Intensity Of Immunostaining Research Articles

E-cadherin as a Prognostic Biomarker in Oral Squamous Cell Carcinoma: A Pilot Study at Tertiary Care Hospital

Introduction: In a recent survey of cancer mortality in India, oral cavity cancer was listed as the leading cause of mortality in men, causing 29% of cancer-related deaths. Objective: The aim is to investigate the expression of E-cadherin to elucidate its role in determining the biological activity of the disease as a reliable and potential marker. Materials and Methods: E-cadherin immune-reactivity was evaluated in ten randomly selected fields in consecutive 50 cases diagnosed with oral squamous cell carcinoma (OSCC) and in each case, its mean was taken for the evaluation of E-cadherin expressing cells. The distribution and intensity of immunostaining E cadherin were also semi-quantitatively evaluated. Results: The most prevalent age group involved was the fifth decade and alveolus was the main site. The most common histological subtype was found to be well-differentiated squamous cell carcinoma and stage IV was the most common subtype in our study. The P value was significant for association between differentiation of tumor, staining location, and expression of E-cadherin (P value-0.002). E cadherin expression was compared with mortality; we found five out of seven death cases showing strong E cadherin expression, however, this was not statistically significant. Conclusions: Clinical and histopathological characteristics of malignancy, such as metastasis, recurrence, low survival, and poor tumor differentiation, have been associated with low expression of E-cadherin in OSCC.

C-FOS Expression Analysis in Pterygia Cell Spot Arrays.

Mechanisms of c-FOS activation in the onset and progression of pterygia remain under investigation. This study aimed to comparatively analyze c-FOS proto-oncogene expression levels in neoplastic pterygia and normal epithelia. We used a liquid-based cytology assay on thirty (n=30) pterygia cell populations and normal epithelia (n=10) extracted by a smooth scraping of conjunctiva epithelia. Applying a cell spot-based technique, we constructed five (n=5) slides, each containing eight (n=8) cell spots. A modified immune-cytochemistry (ICC) assay for c-FOS protein was used. Additionally, digital image analysis was implemented to calculate c-FOS immunostaining intensity levels. High staining intensity levels of c-FOS were detected in 12/30 (40%), whereas the rest 18/30 (60%) demonstrated moderate expression. c-FOS levels were statistically significantly higher in the pterygia compared to control tissues (p=0.001). c-FOS levels in the pterygia were not associated with the sex of patients (p=0.678), the presence of recurrent lesion (p=0.390) or the location of the lesion (p=0.158). The levels of c-FOS, however, were marginally significantly affected by the morphology of the pterygia (p=0.005). More precisely, the c-FOS levels were significantly higher in pterygia with a fleshy morphology. c-FOS over-expression is observed frequently in pterygia. It seems to be critically involved in the molecular mechanism of the lesion by its over-expression affecting partially their morphological features. Cell spot liquid - based array analysis is an innovative, easy to implement technique for simultaneously evaluating a broad spectrum of molecules in multiple specimens on the same slide.

There is reduced immunohistochemical staining of placental aromatase in severe neonatal opioid withdrawal syndrome

Background Placental cytochrome p450 (CYP450) enzymes and efflux transporters, P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), are critical for transfer of drugs from the placenta to maternal circulation. CYP19A1 (aromatase) is the enzyme responsible for metabolizing methadone and buprenorphine in the human placenta. Objective We sought to determine if differences exist in CYP19A1 and efflux transporter immunostaining intensity and density within the syncytiotrophoblast in opioid-exposed and unexposed pregnancies. Additionally, we sought to investigate whether CYP19A1 and efflux transporter expression was different in placentas of infants who developed severe neonatal opioid withdrawal syndrome (NOWS) and those who did not. Study design This was a retrospective nested case control study from 2014 to 2019 at a single tertiary care center. The opioid-exposed cohort included pregnant women aged ≥18 years on maintenance methadone or buprenorphine with non-anomalous singleton fetuses and gestational age ≥33 weeks. Controls included pregnant women with no medication exposure delivering at ≥37 weeks. De-paraffinized placental sections, inclusive of the apical syncytiotrophoblast membrane, were labeled with monoclonal antibodies for aromatase, P-gp, and BCRP. Placentas were scored for the presence and intensity of staining using the Allred scoring schema. Data were analyzed using descriptive, parametric, and nonparametric statistics. p < .05 was considered significant. Results One hundred and ten opioid-exposed neonates were included in this analysis (51 opioid-exposed cases and 59 opioid-exposed controls), with 68/110 delivering at term. Ten unexposed controls delivering at term were also included. The median placental Allred scores for aromatase were significantly lower in the opioid-exposed cohort compared with the unexposed controls (exposed 6.8 ± 1.4 vs. unexposed 7.5 ± 0.7, p = .03). The median placental Allred scores for aromatase were significantly lower in opioid-exposed cases that developed severe NOWS compared to opioid-exposed controls (p = .03) that did not develop severe NOWS. There were no differences in P-gp and BCRP scores between groups. Conclusions Syncytiotrophoblast aromatase immunostaining scores were reduced in opioid-exposed cases compared to unexposed controls. Additionally, infants who developed severe NOWS had significantly lower placental aromatase in the apical syncytiotrophoblast compared with those without severe NOWS.

Nonspecific binding of common anti-CFTR antibodies in ciliated cells of human airway epithelium

There is evidence that the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel is highly expressed at the apical pole of ciliated cells in human bronchial epithelium (HBE), however recent studies have detected little CFTR mRNA in those cells. To understand this discrepancy we immunostained well differentiated primary HBE cells using CFTR antibodies. We confirmed apical immunofluorescence in ciliated cells and quantified the covariance of the fluorescence signals and that of an antibody against the ciliary marker centrin-2 using image cross-correlation spectroscopy (ICCS). Super-resolution stimulated emission depletion (STED) imaging localized the immunofluorescence in distinct clusters at the bases of the cilia. However, similar apical fluorescence was observed when the monoclonal CFTR antibodies 596, 528 and 769 were used to immunostain ciliated cells expressing F508del-CFTR, or cells lacking CFTR due to a Class I mutation. A BLAST search using the CFTR epitope identified a similar amino acid sequence in the ciliary protein rootletin X1. Its expression level correlated with the intensity of immunostaining by CFTR antibodies and it was detected by 596 antibody after transfection into CFBE cells. These results may explain the high apparent expression of CFTR in ciliated cells and reports of anomalous apical immunofluorescence in well differentiated cells that express F508del-CFTR.

CBIO-08. ENDOGENOUS DNA DOUBLE STRAND BREAKS ACTIVATE HETEROGENOUS DNA DAMAGE SIGNALING IN IDH1/2 MUTANT GLIOMAS

Abstract Isocitrate dehydrogenase 1/2 (IDH1/2) mutations are common in astrocytic glioma and are frequently coupled with TP53 and ATRX mutations. Collectively, these alterations cause genomic instability leading to high basal DNA double strand breaks (DSBs). Understanding how IDH/TP53/ATRX mutant cells process endogenous DSBs may help exploit inhibitors of DNA damage response (DDR) for the treatment of patients with IDH mutant gliomas. Through systematic effort to uncover the mechanisms involved in repair of endogenous DSBs in IDH1/2 mutant GBMs, we have discovered that high basal phosphorylated DNA-PK (p-DNA-PK) was characteristic of an IDH1/TP53/ATRX mutant GBM164 patient derived xenograft (PDX) but not in another IDH1 mutant GBM196 PDX. Immunofluorescence (IF) studies in patient specimen from which GBM164 was derived showed that p-DNA-PK co-localized with g-H2AX, 53BP1 or H4K20me2 (but not p-RPA) the known surrogates of DSBs. In contrast, p-DNA-PK was absent in the patient specimen from which GBM196 was derived, which otherwise had equally intense g-H2AX immunostaining colocalized with p-RPA. An independent IF study involving 11 IDH1 wild-type (WT) and 11 IDH1 mutant GBM patient samples, the p-DNA-PK was observed in 3 (27%) of 11 IDH1 mutant samples while IDH1 WT tumors were negative for p-DNA-PK. A telomere specific fluorescence in situ hybridization (Tel-FISH) confirmed elevated alternative lengthening of telomere (ALT) activity in GBM196 (but not in GBM164) indicative of HR proficiency. Consistently, HR related genes, including BRCA1 and MRE11A, were found upregulated in ALT-positive GBM196 as compared to those in GBM164. Interestingly, ALT+ GBM196 cells were highly vulnerable to inhibitors of ATM and ATR pathways. In conclusion, IDH1/TP53/ATRX mutant gliomas can be subdivided into HR-mediated ALT-positive group, which repairs the endogenous DSBs by HR (e.g. GBM196) and an ALT-negative/p-DNA-PK group, which repairs DSBs by c-NHEJ (e.g. GBM164) and this subdivision can be developed as a prescient biomarker of sensitivity to DDR inhibitors.

B Cells in the CNS at Postmortem Are Associated With Worse Outcome and Cell Types in Multiple Sclerosis.

Background and ObjectivesTo define the clinical and pathologic correlations of compartmentalized perivascular B cells in postmortem progressive multiple sclerosis (MS) brains.MethodsBrain slices were acquired from 11 people with secondary progressive (SP) MS, 5 people with primary progressive (PP) MS, and 4 controls. Brain slices were immunostained for B lymphocytes (CD20), T lymphocytes (CD3), cytotoxic T lymphocytes (CD8), neuronal neurofilaments (NF200), myelin (SMI94), macrophages/microglia (CD68 and IBA1), astrocytes (glial fibrillary acidic protein [GFAP]), and mitochondria (voltage-dependent anion channel and cytochrome c oxidase subunit 4). Differences in CD20 immunostaining intensity between disease groups and associations between CD20 immunostaining intensity and both clinical variables and other immunostaining intensities were explored with linear mixed regression models and Cox regression models, as appropriate.ResultsCD20 immunostaining intensity was higher in PPMS (Coeff = 0.410; 95% confidence interval [CI] = 0.046, 0.774; p = 0.027) and SPMS (Coeff = 0.302; 95% CI = 0.020, 0.585; p = 0.036) compared with controls. CD20 immunostaining intensity was higher in cerebellar, spinal cord, and pyramidal onset (Coeff = 0.274; 95% CI = 0.039, 0.510; p = 0.022) compared with optic neuritis and sensory onset. Higher CD20 immunostaining intensity was associated with younger age at onset (hazard ratio [HR] = 1.033; 95% CI = 1.013, 1.053; p = 0.001), SP conversion (HR = 1.056; 95% CI = 1.022, 1.091; p = 0.001), wheelchair dependence (HR = 1.472; 95% CI = 1.108, 1.954; p = 0.008), and death (HR = 1.684; 95% CI = 1.238, 2.291; p = 0.001). Higher immunostaining intensity for CD20 was associated with higher immunostaining intensity for CD3 (Coeff = 0.114; 95% CI = 0.005, 0.224; p = 0.040), CD8 (Coeff = 0.275; 95% CI = 0.200, 0.350; p < 0.001), CD68 (Coeff = 0.084; 95% CI = 0.023, 0.144; p = 0.006), GFAP (Coeff = 0.002; 95% CI = 0.001, 0.004; p = 0.030), and damaged mitochondria (Coeff = 3.902; 95% CI = 0.891, 6.914; p = 0.011).DiscussionPerivascular B cells were associated with worse clinical outcomes and CNS-compartmentalized inflammation. Our findings further support the concept of targeting compartmentalized B-cell inflammation in progressive MS.

The Apelinergic System Immuno-Detection in the Abomasum and Duodenum of Sheep Grazing on Semi-Natural Pasture.

Simple SummaryThe semi-natural pastures in the Apennines represent the feed source for ovine, whose grazing activity helps to preserve the grassland’s biodiversity. Summer drought stress decreases the grassland pastoral value and affects the morpho-functional features of sheep’s digestive systems. A better knowledge of the gastrointestinal system of sheep may contribute to guaranteeing their welfare, a prerequisite for the sustainability of livestock production. This study aimed to immune-localize the apelinergic system in the abomasum and duodenum of sheep grazing on semi-natural pasture during the spring–summer season and to compare its behavior among animal groups fed with or without supplementation. The apelinergic system, composed of apelin and its receptor, is involved in foodintake and the secretion and absorption activities of the digestive apparatus. Apelinergic system molecules were localized at the abomasum lining epithelium and fundic glands level and at the duodenum lining and crypt epithelium, in addition to the neuroendocrine cells. Variations in reactivity were observed in the different feed groups; feed supplementation seemed to maintain the functionality of the apelinergic system in the organs near the status related to the better pasture phase, suggesting that it may be a suitable solution able to counteract the harmful effects of summer drought stress.Apelin (APLN) is an adipokine mainly produced by adipose tissue and related to an individual’s nutritional status as well as digestive apparatus functions. In this work, APLN and its receptor (APLNR) were investigated, by immunohistochemistry, in the abomasum and duodenum of 15 Comisana × Appenninica adult sheep reared in a semi-natural pasture. Organ samples were collected after maximum pasture flowering (M × F group) and after maximum pasture dryness (M × D group); the experimental group (E × p group) received a feed supplementation of 600 grams/day/head of barley and corn in addition to M × D group feeding. APLN and APLNR were identified in the lining epithelium and the fundic gland chief cells of the abomasum. APLNR was observed in the lining epithelium, in the crypts and the serotonin secreting cells of the duodenum. Similar reactivity was observed between the M × F and E × p groups, while the M × D group showed a lower intensity of immunostaining for both APLN and APLNR in all positive structures but the duodenal serotonin neuroendocrine cells. Hence, our findings show that the E × p group presents a picture quite overlapped with M × F and suggest that food supplementation has a maintaining effect on the apelinergic system expression in the investigated digestive tracts of the sheep.

Aluminum Chloride-Induced Reproductive Toxicity in Rats: the Protective Role of Zinc Oxide Nanoparticles.

Reproductive toxicity is a major challenge associated with aluminum (Al) exposure. Therefore, this study aimed to investigate the effects of zinc oxide nanoparticle (ZnONP) treatment on Al-induced reproductive toxicity in rats. Thirty-two adult male albino rats were allocated into four equal groups as follows: control, AlCl3 orally administered group (100mg/kg bwt), ZnONPs injected intraperitoneally (i.p.) group (4mg/kg bwt), and ZnONPs + AlCl3-treated group. The treatment was daily extended for 42 consecutive days. Oral administration of AlCl3 showed an oxidative damage confirmed by an increase in malondialdehyde and nitric oxide levels and superoxide dismutase activity and accompanied by a decrease in glutathione content and catalase activity. Also, AlCl3 administration increased the pro-inflammatory mediator tumor necrosis factor-alpha. Furthermore, significant declines in the levels of serum male reproductive hormones testosterone, luteinizing hormone, and follicle-stimulating hormone in AlCl3-intoxicated rats were noticed. In parallel, severe histopathological alterations were observed in testis tissues. Additionally, the immunohistochemical analysis showed that AlCl3 administration potentiates cell death in the testicular tissue by elevating the immunostaining intensity signal for the pro-apoptotic protein, cysteinyl aspartate specific protease-3 (caspase-3) and a marked depletion in the cell proliferation expression marker, Ki-67, in germinal cells of AlCl3-treated group. On the other hand, the daily i.p. injection to rats with ZnONPs before AlCl3 was found to ameliorate the reproductive toxicity induced by Al administration through reducing the testicular oxidative stress and improving the inflammatory, apoptotic, and reproductive markers as well as histopathological alterations in the testis. These results suggest that ZnONPs could be used as an alternative agent to minimize the reproductive toxicity associated with Al exposure through its antioxidant, anti-inflammatory, anti-apoptotic, and reproductive modulatory activities.

Assessment of testicular steroidogenic enzymes expression in experimental animal model following withdrawal of nandrolone decanoate.

Anabolic steroids are frequently used to increase the growth rate of meat-producing animals. Exposure to an anabolic-androgenic steroid, nandrolone decanoate (ND), is associated with expressional reduction of testicular steroidogenic enzymes. However, the effect of withdrawal of ND exposure on the expression of these testicular molecules has not been thoroughly explored. The current research investigated expression changes of testicular steroidogenic enzymes in rats at several recovery periods (2, 6, and 12 weeks) after the stop of ND treatment with different doses (2 and 10 mg/kg body weight) for 12 weeks. Body and testis weights were recorded, and transcript levels of molecules were determined by quantitative real-time polymerase chain reaction (PCR). The immunohistochemistry was used to examine the changes of immuno-intensities of molecules. At 6 and 12 weeks of the recovery period, the 10 mg/kg ND-treated rats were lighter than other experimental groups. The interstitial compartment vanished by ND treatment filled up as the recovery period became longer. The expression of steroidogenic acute regulatory protein was returned to the control level at 12 weeks of the recovery period. Expression levels of cytochrome P450 side-chain cleavage and 17a-hydroxylase were increased in 2 mg/kg ND-treated group at 6 weeks of the recovery period, and transcript levels of these molecules in 2 and 10 mg/kg ND-treated groups at 12 weeks of the recovery period were significantly lower than the control. Expression levels of 3β-hydroxysteroid dehydrogenase (HSD) type I and 17β-HSD type 3 in 2 mg/kg ND-treated group were comparable with those of control at 12 weeks of the recovery period, but not in 10 mg/kg ND-treated group. Expression of cytochrome P450 aromatase (Cyp19) was reverted to the control level at 2 weeks of the recovery period. Except for Cyp19, there was a visible increase of immuno-staining intensity of other testicular steroidogenic enzymes in the Leydig cells as the recovery period progressed. This research has demonstrated that the cease of ND administration could restore the expression of testicular steroidogenic enzymes close to the normal level. Nevertheless, a relatively long recovery period, compared to the ND-exposure period would be required to retrieve normal expression levels of testicular steroidogenic enzymes.

The Role of Bone Grafts in Preventing Medication-Related Osteonecrosis of the Jaw: Histomorphometric, Immunohistochemical, and Clinical Evaluation in Animal Model.

To evaluate the effects of inorganic bovine bone graft (Lumina Bone, Criteria, Brazil) and beta-tricalcium phosphate (β-TCP) graft (ChronOS, Synthes, Brazil) in rats with the risk of developing post-extraction medication-related osteonecrosis of the jaw (MRONJ). Eighteen male Wistar rats weighing 350 to 450 g were induced to develop MRONJ using zoledronic acid for 5 weeks. In the sixth week, the right maxillary first molar was extracted. The animals in Group I (G1) did not receive bone grafts after tooth extraction, while Group II (G2) animals received inorganic bovine bone grafts, and Group III (G3) animals received beta-tricalcium phosphate (β-TCP) grafts. Clinical evaluation and histomorphometric and immunohistochemical analyses were performed. ANOVA and Tukey's statistical tests were used and a level of significance was considered to be 5%. In the clinical evaluation, animals from G2 and G3 did not present clinical manifestations of osteonecrosis, unlike the control group (G1) animals, which presented necrotic bone tissue exposure in all samples. In the histomorphometric evaluation, animals in G3 showed greater formation of bone tissue (66%) and less formation of bone lacuna (18%) than animals in G1 (58%/32%) and in G2 (59%/27%) (P < 0.05). Moderate (++) immunostaining was observed in G2 and G3 for RANKL, TRAP, and OC, while G1 showed moderate (++) labeling for OC and mild (+) immunostaining for TRAP and RANKL. Greater formation of bone tissue and fewer bone lacunae were found in animals treated with β-TCP. In clinical evaluation, bone graft groups presented with the clinical manifestation of MRONJ and showed higher intensity of immunostaining for TRAP and RANKL. Despite the limitations of experimental animal studies, the results of this work may assist in the development of future clinical research for the prevention of MRONJ.

Transcriptomic Analysis of STAT1/3 in the Goat Endometrium During Embryo Implantation.

Interferon tau (IFNT), a pregnancy recognition signal in ruminants, promotes the establishment of embryo implantation by inducing the expression of interferon-stimulated genes (ISGs) via the Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling pathway. However, the precise regulatory mechanism of IFNT in goat embryo implantation remains largely unknown. In this study, we performed RNA sequencing of goat endometrial epithelial cells (gEECs) with or without 20 ng/mL IFNT treatment. Differential comparison showed that there were 442 upregulated differentially expressed genes (DEGs) and 510 downregulated DEGs. Bioinformatic analyses revealed that DEGs were significantly enriched in immune-related functions or pathways. The qRT-PCR validation results showed that the expression levels of STAT family members (STAT1, STAT2, and STAT3) were significantly upregulated in gEECs after IFNT treatment, which is in agreement with the RNA-seq data. Meanwhile, the protein levels of p-STAT1 and p-STAT3 increased significantly in gEECs after 6 and 24 h of IFNT treatment, respectively. Further in vivo experiments also confirmed that both mRNA and protein phosphorylation levels of STAT1 and STAT3 in the uterus on day 18 of pregnancy (P18) were significantly increased compared to those on day 5 (P5) and day 15 of pregnancy (P15). On P5, STAT1 and STAT3 proteins were primarily located in the uterine luminal epithelium (LE) and glandular epithelium (GE), and were also detected in the stromal cells. The intense immunostaining of STAT1 and STAT3 proteins were decreased on P15 and then increased on P18, especially in the superficial GE and subepithelial stromal cells. Moreover, p-STAT1 and p-STAT3 were highly expressed in the deep GE on P18. Collectively, these results highlight the role of IFNT in regulating endometrial receptivity in gEECs and uncover the temporal and spatial changes in the expression of STAT1/3 during embryo implantation in the goat endometrium.

The immunoexpression of epidermal growth factor receptor in cutaneous squamous cell carcinoma.

Although cutaneous squamous cell carcinomas (cSCCs) account for only 20–25% of non-melanoma skin cancers (NMSCs), they are responsible for most deaths attributable to NMSCs. Apart from SCC seric level, which increases in late-stage disease, no other predictive biomarker for cSCC exists. Epidermal growth factor receptor (EGFR) serves as a predictive biomarker and therapeutic target in numerous malignancies. EGFR immunoexpression is highly elevated in head and neck mucosal SCC. However, its immunoexpression pattern, its relationship with prognosis and survival, and the effect of EGFR targeted therapy in advanced cSCC have not been clarified. We assessed EGFR immunoexpression in 18 cases of cSCC and correlated our findings with the clinicopathological features. Immunohistochemical stainings with anti-EGFR monoclonal antibodies were practiced and the membrane and cytoplasmic immunostaining intensity and quality in the tumors and the non-lesional epithelium were analyzed. Membrane EGFR immunoexpression within the tumors increased with the tumor grade. EGFR overexpression was more frequently found in head and neck cSCCs. We did not find a direct relationship between cytoplasmic EGFR immunoexpression and clinicopathological findings and prognosis. Our results confirm that increased EGFR immunoexpression correlates with aggressive cSCC phenotypes and underline the need for novel treatments for these patients.

Probiotic supplementation affects IGF-1 and leptin levels in Nile tilapia hepatopancreatic tissue

ABSTRACT This work aimed to assess the effect of the probiotic strain, Lactobacillus plantarum, on the levels of leptin, IGF-1 and their receptors on the hepatopancreatic tissues of Nile tilapia (Oreochromis niloticus) and then correlate fish growth performance and gut microbiological parameters. Fish juveniles (±23g) were reared in a recirculation system with constant aeration and temperature (25°C). They were distributed into six polyethylene tanks (45L) and fed twice a day at 5% of the tank biomass with the respective diets: control (commercial diet without probiotic) and supplemented with L. plantarum inoculum (1 x 108 CFU mL-1), both in triplicate. After 30 days of feeding, L. plantarum-fed fishes showed greater weekly growth rate, final weight, and feed conversion rate, in addition to higher count of lactic-acid bacteria and lower count of pathogenic bacteria in the intestinal tract, when compared to the control group. The immunostaining intensity for IGF-1 and leptin hormones was lower after L. plantarum supplementation than in the control group, with no change in the level for receptors. This reduction could implicate important changes in fish metabolism and homeostasis.

Altered Hypoxia-Induced and Heat Shock Protein Immunostaining in Secondary Hair Follicles Associated with Changes in Altitude and Temperature in Tibetan Cashmere Goats.

Simple SummaryCashmere goats in Tibet are adapted to a high altitude, cold climate, high solar radiation, and hypoxia. The aim of the present study was to compare the morphology of the secondary hair follicles and immunostaining of hair follicle regulatory proteins in Tibetan cashmere goats from a high altitude and low temperature (Rikaze) to goats from a lower altitude and comparatively warm temperature (Huan). We conclude that, at the same time of the year, the secondary hair follicles were at different development stages. HIF-1a protein immunostaining in the inner root sheath (IRS) and hair shaft (HS) was higher than the immunostaining in the outer root sheath (ORS). In contrast, immunostaining for HIF-2a protein in the ORS and IRS was higher than that present in the HS. Immunostaining for HIF-3a protein was higher in the ORS than the IRS while HOXC13 protein immunostaining was higher in the ORS than the IRS and HS. Immunostaining in secondary hair follicles for HIF-1a, HIF-2a, and HSP27 protein in the cashmere goats living in Rikaze was significantly higher than that in the secondary hair follicles of cashmere goats from Huan. In contrast, HOX13 protein immunostaining was significantly higher in cashmere goats from Huan than from Rikaze. These results are useful in understanding how altitude and temperature influence secondary hair follicle development.This experiment compared secondary hair follicles (SFs) in Tibetan cashmere goats from two different steppes that were at different altitudes and had different temperatures. Twenty-four 2-year-old goats were studied. Twelve goats were from Rikaze in Tibet which is at an altitude of above 5000 m with an average temperature of 0 °C. The other 12 studied goats were from Huan County of Gansu Province which is around 2000 m above sea level with an average temperature of 9.2 °C. The structural features of SFs were assessed using light microscopy and transmission electron microscopy. The presence of HIF-1a, HIF-2a, HIF-3a, HSP27, and HOXC13 proteins was studied using immunohistochemistry and immunofluorescence. Light and electron microscopy revealed that the SFs of the Tibetan cashmere goats that lived in the Rikaze Steppe were in the proanagen stage in May. However, the SFs of the goats from the lower warmer Huan County were in the anagen stage at the same time. Immunohistochemistry revealed intense immunostaining for HIF-1a protein in the inner root sheath (IRS) and hair shaft (HS); immunostaining against HIF-2a in the outer root sheath (ORS) and IRS; HIF-3a protein immunostaining in the ORS; HSP27 immunostaining in the ORS, IRS, and HS; and HOXC13 immunostaining in the ORS and HS. HIF-1a protein expression in the IRS and HS was higher than the expression in the ORS (p < 0.05) while the expression of HIF-2a protein was higher in the ORS and IRS than the HS (p < 0.05). The expression of HIF-3a protein was higher in the ORS than in the IRS (p < 0.05). Expression of HOXC13 protein was higher in the ORS than in the IRS and HS (p < 0.05). Immunostaining of HIF-1a, HIF-2a, and HSP27 protein was significantly higher in SFs from cashmere goats from Rikaze than in goats from Huan (p < 0.05). In contrast, HOX13 protein immunostaining was significantly higher in cashmere goats from Huan than from Rikaze (p < 0.05). Significant differences were observed in the SFs of cashmere goats from two locations that differ in altitude and temperature. This suggests the differences in the secondary hair follicles could be due to the hypoxia and lower temperatures experienced by the goats in Rikaze. These results are useful in understanding how altitude and temperature influence SF development. Hair produced by the SFs are used for down fiber. Therefore, understanding of the factors that influence SF development will allow the production and harvest of these valuable fibers to be maximized.

Cortical Serotonergic and Catecholaminergic Denervation in MPTP-Treated Parkinsonian Monkeys.

Decreased cortical serotonergic and catecholaminergic innervation of the frontal cortex has been reported at early stages of Parkinson's disease (PD). However, the limited availability of animal models that exhibit these pathological features has hampered our understanding of the functional significance of these changes during the course of the disease. In the present study, we assessed longitudinal changes in cortical serotonin and catecholamine innervation in motor-symptomatic and asymptomatic monkeys chronically treated with low doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). Densitometry and unbiased stereological techniques were used to quantify changes in serotonin and tyrosine hydroxylase (TH) immunoreactivity in frontal cortices of 3 control monkeys and 3 groups of MPTP-treated monkeys (motor-asymptomatic [N = 2], mild parkinsonian [N = 3], and moderate parkinsonian [N = 3]). Our findings revealed a significant decrease (P<0.001) in serotonin innervation of motor (Areas 4 and 6), dorsolateral prefrontal (Areas 9 and 46), and limbic (Areas 24 and 25) cortical areas in motor-asymptomatic MPTP-treated monkeys. Both groups of symptomatic MPTP-treated animals displayed further serotonin denervation in these cortical regions (P<0.0001). A significant loss of serotonin-positive dorsal raphe neurons was found in the moderate parkinsonian group. On the other hand, the intensity of cortical TH immunostaining was not significantly affected in motor asymptomatic MPTP-treated monkeys, but underwent a significant reduction in the moderate symptomatic group (P<0.05). Our results indicate that chronic intoxication with MPTP induces early pathology in the corticopetal serotonergic system, which may contribute to early non-motor symptoms in PD.

Структурна трансформація протокової системи підшлункової залози у хворих на хронічний панкреатит

Актуальність. Клітини проток становлять до 30 % від маси підшлункової залози (ПЗ) людини, а збільшення кількості бранчів проток відноситься до одних із перших сонологічних ознак хронічного панкреатиту (ХП). Мета дослідження — визначення морфологічних й імуногістохімічних особливостей протокової системи ПЗ хворих на ХП, яким були виконані резекційні та дренуючі операції з приводу ускладнень хронічного панкреатиту. Матеріали та методи. Проведено імуногістохімічне дослідження операційного матеріалу 40 хворих з ускладненим хронічним панкреатитом з використанням реакцій на a-ізоформу гладком’язового актину (a-SMA), десмін, фібронектин й колаген I типу. Результати. Характерна морфологічна картина розвитку ХП є поєднанням фіброзування з деформацією протокової системи за рахунок перидуктального фіброзу з розширенням міждолькових фіброзних септ, потовщенням інтралобулярних сполучно­тканинних тяжів, а також значною звивістістю і розширенням проток з ретенційними кістами. Значна структурна перебудова протокової системи ПЗ у хворих на ХП спостерігалася в 87,5 % випадків, в 17,5 % випадків картина доповнювалася явищами ацинарно-протокової метаплазії з трансформацією ацинарних клітин у протокові і формуванням дрібних дуктулоподібних структур, так званих тубулярних комплексів. У 7,5 % випадків в головній панкреатичній протоці визначалися вогнища атрофії протокового епітелію, які чергувалися з вогнищами папілярної інтраепітеліальної гіперплазії епітелію (PanIN). Розвитку панкреатичного фіброзу при ХП сприяє накопичення a-SMA+-активованих панкреатичних зірчастих клітин, що синтезують надлишок компонентів екстрацелюлярного матриксу, зокрема колагену I типу. Середня площа колагену І типу та інтенсивність його експресії були вірогідно нижче при зниженій експресії a-SMA (p &lt; 0,05) і вірогідно вище у випадках вираженої експресії фібронектину (p &lt; 0,05). Висновки. Вивчення структурних змін протокової системи ПЗ з використанням методів імуногістохімії є одним із способів покращення ранньої діагностики ускладнень ХП.

Efficacy of expressions of Arg-1, Hep Par-1, and CK19 in the diagnosis of the primary hepatocellular carcinoma subtypes and exclusion of the metastases.

A paraffin-embedded block including 62 cases of primary HCC, and 18 cases diagnosed as metastatic tumors, were subjected for this study using Anti-liver Arginase antibody (ab125134 Cambridge, USA, polyclonal antibody, 3.75 µg/ml), HepPar-1 (polyclonal mouse antibody OCH1E5; 1:600; DAKO, CA, USA), and CK 19 Anti-Cytokeratin 19 antibody (ab15463, rabbit polyclonal antibody; 1:100; Cambridge, USA). The intensity of immunostaining was scored (0 to 3+). Nuclear and cytoplasmic staining with Arg-1 and cytoplasmic for both HepPar-1 and CK 19 are reported. The histopathological patterns were mainly trabecular no= (24, 38.7%), and pseudoglandular (no=14, 22.5%), mixed hepatocellular cholangiocarcinoma was observed in one case (1.6%). Arginase-1 positivity was in 55 cases (88.7%) opposite to 46 (74.19%) and 8 (12.9%) for HepPAr.1% -1 and CK 19, respectively. The intensity of expression was marked in well and moderate differentiation for Arg-1 and HepPar-1and in poorly differentiated for CK 19. Metastatic carcinoma cases revealed two cases positive for Arg-1 (11.1%), 4 cases (22.2%) positive for HepPar-1, and 13 cases (72.2%) positive for CK 19. Arg-1 and HepPar-1 are confirmative in the diagnosis of primary HCC in most cases, either separately or collectively but the priority of selection leans more towards Arg-1. Arg-1 and HepPar-1 positive with negative CK 19 expressions give more support to diagnosis of primary HCC while the reverse will support the diagnosis of tumour of biliary origin or liver metastasis.

Immunolocalization of stem/progenitor cell biomarkers Oct-4, C-kit and Musashi-1 in endometriotic lesions.

Human endometrium harbors stem/progenitor cells (SPCs) that may contribute to the establishment of endometriosis when seeded outside the uterus. Oct-4, C-kit and Musashi-1 are some of the many proteins used to characterize SPCs, but their association with endometriosis is uncertain. In this study, specimens of normal endometrium (n = 12), eutopic endometrium from women with endometriosis (n = 9), superficial peritoneal endometriosis (SUP, n = 12) and deep endometriosis (DE, n = 13) lesions were evaluated for localization and intensity of immunostaining for Oct-4, C-kit and Musashi-1. The three markers were abundantly expressed in normal endometrium, eutopic endometrium from endometriosis patients, SUP and DE specimens. Oct-4 and C-kit expression did not vary across groups as regards intensity or frequency. C-kit staining signal was seldom detected in vascular endothelium of normal or eutopic endometrium from endometriosis patients; however, it was positive in 67% of the SUP lesions and in 25% of the DE lesions (p = 0.042). Musashi-1 was expressed in some endometriotic glands as cell clusters, but its signal was similar between the four types of tissue (p = 0.971) CONCLUSION: The wide distribution of Oct-4, C-kit and Musashi-1 in endometria of patients with and without endometriosis and in SUP and DE endometriotic lesions suggests that these markers are not suitable for the in situ characterization of endometrial SPCs and should not be taken as surrogates for the study of SPCs in the pathogenesis of endometriosis.

The AAV9 Variant Capsid AAV-F Mediates Widespread Transgene Expression in Nonhuman Primate Spinal Cord After Intrathecal Administration.

Intrathecal delivery of AAV9 into the subarachnoid space has been shown to transduce spinal cord and brain and be less affected by preexisting antibodies, which are lower in cerebral spinal fluid. Still, efficiency of transduction needs to be improved. Recently, we identified a new capsid from a library selection in mice, called AAV-F, that allowed robust transduction of the spinal cord gray matter after lumbar injection. In this study, we test transduction of spinal cord by AAV-F (n = 3) compared to AAV9 (n = 2), using a reporter gene, in cynomolgus monkeys after lumbar intrathecal injection. Using an automated image analysis (IA) approach to sensitively quantitate reporter gene expression in spinal cord, we found that AAV-F capsid mediated slightly higher transgene expression (both in percentages of cells and intensity of immunostaining) in motor neurons and interneurons, in the lumbar and thoracic regions, compared to AAV9. Interestingly, although AAV-F mediated higher transgene expression in spinal cord, the number of genomes in spinal cord and periphery were on average lower for AAV-F than AAV9, which suggest that lower numbers of genomes were able to mediate higher transgene expression in spinal cord with this capsid. In contrast, dorsal root ganglion transduction efficiency was lower for AAV-F compared to AAV9 on average. Interestingly, we also observed transduction of Schwann cells in sciatic nerve in two nonhuman primates injected with AAV-F, but none with AAV9. Overall, our data demonstrate the utility of automated IA for quantitation of AAV transduction in the spinal cord and the favorable on-target:off-target transduction profile suggests that the AAV-F capsid be considered for gene therapy applications focused on treating the spinal cord after intrathecal delivery.

HDAC8 drives spindle organization during meiotic maturation of porcine oocytes.

ObjectivesHistone deacetylase 8 (HDAC8) is one of the class I HDAC family proteins, which participates in the neuronal disorders, parasitic/viral infections, tumorigenesis and many other biological processes. However, its potential function during female germ cell development has not yet been fully understood.Materials and methodsHDAC8‐targeting siRNA was microinjected into GV oocytes to deplete HDAC8. PCI‐34051 was used to inhibit the enzyme activity of HDAC8. Immunostaining, immunoblotting and fluorescence intensity quantification were applied to assess the effects of HDAC8 depletion or inhibition on the oocyte meiotic maturation, spindle/chromosome structure, γ‐tubulin dynamics and acetylation level of α‐tubulin.ResultsWe observed that HDAC8 was localized in the nucleus at GV stage and then translocated to the spindle apparatus from GVBD to M II stages in porcine oocytes. Depletion of HDAC8 led to the oocyte meiotic failure by showing the reduced polar body extrusion rate. In addition, depletion of HDAC8 resulted in aberrant spindle morphologies and misaligned chromosomes due to the defective recruitment of γ‐tubulin to the spindle poles. Notably, these meiotic defects were photocopied by inhibition of HDAC8 activity using its specific inhibitor PCI‐34051. However, inhibition of HDAC8 did not affect microtubule stability as assessed by the acetylation level of α‐tubulin.ConclusionsCollectively, our findings demonstrate that HDAC8 acts as a regulator of spindle assembly during porcine oocyte meiotic maturation.