Increase In Staining Intensity Research Articles

Staining patterns of PNA and UEA-I lectins in the postnatal developing male genital excurrent duct epithelium in mice.

The research was aimed at determining the staining patterns of Peanut agglutinin (PNA) lectin and Ulex europaeus agglutinin I (UEA-I) lectin in postnatal developing (day 1 to 8 weeks) male genital excurrent duct epithelium in mice. Lectin staining was performed on testis and epididymis of 1-day-old and 1-, 2-, 3-, 4-, 5-, 6-, and 8-week-old ICR mice. Rete testis epithelia were UEA-I- and PNA-negative at all postnatal ages. PNA lectin unclearly stained the epithelial apical surface in efferent ductules (ED) and ductus epididymis up to 2 weeks, while UEA-I was negative in those epithelia. By contrast, at 3 weeks of age, PNA and/or UEA-I turned to clearly label epithelia in the excurrent duct system. At 5 weeks, efferent ductules epithelium was UEA-I-negative but strongly PNA-positive; segment I was PNA-negative but faintly UEA-I positive; segment II was weakly PNA-positive but strongly UEA-I-positive; segment III was strongly-PNA- and UEA-I-positive; segment IV was weakly UEA-I-positive but strongly PNA-positive; and segment V was faintly UEA-I-positive but strongly PNA-positive. At 5 and 8 weeks (the adult), the staining patterns of PNA and UEA-I lectins were nearly identical. Moreover, an increasing staining intensity was observed in both lectins with the advancement of age until 5 weeks of age. Additionally, it was noted that, with the exception of segment I, the staining intensity of PNA gradually increased while that of UEA-I gradually decreased towards the distal section of the ductus epididymis. Lectin PNA and UEA-I staining, in conclusion, indicated epithelial segments in the male genital duct of mice from the postnatal developing age of 3 weeks, and up until 5 weeks, their staining patterns were nearly identical to those in adults.

A note on the possibility of identifying Leydig and Sertoli cells by immunohistochemistry in bowhead whales (Balaena mysticetus)

Leydig cells have been found to be either unidentifiable or at apparent low numbers during routine histologic examination of bowhead whale testes. Therefore, formalin-fixed, paraffin-embedded testicular tissues from 14 bowhead whales were retrospectively examined to determine if immunohistochemical staining could aid in identification of Sertoli and Leydig cells. Multiple intratesticular samples were examined when available. Sertoli and Leydig cells were differentiated using inhibin and calretinin stains. Significant whale length and seasonal differences were not found; however, a trend toward increased staining intensity was noted for autumn harvested whales.

Cell-by-cell quantification of the androgen receptor in benign and malignant prostate leads to a better understanding of changes linked to cancer initiation and progression.

The androgen receptor (AR) plays a crucial role in the development and homeostasis of the prostate and is a key therapeutic target in prostate cancer (PCa). The gold standard therapy for advanced PCa is androgen deprivation therapy (ADT), which targets androgen production and AR signaling. However, resistance to ADT develops via AR-dependent and AR-independent mechanisms. As reports on AR expression patterns in PCa have been conflicting, we performed cell-by-cell AR quantification by immunohistochemistry in the benign and malignant prostate to monitor changes with disease development, progression, and hormonal treatment. Prostates from radical prostatectomy (RP) cases, both hormone-naïve and hormone-treated, prostate tissues from patients on palliative ADT, and bone metastases were included. In the normal prostate, AR is expressed in >99% of luminal cells, 51% of basal cells, and 61% of fibroblasts. An increase in the percentage of AR negative (%AR-) cancer cells along with a gradual loss of fibroblastic AR were observed with increasing Gleason grade and hormonal treatment. This was accompanied by a parallel increase in staining intensity of AR positive (AR+) cells under ADT. Staining AR with N- and C-terminal antibodies yielded similar results. The combination of %AR- cancer cells, %AR- fibroblasts, and AR intensity score led to the definition of an AR index, which was predictive of biochemical recurrence in the RP cohort and further stratified patients of intermediate risk. Lastly, androgen receptor variant 7 (ARV7)+ cells and AR- cells expressing neuroendocrine and stem markers were interspersed among a majority of AR+ cells in ADT cases. Altogether, the comprehensive quantification of AR expression in the prostate reveals concomitant changes in tumor cell subtypes and fibroblasts, emphasizing the significance of AR- cells with disease progression and palliative ADT.

Abstract 4499: The peptide-drug conjugate sudocetaxel zendusortide (TH1902) potentiates anti-tumoral activity of the anti-PD-L1 checkpoint inhibitor and induces immune cell infiltration in a B16-F10 syngeneic melanoma model

Abstract Tumor-infiltrating immune cells are involved in the control of cancer and are closely related to clinical outcomes. Sudocetaxel Zendusortide (TH1902), a peptide-drug conjugate (PDC) of the sortilin (SORT1)-binding peptide TH19P01 ester-linked to two docetaxel moieties, has been shown to exert superior anti-cancer activities in multiple cancer models including melanoma syngeneic and xenograft murine models. Melanomas express elevated levels of SORT1 and are considered as one of the most immunogenic tumors where immune checkpoint inhibitors are among the standard of care treatment used. Here, we first questioned whether TH1902 anti-cancer effects involved infiltration of immune cells in a murine SORT1-positive syngeneic, non-immunogenic and highly aggressive B16-F10 melanoma model in C57BL/6 mice. Weekly administration of docetaxel reduced by half the growth of B16-F10 tumor allografts, while administration of TH1902 as a single agent and at equivalent docetaxel doses, was well tolerated and resulted in tumor regression after only 2 treatments. Surprisingly, in this immunologically cold tumor, immunohistochemistry analysis of tumors for immune cell infiltration showed a net increase in total leukocytes (CD45+) infiltration within TH1902-treated tumors compared with docetaxel-treated tumors, especially with for tumor-infiltrating lymphocytes (TILs) and tumor-associated macrophages (TAMs). Furthermore, the increased staining intensities for Perforin and Granzyme B were indicative of the elevated cytotoxic T and natural killer (NK) cells activity in TH1902-treated tumors. This corroborated with increased caspase-3 apoptotic activity. TH1902 and docetaxel doses were next halved and combined with the checkpoint inhibitor anti-PD-L1. In a first study, the TH1902/anti-PD-L1 combination resulted in increased tumor growth inhibition when compared with both agents alone. Interestingly, TH1902 as a single agent showed better tumor growth inhibition than docetaxel or docetaxel/anti-PD-L1 combination. In a second study, the TH1902/anti-PD-L1 combination significantly increased animal survival over either anti-PD-L1 or TH1902 as single agents (21 days increased median survival compared to 2.5 and 12.5 days respectively). We conclude that the superiority of TH1902 anticancer activity over docetaxel involves, in part, the modulation of infiltrating immune cells within the tumor microenvironment. This is the first demonstration that immune cell infiltration patterns play a pivotal role in the TH1902-associated anti-tumoral response. Combination of TH1902 with checkpoint inhibitors (anti-PD-L1) further reveals that this may lead to improved clinical outcomes in future immunotherapy translational approaches. Citation Format: Michel Demeule, Jean-Christophe Currie, Cyndia Charfi, Alain Zgheib, Isabelle Cousineau, Richard Béliveau, Christian Marsolais, Borhane Annabi. The peptide-drug conjugate sudocetaxel zendusortide (TH1902) potentiates anti-tumoral activity of the anti-PD-L1 checkpoint inhibitor and induces immune cell infiltration in a B16-F10 syngeneic melanoma model. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4499.

The integrated stress response is activated in the salivary glands of Sjögren's syndrome patients.

Primary Sjögren's syndrome (SS) is an autoimmune exocrinopathy that affects the structure and function of salivary and lachrymal glands. Labial salivary gland (LSG) acinar cells from SS patients lose cellular homeostasis and experience endoplasmic reticulum and oxidative stress. The integrated cellular stress response (ISR) is an adaptive pathway essential for restoring homeostasis against various stress-inducing factors, including pro-inflammatory cytokines, and endoplasmic reticulum and oxidative stress. ISR activation leads eIF2α phosphorylation, which transiently blocks protein synthesis while allowing the ATF4 expression, which induces a gene expression program that seeks to optimize cellular recovery. PKR, HRI, GCN2, and PERK are the four sentinel stress kinases that control eIF2α phosphorylation. Dysregulation and chronic activation of ISR signaling have pathologic consequences associated with inflammation. Here, we analyzed the activation of the ISR in LSGs of SS-patients and non-SS sicca controls, determining the mRNA, protein, and phosphorylated-protein levels of key ISR components, as well as the expression of some of ATF4 targets. Moreover, we performed a qualitative characterization of the distribution of ISR components in LSGs from both groups and evaluated if their levels correlate with clinical parameters. We observed that the four ISR sensors are expressed in LSGs of both groups. However, only PKR and PERK showed increased expression and/or activation in LSGs from SS-patients. eIF2α and p-eIF2α protein levels significantly increased in SS-patients; meanwhile components of the PP1c complex responsible for eIF2α dephosphorylation decreased. ATF4 mRNA levels were decreased in LSGs from SS-patients along with hypermethylation of the ATF4 promoter. Despite low mRNA levels, SS-patients showed increased levels of ATF4 protein and ATF4-target genes involved in the antioxidant response. The acinar cells of SS-patients showed increased staining intensity for PKR, p-PKR, p-PERK, p-eIF2α, ATF4, xCT, CHOP, and NRF2. Autoantibodies, focus score, and ESSDAI were correlated with p-PERK/PERK ratio and ATF4 protein levels. In summary, the results showed an increased ISR activation in LSGs of SS-patients. The increased protein levels of ATF4 and ATF4-target genes involved in the redox homeostasis could be part of a rescue response against the various stressful conditions to which the LSGs of SS-patients are subjected and promote cell survival.

MitoQ demonstrates connexin- and p53-mediated cancer chemoprevention in N-nitrosodiethylamine-induced hepatocarcinogenesis rodent model

Cancer chemoprevention is an approach that offers huge potential for preventing/retarding carcinogenesis. MitoQ is well-known and extensively studied mitochondria-targeted antioxidants for its applications in diseases linked with oxidative stress. In the present study chemopreventive potential of mitoQ was studied with a focus on the role of gap-junctions and p53 at an advanced stage of HCC. BALB/c mice model of hepatocarcinogenesis was established using N-nitrosodiethylamine as a carcinogen (200 mg/kg b. w., cumulative dose, intraperitoneally). The chemopreventive effect of mitoQ was studied by pre-protecting animals with mitoQ (0.125 mg/kg b. w., orally once a week) till the termination of the study. The tumors developed in the course of the study were histopathologically analyzed and statistically evaluated. The mechanistic role of mitoQ was investigated in terms of mitochondrial oxidative stress, expression of 8-OHdG, Cx26, Cx32, p53 and status of gap-junctional intercellular communication (GJIC) in tumors. Chemopreventive activity of mitoQ was evident from improved survival of animals, significantly (p ≤ 0.05) lower tumor multiplicity, tumor incidence and a total number of tumors. MitoQ treatment significantly (p ≤ 0.05) decreased mitochondrial oxidative stress as indicated by reduced mtROS and mtLPO. Increased staining intensity of 8-OHdG and internalization of Cx26, Cx32 which was observed in hepatic tumors was reduced upon mitoQ treatment. Furthermore, the expression of Cx26, Cx32 and p53 was significantly increased along with improvement in GJIC in mitoQ treatment group. MitoQ demonstrated its chemopreventive potential probably by regulating mtROS, connexins and p53 in hepatocarcinogenesis.

Responses of injured nerve in CD137L knockout mice following sciatic nerve crush-induced neuropathic pain

Abstract Previously, we showed that CD137L, a costimulatory molecule is involved in the development of neuropathic pain following sciatic nerve crush (SNC). CD137L knockout (KO) mice displayed reduced SNC-induced pain-like behaviors and faster recovery following SNC. To determine the responses at the nerve crush site, we further examined the phenotypic and inflammatory changes of injured sciatic nerve in both CD137L KO mice and wild type (WT) C57BL/6 mice. Both CD137L KO and WT mice underwent either SNC or sham surgery. Sample collection occurred at days 0 (naïve), 3, 7, 14, 28, and 56 post-surgery. First, the femur-level segment of sciatic nerve containing the injury site was analyzed via whole mount immunohistochemistry. At the baseline, CD137L KO mice showed higher staining intensity for IB4 (non-peptidergic c-fibers) compared to WT mice. In WT mice but not CD137L KO mice, SNC significantly increased staining intensity for CGRP (all times; peptidergic c-fibers) and IB4 (Days 3 and 28). These increases were not observed in the corresponding CD137L KO mice. Second, selected inflammatory markers were measured via real time qRT-PCR. At baseline, CD137L KO mice expressed significantly lower levels of TNFα, yet higher levels of IL-10. SNC induced higher levels of elevation of IL-6, CCL3, CCL4, and Arg-1 (all peaked at day 3) in CD137L KO mice compared to WT mice. WT but not CD137L KO mice, showed significant up-regulation of CCL5 (throughout the time course) and CXCL1 (peaked at day 14). Therefore, we propose that CD137L-mediated local inflammatory responses may contribute to SNC induced pain-like behaviors in part by affecting the phenotype of injured nerve. Supported by NIH/NINDS R01NS098426 (Cao), COBRE Histology and Imaging Core funded by NIH/NIGMS P20GM103643 (Meng), and the Kahn Family Foundation through the Carmen Pettapiece Student Research Fellowship (Souza Di Nucci).

Exendin‐4 enhances osteogenic differentiation of adipose tissue mesenchymal stem cells through the receptor activator of nuclear factor‐kappa B and osteoprotegerin signaling pathway

The capability of mesenchymal stem cells (MSCs) to repair bone damage and defects has long been investigated. The receptor activator of nuclear factor-kappa B (RANK), its ligand (RANKL) and the decoy receptor osteoprotegerin (OPG) axis is crucial to keep the equilibrium between osteoblastic and osteoclastic activity. Exendin-4 utilization increased bone formation and enhanced bone integrity. This study aimed to investigate the mentioned axis and determine the effect of exendin-4 upon adipose mesenchymal stem cells (Ad-MSCs) osteogenic differentiation. Ad-MSCs were isolated from rat epididymal fat, followed by characterization and then differentiation into osteocytes both in the presence or absence of exendin-4. Osteogenic differentiation was evaluated by alizarin red staining and the expression of osteogenic markers; using reverse transcriptase-quantitative polymerase chain reaction, western blotting and enzyme-linked immunoassay. MSCs derived from rat epididymal fat were isolated and characterized, along with their differentiation into osteocytes. The differentiated cells were alizarin red-stained, showing increased staining intensity upon addition of exendin-4. Moreover, the addition of exendin-4 elevated the messenger RNA expression levels of osteogenic markers; runt-related transcription factor-2 (RUNX-2), osteocalcin, and forkhead box protein O-1while reducing the expression of the adipogenic marker peroxisome-proliferator-activated receptor-gamma. Exendin-4 addition elevated OPG levels in the supernatant of osteogenic differentiated cells. Moreover, exendin-4 elevated the protein levels of glucagon-like peptide-1 receptor and RUNX-2, while decreasing both RANK and RANKL. In conclusion, osteogenic differentiation of Ad-MSCs is associated with increased osteoblastic rather than osteoclastic activity. The findings of this study suggest that exendin-4 can enhance Ad-MSCs osteogenic differentiation partially through the RANK/RANKL/OPG axis.

The Acute Effects of Furosemide on Na-K-Cl Cotransporter-1, Fetuin-A and Pigment Epithelium-Derived Factor in the Guinea Pig Cochlea.

BackgroundFurosemide is a loop diuretic used to treat edema; however, it also targets the Na-K-Cl cotransporter-1 (NKCC1) in the inner ear. In very high doses, furosemide abolishes the endocochlear potential (EP). The aim of the study was to gain a deeper understanding of the temporal course of the acute effects of furosemide in the inner ear, including the protein localization of Fetuin-A and PEDF in guinea pig cochleae.Material and MethodAdult guinea pigs were given an intravenous injection of furosemide in a dose of 100 mg per kg of body weight. The cochleae were studied using immunohistochemistry in controls and at four intervals: 3 min, 30 min, 60 min and 120 min. Also, cochleae of untreated guinea pigs were tested for Fetuin-A and PEDF mRNA using RNAscope® technology.ResultsAt 3 min, NKCC1 staining was abolished in the type II fibrocytes in the spiral ligament, followed by a recovery period of up to 120 min. In the stria vascularis, the lowest staining intensity of NKCC1 presented after 30 min. The spiral ganglion showed a stable staining intensity for the full 120 min. Fetuin-A protein and mRNA were detected in the spiral ganglion type I neurons, inner and outer hair cells, pillar cells, Deiters cells and the stria vascularis. Furosemide induced an increased staining intensity of Fetuin-A at 120 min. PEDF protein and mRNA were found in the spiral ganglia type I neurons, the stria vascularis, and in type I and type II fibrocytes of the spiral ligament. PEDF protein staining intensity was high in the pillar cells in the organ of Corti. Furosemide induced an increased staining intensity of PEDF in type I neurons and pillar cells after 120 min.ConclusionThe results indicate rapid furosemide-induced changes of NKCC1 in the type II fibrocytes. This could be part of the mechanism that causes reduction of the EP within minutes after high dose furosemide injection. Fetuin-A and PEDF are present in many cells of the cochlea and probably increase after furosemide exposure, possibly as an otoprotective response.

The Predictive Efficacy of Serum Exosomal microRNA-122 and microRNA-148a for Hepatocellular Carcinoma Based on Smart Healthcare.

Objective Hepatocellular carcinoma (HCC) remains a devastating tumor globally. Serum exosomes are reliable biomarkers for tumors, including HCC. Hence, this study explored the efficacy and mechanism of serum exosomes in HCC. Methods microRNA (miR)-122 and miR-148a expressions in serum exosomes from HCC patients and healthy subjects and their predictive efficacy for HCC were detected. Correlation between serum exosomal miR-122/148a expressions with survival rate, clinical stage, lymph node metastasis, and tumor differentiation level and levels of HCC-related serum markers (CA199, FucAFP, ALD-A, and AFu) were detected. PAX2 staining intensity and expression in HCC were measured. PAX2 predictive efficacy for HCC and its correlation with clinical stage, lymph node metastasis, tumor differentiation level, and HCC-related serum marker levels were analyzed. The targeted binding relationship between miR-122 and miR-148a and PAX2 was predicted and verified. Results Serum exosomal miR-122 and miR-148a expressions were downregulated in HCC, showing potent predictive efficacy for HCC, which was negatively related to clinical stage and lymph node metastasis and positively related to tumor differentiation level, patient survival rate, and HCC-related serum marker levels. PAX2 showed increased staining intensity and expression in HCC, together with high predictive efficacy for HCC. PAX2 expression showed a positive correlation with clinical stage and lymph node metastasis and a negative correlation with tumor differentiation level and HCC-related serum marker levels. miR-122 and miR-148a conjointly targeted PAX2 in HCC. Conclusion We demonstrated that serum exosomal miR-122 and miR-148a played a predictive role and were linked to prognosis in HCC via interactions with PAX2.

Substance P failed to reverse dextran sulfate sodium-induced murine colitis mediated by mitochondrial dysfunction: implications in ulcerative colitis.

As controversy exists about the efficacy of substance P (SP) in treating ulcerative colitis (UC) with no previous study highlighting the impact of SP on mitochondrial dysfunction in this diseased condition, it became logical to perform the present study. C57BL/6J mice were administered with DSS @ 3.5%/gm body weight for 3 cycles of 5days each followed by i.v. dose of SP @ 5nmole per kg for consecutive 7days. Histopathological features were noticed in the affected colon along with colonic mitochondrial dysfunction, alterations in mitochondrial stress variables and enhanced colonic cell death. Interestingly, SP failed to reverse colitic features and proved ineffective in inhibiting mitochondrial dysfunction. Unexpectedly SP alone seemed to impart detrimental effects on some of the mitochondrial functions, enhanced lipid peroxidation and increased staining intensities for caspases 3 and 9 in the normal colon. To substantiate in vivo findings and to assess free radical scavenging property of SP, Caco-2 cells were exposed to DSS with or without SP in the presence and absence of specific free radical scavengers and antioxidants. Interestingly, in vitro treatment with SP failed to restore mitochondrial functions and its efficacy proved below par compared to SOD and DMSO indicating involvement of O2 •- and •OH in the progression of UC. Besides, catalase, L-NAME and MEG proved ineffective indicating non-involvement of H2O2, NO and ONOO- in UC. Thus, SP may not be a potent anti-colitogenic agent targeting colonic mitochondrial dysfunction for maintenance of colon epithelial tract as it lacks free radical scavenging property.

Effect of Ethanol Extract of <i>Moringa oleifera</i> Leaf on Nissl Granules, Immunohistochemistry of Inferior Colliculus and Oxidative System Following Quinine Toxicity in Wistar Rats

The metabolite of Quinine, 3-hydroxyquinine is the active substance and more toxic than quinine hence, it can exert pro-oxidant effect in the body. In this study, the effect of ethanol leaf extract of <i>Moringa oleifera</i> was evaluated against quinine toxicity on nissl granules, immunohistochemistry of inferior colliculus and oxidative system of Wistar rats. Fifty Wistar rats weighing 180 – 200g were alloted into 10 groups of 5 rats each. Group 1 served as control while Groups 2 to 10 were the treatment groups. Groups 2 – 4 received 10, 20 and 30 mg/kg body weight of quinine hypochloride injection intramuscularly eight hourly for 7 days. Groups 5 – 7 were orally administered 250, 500 and 750 mg/kg body weight of <i>M. oleifera</i> leaf extract daily for 7 days respectively. Group 8 received 250mg/kg of leaf extract orally and intramuscular (IM) 10mg/kg of quinine; Group 9 was treated with oral 500mg/kg of the extract and IM 20mg/kg of quinine while Group 10 received 750mg/kg of the extract orally and IM 30mg/kg of quinine for 7 days. Results revealed that quinine significantly (p < 0.05) decreased the activities of anti-oxidative enzymes (superoxide dismutase and catalase) and increased lipid peroxidation (malondialdehyde, MDA) in treated rats. However, <i>Moringa oleifera</i> leaf extract showed significant increase in the activities of anti-oxidant enzymes and decreased lipid peroxidation. Histomorphological and immunohistochemical observations revealed that quinine caused neuronal distortions, nuclear degenerations, and depletion of Nissl granules, vascular congestions and vacuolations and increased astrogliosis in the inferior colliculus. Incorporation of the extract to the quinine treated groups revealed neuronal regenerations, increased staining intensity and density of the Nissl granules, reduced astrogliosis and restoration in the inferior colliculuscyto-architectures. In conclusion, <i>Moringa oleifera</i> leaf extract can be used to ameliorate the adverse effects of quinine toxicity on inferior colliculus.

Paracrine roles of hormone receptors in Riehl’s melanosis: A quantitative analysis of oestrogen and progesterone receptor expression patterns

The incidence of Riehl's melanosis (RM) is most common in the fifth or sixth decade of life with a female preponderance. As the skin is regarded a non-reproductive organ on which sex steroid hormones act, a possible relationship between the pathogenesis of RM and sex steroid hormone receptors can be inferred. This study intended to evaluate the expression profile of oestrogen receptor (ER)β and progesterone receptor (PR) in RM. Twelve lesional and perilesional normal-appearing skin samples of RM patients and the skin of 12 healthy controls were retrieved for the analysis. Real-time PCR analysis and immunohistochemical studies were conducted for ERβ and PR, respectively. The lesional and perilesional normal-appearing skin of 12 patients with RM and the skin of 12 healthy controls were retrieved for the analysis. Interestingly, the dermal ERβ immunostaining intensity was increased more in lesional skin than in perilesional skin. When compared to healthy controls, increased expression of ERβ and PR mRNAs was observed in the lesional skin of patients with RM. Of note, epidermal and dermal ERβ and dermal PR expressions showed increased staining intensities in the lesional skin of RM patients compared with healthy controls. The altered expression of ERβ and PR in RM supports the possible role of these hormone receptors in the pathogenesis of RM.

P63 expression profile in uterine cervical lesions

Background and objective: P63 is a member of the p53 gene family. It plays an essential role in the normal development of the cervix. This study aimed to assess p63 expression in different cervical lesions and the correlation between their expression in cervical intraepithelial neoplastic changes and invasive cervical cancer. Methods: A total of 62 cervical tissues were performed for p63 immunostaining, including 15 samples of non-neoplastic reactive cervicitis, 27 cervical intraepithelial neoplastic changes, and 15 invasive cervical cancer cases as well as five samples of normal cervical tissue as control. Results: P63 in normal cervical squamous epithelium had restricted staining pattern in the nuclei of basal cells. Reactive cervicitis staining ranged from restricted staining pattern in basal cells to mild increase in staining intensity involving upper parabasal cells. P63 was positive in all CIN lesions, and staining intensity increased significantly with increasing severity of cervical intraepithelial lesions compared with non-neoplastic epithelium. All cervical squamous cell carcinoma had high staining intensity while adenocarcinoma and small cell carcinoma were stained negative. Conclusion: P63 can be used as a marker for the distinction between reactive cervicitis and cervical intraepithelial changes. It is also of value for grading of CIN lesions and differentiation between squamous cell carcinoma, adenocarcinoma, and small cell carcinoma.

Antigen Retrieval for Immunostaining of Modified Cytosine Species.

Immunostaining (also called as immunofluorescence) is a fluorescence labeling method to stain one or more epitopes of interest on DNA and/or protein using specific antibodies. Cytosine modifications can be detected quantitatively by immunostaining. The protocol commonly includes sequential steps. These include fixation, permeabilization, antigen retrieval, blocking, incubation with primary and secondary antibodies, and visualization under the microscope followed by image-based intensity analysis of staining. Each step is important, but antigen retrieval is especially necessary for DNA epitopes such as cytosine modifications as antibodies can access cytosines in DNA only once the DNA double-strand is denatured and DNA-packaging proteins have been removed. Hydrochloric acid is commonly used for this purpose. However, there are additional treatments with enzymes to enhance antigen retrieval and improve the detection by increasing staining intensity. This chapter describes current methodology for improving antigen retrieval for the staining of the cytosine modifications 5'-methylcytosine (5meC), 5'-hydroxymethylcytosine (5hmC), 5'-formylcytosine (5fC), and 5'-carboxycytosine (5caC).

Effects of various fixatives and temperature on the quality of glycogen demonstration in the brain and liver tissues

The visualization of glycogen deposits in cells and tissues is important for studying glycogen metabolism as well as diagnosis of glycogen storage diseases. Evidence suggests that the demonstration of glycogen can better be enhanced by factors such the choice of fixative and temperature during fixation. Here, we assessed efficacy of neutral buffered formalin (NBF), alcoholic formalin (AF) and paraformaldehyde (PFA) at 4 °C, 37 °C and 40 °C using Periodic Acid Schiff's staining method. Each liver specimen was fixed in NBF and AF while the brain tissues were fixed in NBF, AF and PFA. We found that there was a better PAS staining intensity with the liver tissues fixed in AF compared with NBF. Also, there was no difference in the quality of the staining for tissues fixed in AF at 37 °C, 4 °C and 40 °C, but fixation with NBF at 4 °C gave the best staining quality when compared with 40 °C and 37 °C. Furthermore, hippocampal tissues fixed in AF showed better quality of PAS staining compared with NBF and PFA. A significant increase in staining intensity was observed for PFA when compared with NBF. Superior staining intensity for PAS was observed at 4 °C for hippocampal tissues fixed with NBF, AF and PFA. Taken together our results show that AF at a temperature of 4 °C gave the best result. Hence, glycogen demonstration can better be enhanced by the choice of fixative and temperature during fixation.

Differentiated thyroid carcinoma in sporadic and familial presentations of acromegaly: A case series

BackgroundIn acromegaly, chronic growth hormone (GH) and insulin-like growth factor-1 (IGF-1) exacerbate comorbidities in multiple organs. Differentiated thyroid carcinoma (DTC) has been reported as being a comorbid condition in acromegaly. Acromegaly is usuallysporadic, but 5% of cases may be genetic. The most frequent inheritable form of acromegaly is related to germline mutations in the aryl hydrocarbon receptor interacting protein (AIP) gene. Epidemiological data on the relationship between active acromegaly, its familial forms and DTC are sparse. We present the investigation of a FIPA family (familial isolated pituitary adenoma) with homogeneous acromegaly and 6 sporadic acromegaly patients with DTC. Patients and methodsA study of 59 acromegaly patients assessed thyroid nodules on ultrasound and fine-needle aspiration biopsy following the ATA 2015 criteria. We diagnosed 7 differentiated thyroid carcinomas. Resected thyroid carcinoma tissues were stained using an anti-AIP antibody. Analysis of germline and tumor-derived DNA for variants in the AIP and MEN1 genes were performed in the FIPA kindred. ResultsWe describe one FIPA patient and 6 sporadic acromegaly cases with DTC. The FIPA family (AIP mutation negative) consisted of two sisters, one of whom had a DTC with intermediate risk and incomplete structural response to therapy. In our study, DTC in sporadic acromegaly had a low recurrence rate (6/6), and excellent response to therapy (6/6). Immunohistochemistry for AIP showed similar or increased staining intensity in DTC versus normal thyroid tissue. ConclusionIn our cohort of sporadic and familial forms of acromegaly with DTC, AIP did not appear to influence thyroid cancer progression.

Decreased neural expression of the noradrenaline transporter in the papillary dermis after partial sciatic nerve lesion

After peripheral nerve injury, regeneration or collateral sprouting of noradrenergic nerve fibres in the papillary dermis of the injured limb may contribute to sympathetically-maintained pain. The aim of this study was to determine whether noradrenergic nerve fibre regeneration after partial sciatic nerve ligation (PSL) in Wistar rats was accompanied by parallel shifts in expression of the noradrenaline transporter (NAT). Four or 28 days after PSL surgery, immunohistochemistry was used to examine NAT expression in plantar hind paw skin in relation to pan-neuronal markers (class III beta-tubulin and protein gene product 9.5), peptidergic afferents containing calcitonin gene-related peptide (CGRP), nonpeptidergic afferents labelled by isolectin B4 (IB4), and tyrosine hydroxylase (TH), a marker for cutaneous noradrenergic nerve fibres. Most dermal nerve fibre populations decreased shortly after PSL. However, four weeks after PSL, an increase in staining intensity of CGRP and novel expression of TH were observed in the papillary dermis on the injured side. In contrast, neural expression of NAT was reduced in this region. Loss of NAT might have implications for sympathetically-maintained pain, as failure to rapidly clear noradrenaline could exacerbate aberrant sympathetic-sensory signalling between closely apposed noradrenergic and peptidergic nerve fibres.

Phenotypic Differences in Primary Murine Microglia Treated with NOD1, NOD2, and NOD1/2 Agonists.

The purpose of the study was studying the influence of different NOD agonists on the morphological phenotype of primary murine microglia and to examine their influence on characteristic cytokines. Primary CD11b-positive cells were isolated from the brain of neonatal mice. The microglial phenotype of the cells was examined by ionized calcium-binding adapter molecule (Iba)1 staining. After14 days in culture, these cells were stimulated by iE-DAP, L18-MDP, or M-TriDAP as NOD1, NOD2, and NOD1/2 agonists, respectively. The cellular morphology was recorded and compared to the phenotype of cells cultured in medium alone or after LPS stimulation. The cells developed a specific phenotype only after treatment with the NOD2 agonist L18-MDP. These cells were characterized by straight extensions carrying tiny spikes and had a high ramification index. This was in sharp contrast to all other treatments, which always resulted in an amoeboid phenotype typically shown by activated microglia in vivo and by cultured microglia in vitro. The staining intensity of IL-6 and TNF-α did not reveal any clear difference independent of the NOD agonist treatment. In contrast, an increased staining intensity was observed for IL-10 after L18-MDP treatment. The NOD2 agonist L18-MDP induced a morphologically distinct phenotype characterized by microspike-decorated dendritiform extensions and a high degree of ramification in primary murine microglia. Increased ramification index and elevated staining intensity of anti-inflammatory IL-10 as hallmarks suggest that a M2-like phenotype of microglia was induced.

Steroid hormones and hormone antagonists regulate the neural marker neurotrimin in uterine leiomyoma

To characterize the role of steroid hormone and antihormone exposure on neurotrimin (NTM) expression in human leiomyoma and myometrial tissue and cells. Laboratory study of placebo and ulipristal acetate (UPA)-treated patient tissue. Invitro assessment of immortalized myometrial and leiomyoma cell lines after hormone and antihormone exposure. Academic research center. Not applicable. Exposure of leiomyoma cell lines to 17β-E2, medroxyprogesterone acetate (MPA), UPA, and fulvestrant. Messenger RNA expression quantified with the use of RNASeq analysis and quantitative real-time polymerase chain reaction (qRT-PCR). Protein levels quantified by means of Western blot analysis. Immunohistochemistry (IHC) on placebo- and UPA-treated patient uterine tissue specimens. Expression of NTM in human uterine leiomyoma specimens according to RNASeq was increased compared with myometrium (5.22 ± 0.57-fold), which was confirmed with the use of qRT-PCR (1.95 ± 0.05). Furthermore, NTM protein was elevated in leiomyoma tissue compared with matched myometrium (2.799 ± 0.575). IHC revealed increased staining intensity in leiomyoma surgical specimens compared with matched myometrium of placebo patients. Western blot analysis in immortalized leiomyoma cell lines demonstrated an up-regulation of NTM protein expression (2.4 ± 0.04). Treatment of leiomyoma cell lines with 17β-E2 yielded a 1.98 ± 0.11-fold increase in NTM protein expression; however, treatment with fulvestrant showed no significant change compared with control. Leiomyoma cell lines demonstrated a 1.91 ± 0.97-fold increase in NTM protein expression after progesterone treatment. RNASeq analysis demonstrated a reduced expression in patient leiomyoma after UPA treatment (0.75 ± 0.14). Treatment of leiomyoma cells with UPA demonstrated a reduced total NTM protein amount (0.54 ± 0.31) in patients, which was confirmed with the use of IHC (UPA10 147.2 ± 9.40, UPA20 182.8 ± 8.98). Invitro studies with UPA treatment revealed a concentration-dependent effect that supported these findings. NTM, a neural cell adhesion molecule, is increased in leiomyoma compared with myometrium in patient tissue and invitro models after estrogen and progesterone treatment. Down-regulation of expression occurs after UPA treatment, but not after fulvestrant exposure. NCT00290251.