the cell proliferation marker topoisomerase II alpha as prognostic factor in advanced stage (cHL). Patient and Methods: This study included 82 patients with Advanced stage classic Hodgkin lymphoma presented to Medical Oncology Departments of the National Cancer Institute, Cairo University and Zagazig University hospitals in the period from 2012 to 2017. All patients received ABVD regimen as induction. Immunohistochemical (IHC) analysis was performed for all cases using Tissue microarrays (TMAs) sections and stained with anti‐ CD68 (clones KP1 and PGM1, Dako, Glostrup,Denmark). For Topo II testing, IHC staining was performed on deparaffinized sections using the avidin-biotin complex immunoperoxidase technique with separate antibodies recognizing CD30 and the isoform of Topo II. The percentage of cells expressing cytoplasmic CD30 and nuclear Topo II staining simultaneously was determined for each case. We examined four cutoff points of CD68 expression (<= 20% versus >20%), <5% low (score 1), 5-15% intermediate (score 2) & > 15% high (score 3), <5% low (score 1), 5-25% intermediate (score 2) & > 25% high (score 3) and <= 50% versus > 50%. For Topo II expression, two cut off points were analyzed (<= 30% versus >30%) and (<= 75% versus >75%). Results: The study included 41 males and 41 females with a ratio of 1: 1. The median age was 30.5 years (range: 18-60 years). CRwas achieved in 64.6% (53/82 of patients) and 35.4% of all patients (29/82) were refractory. Relapse was reported in17% of the CR cases (9/53). The significant statistical difference in disease-free survival (DFS) for CD68+was observed at a cut off value of 50%, where 60 patients had CD68 <= 50% and 22 patients had CD68 >50%. The mean DFS for patients with CD68 <= 50% was 49.33 months ± 2.18 months (confidence interval: 45.05 to 53.60 months) vs. 35.255 ± 4.94 months (confidence interval: 25.55 to 44.95 months) in patients with CD68 > 50% (p = 0.015). The low CD68 group included more patients in CR (67.9%, n=36 vs. 32.1%, n=17) , male to female ratio was equal(1:1) in both groups (<= 50% ,n=30:30) and (> 50% ,n=11:11), B symptoms also was equal in both groups (<= 50% , 81% ,n=49) and (> 50% 81% ,n=18), patients with CD68<= 50% had more extra nodal disease (22%, n=13 vs. 18%, n=4) and also more bulky disease (20%, n=12 vs. 4.5% n=1) compared with high (CD68>50%) group. The overall survival (OS) was not significant for all CD68 cutoff points. Higher CD68 is associated with decreased OS: 48 vs. 52 month (CI: 42 to 53 month, p=0.42) at cut off 50%. Topo II expression using the two cut off points was not statistically significant in relation to both DFS and OS , the higher topoII group included more patients in CR (80% ,n=42 topo II >30% vs. 20%, n=10 topo II<=30%) also,(67%, n=53 Topo II >= 75% vs. 33%, n=17 Topo II < 75%). Higher Topo II is associated with prolonged DFS (45 months vs. 36 month) confidence intervals: 40 to 50 months vs 27 to 46. Months) at cut off 30% (p=0.8). At cutoff 75%, the DFS was 46 vs. 38 months (Confidence intervals: 41.73 to 52.10 months vs. 30 to 47.7 months, p= 0.36). Higher Topo II is associated with decreased OS: 50 vs.54 months (CI: 46 to 54 month) at cutoff 75% (p=0.19). CD68 was only statistically significant in correlation to patient age (p = 0.05), and non significant to serum albumin ,hemoglobin level , white blood cells count ,international prognostic score (IPS). Topo II was not statistically significant with the previous patient characteristics. Conclusion : High level of CD68 expression in patients with advanced stage HL (>50%) was significantly associated with inferior DFS but not OS, Topo II does not have significant prognostic value in relation to both DFS or OS of advanced HL cases. DisclosuresNo relevant conflicts of interest to declare.

Specific neuronal subpopulations in the rat basolateral amygdala express high levels of nonphosphorylated neurofilaments.

Cortical pyramidal neurons (PNs) containing nonphosphorylated neurofilaments (NNFs) localized with the SMI-32 monoclonal antibody have been shown to be especially vulnerable to degeneration in Alzheimer's disease (AD). The present investigation is the first to study the expression of SMI-32+ NNFs in neurons of the basolateral nuclear complex of the amygdala (BNC), which contains cortex-like PNs and nonpyramidal neurons (NPNs). We observed that PNs in the rat basolateral nucleus (BL), but not in the lateral (LAT) or basomedial (BM) nuclei, have significant levels of SMI-32-ir in their somata with antibody diluents that did not contain Triton X-100, but staining in these cells was greatly attenuated when the antibody diluent contained 0.3% Triton. Using Triton-containing diluents, we found that all SMI-32+ neurons in all three of the BNC nuclei were NPNs. Using a dual-labeling immunoperoxidase technique, we demonstrated that most of these SMI-32+ NPNs were parvalbumin-positive (PV+) or somatostatin-positive NPNs but not vasoactive intestinal peptide-positive or neuropeptide Y-positive NPNs. Using a technique that combines retrograde tracing with SMI-32 immunohistochemistry using intermediate levels of Triton in the diluent, we found that all BNC neurons projecting to the mediodorsal thalamic nucleus (MD) were large NPNs, and most were SMI-32+. In contrast, BNC neurons projecting to the ventral striatum or cerebral cortex were PNs that expressed low levels of SMI-32 immunoreactivity (SMI-32-ir) in the BL, and no SMI-32-ir in the LAT or BM. These data suggest that the main neuronal subpopulations in the BNC that degenerate in AD may be PV+ and MD-projecting NPNs.

Neuronal localization of m1 muscarinic receptor immunoreactivity in the monkey basolateral amygdala.

The basolateral nuclear complex (BNC) of the amygdala plays an important role in the generation of emotional/motivational behavior and the consolidation of emotional memories. Activation of M1 cholinergic receptors (M1Rs) in the BNC is critical for memory consolidation. Previous receptor binding studies in the monkey amygdala demonstrated that the BNC has a high density of M1Rs, but did not have sufficient resolution to identify which neurons in the BNC expressed them. This was accomplished in the present immunohistochemical investigation using an antibody for the m1 receptor (m1R). Analysis of m1Rs in the monkey BNC using immunoperoxidase techniques revealed that their expression was very dense in the BNC, and suggested that virtually all of the pyramidal projection neurons (PNs) in all of the BNC nuclei were m1R-immunoreactive (m1R+). This was confirmed with dual-labeling immunofluorescence using staining for calcium/calmodulin-dependent protein kinase II (CaMK) as a marker for BNC PNs. However, additional dual-labeling studies indicated that one-third of inhibitory interneurons (INs) expressing glutamic acid decarboxylase (GAD) were also m1R+. Moreover, the finding that 60% of parvalbumin (PV) immunoreactive neurons were m1R+ indicated that this IN subpopulation was the main GAD+ subpopulation exhibiting m1R expression. The cholinergic innervation of the amygdala is greatly reduced in Alzheimer's disease and there is currently considerable interest in developing selective M1R positive allosteric modulators (PAMs) to treat the symptoms. The results of the present study indicate that M1Rs in both PNs and INs in the primate BNC would be targeted by M1R PAMs.

Natural Infection of Goats with Orf (Contagious ecthyma) and Its Diagnosis

Background: The Study was intended to evaluate some common diagnostics that could supplement the clinical and histological identification of orf in goats. Methods: Samples from suspected clinical cases of orf (Contagious ecthyma) were collected from various organized and unorganized goat herds around Guwahati, Assam. Presumptive diagnosis was based on the typical signs and lesions. For confirmatory diagnosis, various molecular and immunoassays were employed for the detection of orf virus or circulating antibodies. Result: Cutaneous lesions observed included solitary to multifocal erythema, papules, vesicles, pustules and scab stages on the lips, ears, gums, tongue, udder and perineal region. No mortality was observed and the morbidity rate varied between 35-60%. Microscopic lesions in skin biopsies were typically marked by epidermal hyperplasia and ballooning degeneration of the keratinocytes along with other changes. Eosinophilic intracytoplasmic inclusion bodies were demonstrable within keratinocytes only in early papulo-vesicular stages. Dermis was infiltrated with polymorphonuclear and mononuclear cells in varying proportions. Initial screening of samples was done with PCR systems to specifically detect parapoxvirus DNA employing a semi-nested PCR targeted against the partial B2L gene (with reported primers yielding 594 bp and 235 bp amplified products) and an uniplex PCR targeting the whole B2L gene (with reported primer set to yield an amplified product of 1206 bp). Agar gel immuno-diffusion (AGID) failed to develop positive immunoprecipitation with hyperimmune serum against scab lysates; however, counter-immuno-electrophoresis (CIE) showed positive immunoprecipitation in the same samples. For rapid and in situ detection of ORFV antigen in tissues, immunofluorescent and immunoperoxidase techniques were successfully employed both on cryosections and formalin fixed skin biopsies. Immunofluorescent technique on cryosections was found to be easy, rapid and more specific. A dot-ELISA was also developed for successful confirmation of orf virus from clinical samples. For antibody detection in convalescing goats, an indirect-ELISA and a dot-ELISA was also successfully tested to demonstrate for antibody detection in convalescing goats, but protective titres later after infection was not addressed. Monoclonal antibody employed in the assays was found to be specific, sensitive and versatile for virus detection from direct clinical samples. It is contemplated that assays employing hyperimmune sera or detection of circulating antibody against orf virus may have limited diagnostic applications owing to its partial and transient humoral immunity and the inherent property of the virus to modulate and interfere with the host response and evade immune mechanisms.