Intense Immunoreactivity Research Articles

Starburst amacrine cells, involved in visual motion perception, lose their synaptic input from dopaminergic amacrine cells and degenerate in Parkinson’s disease patients

BackgroundThe main clinical symptoms characteristic of Parkinson’s disease (PD) are bradykinesia, tremor, and other motor deficits. However, non-motor symptoms, such as visual disturbances, can be identified at early stages of the disease. One of these symptoms is the impairment of visual motion perception. Hence, we sought to determine if the starburst amacrine cells, which are the main cellular type involved in motion direction selectivity, are degenerated in PD and if the dopaminergic system is related to this degeneration.MethodsHuman eyes from control (n = 10) and PD (n = 9) donors were available for this study. Using immunohistochemistry and confocal microscopy, we quantified starburst amacrine cell density (choline acetyltransferase [ChAT]-positive cells) and the relationship between these cells and dopaminergic amacrine cells (tyrosine hydroxylase-positive cells and vesicular monoamine transporter-2-positive presynapses) in cross-sections and wholemount retinas.ResultsFirst, we found two different ChAT amacrine populations in the human retina that presented different ChAT immunoreactivity intensity and different expression of calcium-binding proteins. Both populations are affected in PD and their density is reduced compared to controls. Also, we report, for the first time, synaptic contacts between dopaminergic amacrine cells and ChAT-positive cells in the human retina. We found that, in PD retinas, there is a reduction of the dopaminergic synaptic contacts into ChAT cells.ConclusionsTaken together, this work indicates degeneration of starburst amacrine cells in PD related to dopaminergic degeneration and that dopaminergic amacrine cells could modulate the function of starburst amacrine cells. Since motion perception circuitries are affected in PD, their assessment using visual tests could provide new insights into the diagnosis of PD.

Diagnostic utility of FOXO1 immunohistochemistry for rhabdomyosarcoma classification.

Rhabdomyosarcomas currently are classified into one of four subtypes (alveolar, embryonal, spindle cell/sclerosing, or pleomorphic) according to their morphological, immunohistochemical, and molecular genetic features. The alveolar subtype is characterised by a recurrent translocation involving PAX3 or PAX7 and FOXO1; identification of this translocation is important for appropriate classification and prognostication. In this study, we aimed to explore the diagnostic utility of FOXO1 immunohistochemistry for rhabdomyosarcoma classification. A monoclonal antibody targeting a FOXO1 epitope retained in the fusion oncoprotein was used to study 105 rhabdomyosarcomas. FOXO1 was positive for expression by immunohistochemistry in all 25 alveolar rhabdomyosarcomas, with 84% showing diffuse expression in greater than 90% of neoplastic cells; the remainder of alveolar rhabdomyosarcomas displayed at least moderate staining in a minimum of 60% of lesional cells. Apart from three spindle cell rhabdomyosarcomas showing heterogeneous nuclear immunoreactivity in 40-80% of tumour cells, the 80 cases of embryonal, pleomorphic, and spindle cell/sclerosing rhabdomyosarcoma were negative for FOXO1 expression (96.3% specific) when using a threshold of nuclear staining in 20% of neoplastic cells to determine positivity. Variable cytoplasmic staining was present in a fraction of all rhabdomyosarcoma subtypes. Nonneoplastic lymphocytes, endothelial cells, and Schwann cells also showed variably intense nuclear anti-FOXO1 immunoreactivity. Taken together, our findings suggest that FOXO1 immunohistochemistry is a highly sensitive and relatively specific surrogate marker of the PAX3/7::FOXO1 fusion oncoprotein in rhabdomyosarcoma. Cytoplasmic immunoreactivity, expression in nonneoplastic tissues, and limited nuclear staining of nonalveolar rhabdomyosarcomas represent potential pitfalls in interpretation.

Liver microsomal cytochrome P450 3A-dependent drug oxidation activities in individual dogs

Drug oxidations are mediated mainly by cytochromes P450 (P450s or CYPs). CYP3As are an important P450 subfamily and include liver-specific CYP3A12 and intestine-specific CYP3A98 in dogs. Individual differences in drug oxidation activities were investigated, including correlations with immunoreactive CYP3A protein intensities and CYP3A mRNA expression levels in livers. Pooled and individual dog liver microsomes showed activities towards nifedipine, midazolam, alprazolam, and estradiol, but the levels of catalytic activities varied approximately twofold among the individual dogs. One dog harboured a CYP1A2 variant causing protein deletion but showed higher activities than the other dogs towards nifedipine oxidation, midazolam 1′-hydroxylation, alprazolam 4-hydroxylation, estradiol 16α-hydroxylation activities, and caffeine C 8-hydroxylation; the latter is used as a reference reaction for CYP1A. In individual dog liver microsomes, the intensities of the immunochemical bands with anti-human CYP3A4 and anti-rat CYP3A2 antibodies along with CYP3A12 and CYP3A26 mRNA expression levels showed good correlations (p < 0.05) with nifedipine oxidation, midazolam 1′- and 4-hydroxylation, alprazolam 1′- and 4-hydroxylation, and estradiol 16α-hydroxylation activities. These results suggest that the oxidation activities of dog liver microsomes towards nifedipine and other typical CYP3A-catalyzed drugs exhibit approximately twofold individual differences and were predominantly mediated by liver-specific CYP3A12 in the dogs.

EXPRESSION OF OSTEOPONTIN IN ORAL SQUAMOUS CELL CARCINOMA – AN IMMUNOHISTOCHEMICAL STUDY

Objectives: To analyze the expression of Osteopontin (OPN) is well differentiated, moderately differentiated, and poorly differentiated oral squamous cell carcinoma (OSCC). Methodology: This study was conducted at the Department of Pathology, Peshawar Medical College, over a pe­riod of 12 months. A total of 50 formalin-fixed paraffin-embedded (FFPE) blocks (n=50) of confirmed cases with varying grades of OSCC were included in the study. Immunostaining was evaluated for intensity and percentage of positive cells and Immunoreactivity of OPN was assessed by the Immunoreactivity scoring system (IRS) method. Results: The age of the patients was 58.44 ± 13.01 years ranging from 30 to 82 years on average. Maximum number of cases belonged to the age group 51 to 70 years having 27 (54%) participants. The most common cat­egory of OPN expression among well differentiated OSCC was moderate (n=16, 66.7%). For poorly differentiated OSCC the strong staining was found in 6 cases (50%) and was the most common category of OPN expression. In males the pattern of staining for OPN expression was not statistically significant among various grades of OSCC (P=0.455) while among females the results were highly significant (P=0.003). The mean IHC staining score by IRS method for OPN expression was highest among well differentiated (9.20±4.31) Conclusion: OPN can be used as a diagnostic marker for OSCC. It can also help in assessing the potential aggres­sive behavior of the tumor pointing at close follow up of such patients for recurrence.

Catfish Epidermal Club Cell Morphologic and Immunologic Attributes: Heterogeneous S-100 Immunoreactivity and Possible Neuroendocrine Function.

The fish epidermis (EP) contains several cell types, and it has several functional roles, though the secretory role prevails. The alarm or epidermal club cells (ECCs) represent a voluminous immune cell secreting an alarm substance. Our work targeted the histochemical and immunologic attributes of the ECCs in catfish. Matrix metalloproteinase (MMP-9) immunostaining revealed strong immunoreactive signals in the cytoplasm of all ECCs, while S-100 protein immunoreactivity selectively marked the ECCs. Individual ECCs exhibit intense S-100 immunoreactivity compared to neighboring ECCs. The difference in the intensity of the S-100 immunostaining was associated with the difference in the ECC size. This was confirmed by the semithin results where several developmental stages of ECCs could be distinguished. Some eosinophilic granular cells with their characteristic metachromatic granules were recorded juxtaposing the ECCs. Moreover, some free electron-dense granules could be demonstrated in the intercellular spaces surrounding the ECCs. Collectively, these findings suggest several functional profiles for the catfish ECCs depending on their developmental stage. Most importantly, S-100 immunoreactivity in the ECCs suggests its neuroendocrine function. Moreover, the difference in the intensity and polar distribution of S-100 staining is a sign of its role in the differentiation of the ECCs.

The HOPX and BLBP landscape and gliogenic regions in developing human brain.

Outer radial glial cells (oRGs) give rise to neurons and glial cells and contribute to cell migration and expansion in developing neocortex. HOPX has been described as a marker of oRGs and possible actor in glioblastomas. Recent years' evidence points to spatiotemporal differences in brain development which may have implications for the classification of cell types in the central nervous system and understanding of a range of neurological diseases. Using the Human Embryonic/Fetal Biobank, Institute of Cellular and Molecular Medicine, Faculty of Health and Medical Sciences, University of Copenhagen, Denmark, HOPX and BLBP immunoexpression was investigated in developing frontal, parietal, temporal and occipital human neocortex, other cortical areas and brain stem regions to interrogate oRG and HOPX regional heterogeneity. Furthermore, usage of high-plex spatial profiling (Nanostring GeoMx® DSP) was tested on the same material. HOPX marked oRGs in several human developing brain regions as well as cells in known gliogenic areas but did not completely overlap with BLBP or GFAP. Interestingly, limbic structures (e.g. olfactory bulb, indusium griseum, entorhinal cortex, fimbria) showed more intense HOPX immunoreactivity than adjacent neocortex and in cerebellum and brain stem, HOPX and BLBP seemed to stain different cell populations in cerebellar cortex and corpus pontobulbare. DSP screening of corresponding regions indicated differences in cell type composition, vessel density and presence of apolipoproteins within and across regions and thereby confirming the importance of acknowledging time and place in developmental neuroscience.

D1- and D2-type dopamine receptors are immunolocalized in pial and layer I astrocytes in the rat cerebral cortex.

Pial astrocytes, a cellular component of the cerebral cortex surface structure, are observed in a wide range of mammalian species. Despite being recognized as such, the functional potential of pial astrocytes has long been overlooked. Our previous research demonstrated that pial astrocytes exhibit stronger immunoreactivity for muscarinic acetylcholine receptor M1 than protoplasmic astrocytes, indicating sensitivity to neuromodulators. Here, we examined whether pial astrocytes express receptors for dopamine, another crucial neuromodulator of cortical activity. We investigated the immunolocalization of each dopamine receptor subtype (D1R, D2R, D4R, D5R) in the rat cerebral cortex, and compared the intensity of immunoreactivity between pial astrocytes, protoplasmic astrocytes, and pyramidal cells. Our findings revealed that pial astrocytes and layer I astrocytes exhibit stronger D1R- and D4R-immunoreactivity than D2R and D5R. These immunoreactivities were primarily localized in the somata and thick processes of pial and layer I astrocytes. In contrast, protoplasmic astrocytes located in cortical layers II-VI displayed low or negligible immunoreactivities for dopamine receptors. D4R- and D5R-immunopositivity was distributed throughout pyramidal cells including somata and apical dendrites. These findings suggest that the dopaminergic system may regulate the activity of pial and layer I astrocytes via D1R and D4R.

P018 Expression of annexin A1 and NLRP3 inflamassome in Chron’s disease

Abstract Background Despite annexin A1 (ANXA1), an anti-inflammatory and pro-resolving protein, regulates the NLRP3 inflammasome activation in inflammatory cells, this relationship has not been explored in inflammatory bowel disease. Then, this study evaluated the expression patterns of ANXA1 and NLRP3 inflammasome in Crohn’s disease (CD). Methods Paraffin-embedded human intestine biopsies, obtained from CD (n=20) and control (n=8) patients were processed for histological and immunohistochemical analyses. The study was approved by the UNIFESP Ethics Committee (CEP 4.491.836/2020). GSE179285 study, which features ileum (Control - n=8, CD non-inflamed - n=48, CD inflamed - n=33) and colon (Control - n=12, CD non-inflamed – n=74, CD inflamed - n=14) transcriptome, was selected from the Gene Expression Omnibus repository. Datasets were analyzed using the GEO2R tool to detect ANXA1, CASP1, NLRP3, and PYCARD expressions. Results Bioinformatics analysis of GSE179285 samples showed a marked increase in the ANXA1 expression in the CD inflamed ileum and colon compared to CD non-inflamed and controls samples (Fig.1A). Regarding the genes associated with the NLRP3 inflammasome activation, transcriptome analysis also revealed a significant increase in NLRP3 and CASP1 in the CD inflamed intestine compared to the other ones (Fig.1B,C). PYCARD expression did not show alterations between the different conditions (Fig.1D). Immunohistochemical analyzes showed intense immunoreactivity for ANXA1 and NLRP3 proteins in the CD ileum and colon, especially in inflammatory infiltrate and epithelium of the mucosal layer (Fig.2C,D,G,H), compared to controls (Fig. 2A,B,E,F). These findings corroborate the transcriptome analyses. Conclusion ANXA1 and NLRP3 expression is increased in CD-inflamed intestines. High levels of ANXA1 may contribute to controlling inflammation and repairing intestinal injuries. High levels of NLRP3 evidence dysfunction in the pro-inflammatory activity of DC, contributing to the release of IL-1β and IL-18. Support: FAPESP (grants 20/03565-2, 21/01541-1 and 21/06059-3). Figure 1. ANXA1, NLRP3, CASP1 and PYCARD expressions. Analysis performed in the healthy or CD terminal ileum and ascending/descending colon of human biopsies from GSE179285 study. Data represent mean ± S.E.M. of the gene expression values. **p&amp;lt;0.01; ***p&amp;lt;0.001; ****p&amp;lt;0.0001 (Kruskal-Wallis, Dunn’s post-test). Figure 2. ANXA1 and NLRP3 expression in human intestine. Representative images of ileum (A, C, E, G) and colon (B, D, F, H) of control (A,B,E,F) and CD patients (C,D,G,H). ANXA1 and NLRP3 expression detected in intestinal epithelium (arrows) and inflammatory infiltrate (asterisks). Counterstaining: Carazzi’s Hematoxylin. Bars: 50 µm.

Nestin expression in intact and hypertrophic myocardium of spontaneously hypertensive rats during aging

Nestin is a unique intermediate filament expressed for a short period in the developing heart. It was also documented in several cell types of the adult myocardium under pathological conditions such as myocardial infarction or fibrosis. However, circumstances of nestin re-occurrence in the diseased or aging heart have not been elucidated yet. In this work we immunohistochemically detected nestin to determine its expression and distribution pattern in the left ventricular myocardium of normotensive Wistar Kyoto (WKY) rats and in the hypertrophic ones of spontaneously hypertensive (SHR) rats, both at the age of 1 and 1.5 year. No nestin+ cells were identified in the intact myocardium of 1-year-old WKY rats, whereas in the aged 1.5-year-old WKY rats nestin+ endothelial cells in some blood vessels were discovered. In the hypertrophic myocardium of all SHR rats, nestin was rarely detected in desmin+ vimentin− cardiomyocytes and in some vimentin+ interstitial cells often accumulated in clusters, varying in intensity of desmin immunoreactivity. Moreover, nestin was infrequently expressed in the endothelial cells of some myocardial blood vessels in 1-year-old SHR rats, but not in 1.5-year-old ones. Quantitative image analysis of nestin expression in the myocardium confirmed significant increase in 1.5-year-old WKY rats and in SHR rats of both ages compared to the intact 1-year-old WKY rats. This study firstly documents nestin re-expression indicating cytoskeletal remodelling in different cell types of the aging intact and chronically pressure over-loaded hypertrophied myocardium. Our findings confirm nestin involvement in complex changes during myocardial hypertrophy and progressive aging.

The Relationship Between Leptin Receptor Expression and Endometrial Carcinoma; A Case-Control Study.

Endometrial carcinoma is one of the most common malignancies in women in developed countries and the fourth malignancy in Iranian women. Therefore, the identification of its causative factors is essential for the prevention, diagnosis, and treatment. This study was aimed to compare the leptin receptor (Ob-R) expression in the endometrial carcinoma cases and non-carcinoma samples. In this case-control study, 89 samples (including 45 carcinoma and 44 non-carcinoma samples) were examined. The carcinomatous samples were selected by the census method and others were selected with random method. The data were obtained from histopathologic diagnosis, immunohistochemistry (negative, positive and intensity of immunoreactivity), age, history of diabetes, and hypertension. Ob-R expression was compared in the studied groups using Chi-square, Fisher tests and Multivariate logistic regression analysis. In all tests the level of significance was set at 0.05. The SPSS 26 was used for data analysis. The frequency of high levels of leptin receptors in the patients with endometrial carcinoma was significantly higher compared to the control group (57.8% vs. 2.3%) (P<0.05). Adjusting the effects of age, history of diabetes mellitus (DM) and hypertension (HTN) revealed that the positive-receptor group had 37.75 (95% CI; 5.18-275.04) odds of having endometrial carcinoma (P<0.001). The leptin receptor may be a risk factor for the endometrial carcinoma among women tested in Kashan. Based on these results, leptin receptor might be considered as a potential biomarker for screening the endometrial carcinoma or targeting the therapeutic purposes.

Novel neuron-Schwann cell co-culture models to study peripheral nerve degeneration and regeneration.

Novel neuron-Schwann cell co-culture models to study peripheral nerve degeneration and regeneration.

The nonsynaptic plasticity in Parkinson's disease: Insights from an animal model

BackgroundThe 6-OHDA nigro-striatal lesion model has already been related to disorders in the excitability and synchronicity of neural networks and variation in the expression of transmembrane proteins that control intra and extracellular ionic concentrations, such as cation-chloride cotransporters (NKCC1 and KCC2) and Na+/K+-ATPase and, also, to the glial proliferation after injury. All these non-synaptic mechanisms have already been related to neuronal injury and hyper-synchronism processes. ObjectiveThe main objective of this study is to verify whether mechanisms not directly related to synaptic neurotransmission could be involved in the modulation of nigrostriatal pathways. MethodsMale Wistar rats, 3 months old, were submitted to a unilateral injection of 24 µg of 6-OHDA, in the striatum (n = 8). The animals in the Control group (n = 8) were submitted to the same protocol, with the replacement of 6-OHDA by 0.9% saline. The analysis by optical densitometry was performed to quantify the immunoreactivity intensity of GFAP, NKCC1, KCC2, Na+/K+-ATPase, TH and Cx36. ResultsThe 6-OHDA induced lesions in the striatum, were not followed by changes in the expression cation-chloride cotransporters and Na+/K+-ATPase, but with astrocytic reactivity in the lesioned and adjacent regions of the nigrostriatal. Moreover, the dopaminergic degeneration caused by 6-OHDA is followed by changes in the expression of connexin-36. ConclusionsThe use of the GJ blockers directly along the nigrostriatal pathways to control PD motor symptoms is conjectured. Electrophysiology of the striatum and the substantia nigra, to verify changes in neuronal synchronism, comparing brain slices of control animals and experimental models of PD, is needed.

Broiler İleumunda Kuluçka Sonrası Farklı Dönemlerin TLR4 ve VEGF Ekspresyon Paternleri Üzerine Etkisi

This study is aimed to evaluate the relationship between VEGF and TLR4 expression in the ileum during broiler post-hatching development. The material for the study was taken from the ileum tissue of 7-, 21-, and 42-day-old broilers. In tissue sections VEGF and TLR4 expression were demonstrated by Streptavidin-biotin complex immunohistochemistry method. Beginning on the 7th day after hatching, the number of stained cells and staining intensity in the epithelial cells lining the villus intestinalis increased in TLR4 immunostaining. On the 7th day following hatching, TLR4 protein expression was not seen in crypt epithelial cells. At day 21, crypt epithelial cells began to stain and gave a more intense immunoreaction at day 42. In VEGF-stained sections, the ileum villus epithelial cells, crypt, and smooth muscle tissue showed a brown intracytoplasmic response. The expression of the VEGF protein in the upper villus epithelial cells started to increase on the 7th day, and it stained intensely, especially on the 42nd day. In addition, it was observed that the staining intensity of the tunica muscularis layer was the same on the 7th and 21st days, and increased on the 42nd day. It was remarkable that goblet cells gave negative results in both immunostaining. In summary it seen that TLR4 and VEGF expression were found to be increase in this study from the 7th to the 42nd day following hatching. Thus, it was concluded that angiogenesis mechanisms and the development of innate and adaptive defense systems continue throughout the post-hatching period.

Effects of estradiol on dopaminergic synapse formation in the mouse olfactory bulb.

Olfactory glomeruli are the sites of initial synaptic integration in olfactory information processing. They are surrounded by juxtaglomerular (JG) cells, which include periglomerular, superficial short axon, and external tufted cells. A subpopulation of JG cells expresses the dopamine synthetic enzymes, tyrosine hydroxylase (TH), and aromatic l-amino acid decarboxylase (AADC). TH cells corelease γ-aminobutyric acid (GABA) and their processes extend to multiple glomeruli forming intra- and interglomerular circuits. It is well established that 17β-estradiol (E2) exerts wide ranging effects in the central nervous system. However, participation of E2 in the modulation of neurotransmission and synaptic plasticity of TH cells in olfactory glomeruli is unknown. To address this, we subcutaneously implanted a 60-day release pellet of E2 or placebo into intact male mice and compared glomerular TH, AADC, and vesicular γ-aminobutyric acid transporter (VGAT) immunoreactivity between them. High-voltage electron microscopy (HVEM) and ultra-HVEM using immunogold revealed significantly increased immunoreactive intensity at the cellular level for TH and AADC after E2 treatment and for VGAT in TH cells. These results indicate that E2 may affect the interplay between dopaminergic and GABAergic systems. Moreover, random-section electron microscopy analysis showed a significant increase in the number of symmetrical synapses from TH cell to mitral/tufted cell dendrites after E2 treatment. This result was supported by quantitative immunofluorescence staining with synapse markers. Together, these data indicate that E2 may regulate inhibition between TH cells and olfactory bulb neurons within the glomerulus via interaction between dopaminergic and GABAergic systems, thereby contributing to neuromodulation of odor information processing.

Increased GLUT1 expression and localization to Golgi apparatus of acinar cells in the parotid gland of Goto-Kakizaki diabetic rats

ObjectivePatients with diabetes are known to have high salivary glucose levels. But the mechanisms are still unclear. We hypothesized that the topological changes of glucose transporters affect the salivary glucose level. MethodsWe used adult Goto-Kakizaki (GK) rats, an animal model of advanced diabetes, and Wistar rats as a control, with or without glucose load. The sections of salivary glands from the animals were processed for standard histological, immunohistochemical, and immunofluorescent staining. ResultsParotid acinar cells of GK rats appeared like mucous filled with low-eosin-stained granules and possessing a flat nucleus located basally, whereas those of Wistar rats appeared as a typical serous gland with eosin-rich cytoplasm and a spherical nucleus. Cytoplasmic granules of GK rat parotid acinar cells showed no reaction of polysaccharide staining. In acinar cell cytoplasm of GK rats, intense GLUT1 immunoreactivity was observed compared to Wistar rats. By double immunostaining for GLUT1 and Golgi apparatus-specific markers, it was determined that GLUT1 was localized to the Golgi apparatus. By glucose loading in starved GK rats, the distribution of GLUT1-immunoreactive signals was spread out clearly from the apical side of the nucleus to the basolateral side. ConclusionsIn rat model of diabetes, highly localized GLUT1 at Golgi apparatus in acinar cells seems to increase taking up cytoplasmic glucose to form exocytotic vesicles. This phenomenon may transform parotid glands from serous to mucous-like and result in saccharide-rich saliva.

Infectivity and virulence of the infectious Macrobrachium rosenbergii nodavirus produced from Drosophila melanogaster cell using Penaeus merguiensis as an infection model

It has been established that baculovirus-insect cell line is applicable for shrimp virus replication, propagation and secretion in the in vitro culture system. We thus aimed to produce Macrobrachium rosenbergii nodavirus (MrNV) clone within S2 cell to improve viral production over the previous model using Sf9 cell. Upon the transfection of genomic RNA1 and RNA2 into S2 cells, the recognizable cellular changes including cytoplasmic swelling and clumping of cells were observed within 24 h. The culture media containing secreted MrNV particles were re-transfected into healthy S2 cells and similar cellular changes as with the first transfection were observed. Immunohistochemistry analysis of the re-infecting S2 cell revealed an intense immunoreactivity against MrNV capsid protein confirming that S2 cell was permissive cells for MrNV. In vivo infectivity test using P. merguiensis as a model animal exposed to the secreted MrNV revealed the presence of RNA2 fragment in shrimp tissue accompanied with the sign of whitish abdominal muscle at 24 h post-infection (p.i.). In addition, the number of shrimp hemocytes decreased at 6–24 h p.i. and returned to the normal level at 48 h p.i., whereas a significant up-regulation of immune-related genes including HSP70 and trypsin was noted. These data suggested that rescued MrNV produced in S2 is practically useful for MrNV infection test in which their natural virion inoculae are difficult to obtain. In addition, the molecular basis of viral pathogenesis can further be investigated which should be beneficial for any antiviral therapy developments in the future.

Pomegranate (Punica granatum) peel alleviates lithium-induced alterations in the thyroid gland of rats by modulating apoptosis and oxidative stress.

Lithium carbonate (LC) is known to alter thyroid gland function. Pomegranate (PG) is a fruit with multiple antioxidant and antiapoptotic properties. Here, we studied the effect of PG on LC-induced morphological and functional alterations in the thyroid glands of rats. Rats were divided into four groups: control, lithium, lithium-PG, and PG. After 8 weeks, the rats were sacrificed, the levels of thyroid hormones and oxidative stress markers were estimated, and thyroid tissues were subjected to histological, immunohistochemical, and ultrastructural evaluations. Compared to the control group, the lithium group showed significant changes in thyroid hormone levels, greater expression of the oxidant marker malondialdehyde, and lower expression of the antioxidant marker superoxide dismutase (SOD). Most of these changes improved upon PG treatment. Histological evaluation of the thyroid in the lithium group showed disorganization and follicle involution. Additionally, the periodic acid Schiff staining intensity and SOD immunoreactivity declined significantly, whereas the collagen fiber content and Bax immunoreactivity increased. The follicular ultrastructure showed marked distortion. These changes were mitigated upon PG treatment. In conclusion, PG alleviated the morphological and functional changes in the thyroid glands induced by LC by modulating apoptosis and oxidative stress.

Immunohistochemical assessment and clinical, histopathologic, and molecular correlates of membranous somatostatin type-2A receptor expression in high-risk pediatric central nervous system tumors.

177Lu-DOTATATE, a radionuclide therapy that binds somatostatin type-2A receptors (SST2A), has demonstrated efficacy in neuroendocrine tumors and evidence of central nervous system (CNS) penetration, supporting potential expansion within pediatric neuro-oncology. Understanding the prevalence of SST2A expression across pediatric CNS tumors is essential to identify patients who may benefit from somatostatin receptor-targeted therapy and to further elucidate the oncogenic role of SST2A. SST2A immunohistochemistry (IHC) was performed on tumor specimens and interpreted by an experienced pathologist (blinded), utilizing semi-quantitative scoring of membranous expression within viable tumor. Immunoreactive cell percentage was visually scored as 0 (none), 1 (<10%), 2 (10-50%), 3 (51-80%), or 4 (>80%). Staining intensity was scored as 0 (none), 1 (weak), 2 (moderate), or 3 (strong). Combined scores for each specimen were calculated by multiplying percent immunoreactivity and staining intensity values (Range: 0-12). A total of 120 tumor samples from 114 patients were analyzed. Significant differences in SST2A IHC scores were observed across histopathologic diagnoses, with consistently high scores in medulloblastoma (mean ± SD: 7.5 ± 3.6 [n=38]) and meningioma (5.7 ± 3.4 [n=15]), compared to minimal or absent expression in ATRT (0.3 ± 0.6 [n=3]), ETMR (1.0 ± 0 [n=3]), ependymoma (grades I-III; 0.2 ± 0.7 [n=27]), and high-grade glioma (grades III-IV; 0.4 ± 0.7 [n=23]). Pineoblastoma (3.8 ± 1.5 [n=4]) and other embryonal tumors (2.0 ± 4.0 [n=7]) exhibited intermediate, variable expression. Among medulloblastomas, SST2A IHC scores were higher in non-SHH (8.5 ± 3.1) than SHH (5.0 ± 3.3) molecular subgroups (p=0.033). In a subset of paired primary and recurrent specimens from four patients, SST2A IHC scores remained largely unchanged. High membranous SST2A expression was demonstrated in medulloblastoma, meningioma, and some rarer embryonal tumors with potential diagnostic, biologic, and therapeutic implications. Somatostatin receptor-targeted therapy such as 177Lu-DOTATATE deserves further investigation in these highly SST2A-expressing pediatric CNS tumors.

Histochemical assessment of accelerated bone remodeling and reduced mineralization in Il-6 deficient mice

ObjectivesInterleukin-6 (IL-6) contributes to the regulation of functions in various tissues and organs. Even though IL-6 has been reported to modulate bone metabolism in previous studies, this finding is controversial. This study aims to evaluate the possible involvement of IL-6 in bone metabolism by examining the histological activity of osteoblasts and osteoclasts in the femora of Il-6 deficient (Il-6−/−) mice. MethodsEight-week-old male Il-6−/− mice and their wild-type littermates were fixed with a paraformaldehyde solution, and their femora were extracted for micro-CT analysis, immunohistochemistry, and real-time PCR analysis. ResultsIl-6−/− femora showed an increased bone volume/tissue volume (TV) but a reduced bone mineral density compared with the wild-type. Furthermore, the tissue-nonspecific alkaline phosphatase positive area/TV ratio, the expression of Runx2, Osterix, and Rankl, and the number of tartrate-resistant acid phosphatase-positive osteoclasts were all increased in the Il-6−/− mice. A considerable number of unmineralized areas within the bone matrix and abundant sclerostin-reactive osteocytes were observed in Il-6−/− femoral metaphyses but not in the wild-type. Interestingly, the gene expression of Cd206 was elevated in Il-6−/− femora, and many F4/80-positive macrophages/monocytes and CD206-immunoreactive macrophages in the primary trabeculae had migrated closer to the growth plate, where intense RANKL immunoreactivity was detected. These results suggest that, in an IL-6-deficient state, CD206-positive macrophages may differentiate into osteoclasts when in contact with RANKL-reactive osteoblastic cells. ConclusionIn a state of IL-6 deficiency, the population and cell activities of osteoblast, osteoclasts, and macrophages seemed to be facilitated, except for the reduced mineralization in bone.

Generation of monoclonal antibodies against heterologously expressed major capsid protein of infectious spleen and kidney necrosis virus (ISKNV)

Infectious spleen and kidney necrosis virus (ISKNV) is one of the detrimental viruses causing economic losses in several fish species including Asian sea bass (Lates calcarifer). In this study, monoclonal antibodies specific to major capsid protein (MCP) of ISKNV were generated. The MCP encoding gene of ISKNV genotype II was cloned into pGEX-6P-1 and transformed into E. coli. After induction, a glutathione-S-transferase (GST)-MCP fusion protein was produced and used for immunization of Swiss mice for monoclonal antibody (MAb) production. Five MAbs specific to MCP were obtained after selection against naturally ISKNV-infected Asian sea bass by dot blotting, Western blotting and immunohistochemistry. All MAbs showed no cross-reaction with fish tissues, fish viruses including tilapia lake virus (TiLV), nervous necrosis virus (NNV), scale drop disease virus (SDDV) and eight bacterial species commonly found in diseased fish. The detection sensitivity of MAbs was inferior to that of one step PCR. However, a combination of two MAbs enhanced the detection limit about two-fold. The intense immunoreactivity staining could be observed in liver, kidney, gill, intestine, stomach, and spleen tissues from ISKNV-infected fish by immunohistochemical analysis. All five MAbs can be used to detect naturally ISKNV-infected fish from two different farms and phylogenetic tree analysis based on MCP gene sequences demonstrated ISKNV genotype I infection in diseased fish. In addition, all MAbs displayed immunoreactivity to red sea bream iridovirus (RSIV) in the GF cell culture fluid by dot blotting. Therefore, the generated MAbs could be used for detection of ISKNV infection by various immunological assays and might be able to detect RSIV infection as well.