Weak Immunostaining Research Articles

Forkhead box N1 is possibly a novel biomarker and prognostic indicator for patients with squamous cell lung carcinoma.

Background: The roles of Forkhead box N1 (FOXN1) in lung squamous cell carcinoma (LUSC) remains elusive. This study was focused on assessing the expression levels of FOXN1 in LUSC and exploring its potential clinical implications. Methods: Utilizing a range of databases, this study conducted an analysis of the FOXN1 gene's expression levels, comparing LUSC samples with those from normal lung tissues. The expression levels of FOXN1 in primary LUSC and corresponding normal lung tissues were assessed using immunohistochemistry (IHC). Histoscore was used to evaluate the staining degree. χ2 test and Fisher's exact test were employed to assess the association between categorical variables that do not possess an ordinal nature. Multivariate survival analysis was conducted using the Kaplan-Meier method, the Wilcoxon test, and the Cox proportional hazards model. Results: In contrast to normal lung tissues, the expression of the FOXN1 gene was found to be significantly elevated in LUSC tissues (P < 0.01). And FOXN1 was expressed in 79 (98.8%) evaluated LUSC tissues, most of which showed compositive IHC-staining intensity, presenting heterogeneously expression. 69 (87.3%) cases were characterized for strong immunostaining intensity, 70 (87.5%) cases showed moderate intensity, and 66 (82.5%) cases presented weak intensity. Only one sample of normal lung tissue, which represents 10% of the total, exhibited weak immunostaining exclusively (P < 0.05). Additionally, the expression of FOXN1 was found to have a significant correlation with the grading of LUSC, the presence of lymph node and distant metastases, the stage of the disease, and the survival outcomes (P < 0.05). Conclusion: The expression of FOXN1 is frequently increased in LUSC, and the patients with high FOXN1 expression have a poorer survival outcome. FOXN1 can be a novel biomarker and prognostic indicator for LUSC patients.

9234 A Case of Gigantism Due to An AIP Gene Mutation: Implications for Genetic Counseling and Preimplantation Genetic Diagnosis

Abstract Disclosure: M. Serebro: None. M. Yacobi Bach: None. N. Even Zohar: None. A. Reches: None. Y. Greenman: None. From the age of 17 the patient experienced accelerated growth, facial changes, and enlargement of hands and feet. Despite having a sister already diagnosed with acromegaly, the patient did not undergo endocrine evaluation until the age of 25, when he was diagnosed with gigantism. Magnetic resonance imaging (MRI) revealed the presence of a macroadenoma with suprasellar extension reaching the optic chiasm. The patient underwent transsphenoidal surgery and excision of a sparsely granulated somatotroph adenoma that was Pit1 positive and exhibited weak immunostaining for growth hormone (GH). Insulin-like growth factor 1 (IGF1) levels normalized after surgery, and a fasting GH level was 0.6 ng/ml. Given the family history and the early onset of the disease, genetic counseling was recommended. Genetic testing revealed a heterozygote likely pathogenic variant in the AIP gene (c.713G&amp;gt;A). This finding prompted the recommendation for genetic testing of all first-degree relatives. The patient and his wife, who desired to have children, were referred to obstetrics and gynecology for pregestational diagnosis. Family segregation testing indicated that the patient's mother also carried the same AIP variant. Preimplantation genetic diagnosis (PGD) was conducted, revealing that 11 out of 20 embryos were carriers of the AIP variant. The non-carrier embryos were selected for implantation, and pregnancy was achieved. Efforts to inform first-degree relatives about the importance of genetic testing were undertaken, and one sister was tested and found to be healthy. Other siblings have not yet been tested. The patient's mother, identified as an AIP variant carrier, underwent endocrine laboratory tests that showed GH, IGF1, and prolactin levels within the normal range. Brain MRI did not reveal a pituitary lesion.This case highlights the significance of genetic counseling and testing in patients with familial endocrine disorders, the potential implications of a molecular diagnosis of a pathogenic AIP variant, and the challenges faced in reproductive decision-making for affected individuals and their families. Presentation: 6/3/2024

Immunohistochemical Changes of Polycystic Ovaries in rats Treated with Asparagus Officinalis L. Roots Extract

Abstract Background: Polycystic ovary syndrome (PCOS) which a series reproductive, metabolic and psychological condition that is characterized by abnormal gonadotropin secretion or hyperandrogenism and associated with insulin resistance and most commonly found in the reproductive age female. Objectives: This research paper were conducted to examine the effects of Asparagus officinalis L. roots extract on letrozole induced PCOS where the effect of 400 mg/kg A. officinalis L. root extract on the PCOS was studied for 28 days in female rats. Materials and Methods: Thirty-five female Albino rats were randomly divided into five groups, including control negative group, positive control, PCOS, treatment and preventive group. During 28 days, the positive control group got an extract of the A. officinalis roots. Letrozole 1 mg/kg dissolve in 0.5% carboxymethyl cellulose solution was used to cause PCOS for 28 days. Following the induction of PCOS, the treatment group received extract A. officinalis roots for 28 days. The preventative group received an extract of asparagus roots during PCOS induction. Results: Immunohistochemical results are shown Ovary slices from of the therapeutic and preventative groups next to the asparagus group were immunostained for Ki 67, which showed high positive statistically (P &lt;0.05) immunological activity in the granulosa nuclei and less in the theca cells. When compared to the PCOS group, both the granulosa and theca layers had very weak positive immunostaining (P &lt;0.05). According to the morphological findings, granulosa cells from the Control group and asparagus group proliferated much more than granulosa cells from the PCOS group statistically significant (P &lt;0.05). Conclusion: The findings show that theca and granulosa cells are quickly stimulated to proliferate by asparagus root extract. Moreover, it therefore speeds up oogenesis and early cell divisions.

Differences in Stromal Immunostaining of Tumour Necrosis Factor-alpha and its Receptors in Neoplastic and Non Neoplastic Ovarian Lesions: A Cross-sectional Study

Introduction: Ovarian cancer has an unknown pathogenesis, and cytokines may play an important role in the aetiology and prognosis. Tumour Necrosis Factor-alpha (TNF-α) and its receptors (TNFR1 and TNFR2) are involved in the biology of ovarian cancer, tumour pathogenesis, and their relationship with prognostic factors. They are involved in biological processes such as immunoregulation, growth modulation, and cell differentiation. Aim: To evaluate stromal immunostaining of TNF-α and its receptors (TNFR1 and TNFR2) in malignant ovarian neoplasms, comparing it with benign ovarian neoplasms and non neoplastic ovarian lesions. Materials and Methods: A cross-sectional study was carried out at the Department of Gynaecology and Obstetrics, Federal University of Triângulo Mineiro, Uberaba, Minas Gerais, Brazil, from January 1997 to December 2020. Patients with ovarian lesions who underwent surgical treatment according to preestablished criteria (n=95) were included in the study. Patients with benign (n=37) and malignant (n=43) ovarian epithelial neoplasms, and non neoplastic ovarian lesions (n=15) were included. Data evaluated included age, parity, hormonal status (menarche or menopause), histological grade, and staging. Immunohistochemical study was performed to evaluate stromal TNF-α, TNFR1, and TNFR2. Data were analysed by GraphPad Prism 6 and IBM Statistical Package for the Social Sciences (SPSS) Statistics 20.0 software. The comparison between non neoplastic tumours, benign and malignant neoplasms was performed by the Fisher’s exact test with a significance level below 0.05. Results: Considering staining intensity 0 and 1 as “weak immunostaining” and 2 and 3 as “strong immunostaining,” TNF-α stromal immunostaining was stronger (2/3) in benign ovarian neoplasms compared to non neoplastic tumours (p-value=0.0016) and in malignant neoplasms compared to non neoplastic tumours (p-value&lt;0.0001). TNFR1 immunostaining was stronger (2/3) in the stroma of malignant neoplasms compared to benign neoplasms (p-value&lt;0.0001) and stronger (2/3) when comparing benign neoplasms with non neoplastic ovarian lesions (p-value=0.0002). For TNFR2, stromal immunostaining was stronger (2/3) in malignant neoplasms compared to benign neoplasms (p-value =0.0091) and stronger in malignant neoplasms compared to non neoplastic lesions (p-value=0.0004). Conclusion: A stronger immunostaining for TNF-α and its receptors was found in ovarian cancer, suggesting that they may be targets for further studies to verify their role in carcinogenesis and the progression of ovarian neoplasms. A better understanding of the role of TNF-α and its receptors in the tumour stroma of ovarian tumours may lead to future studies that may clarify the mechanisms of carcinogenesis and tumour progression.

Solitary fibrous tumor: Two cases report with a focus on the differential diagnosis

Solitary fibrous tumor (SFT) is a neoplasm of fibroblastic/myofibroblastic origin with intermediate biological behavior. We report here two cases of SFT affecting an unusual anatomical site in 58-year-old and 40-year-old female patients and discuss the differential diagnosis of this lesion. In case 01, the lesion showed the clinical appearance of an asymptomatic “blister” with normal color, rubbery consistency, measuring 0.3 cm, and affected the lower lip; while in case 02, a symptomatic red nodular lesion with a soft consistency and measuring 0.5 cm affected the floor of the mouth. Excisional biopsies were performed. Microscopically, two well-delimited benign neoplasms were observed, exhibiting the proliferation of ovoid to spindle-shaped mesenchymal cells, vascular spaces with staghorn arrangement, and the absence of mitosis figures. Immunohistochemistry was performed in case 01 to assist in the diagnosis. Weak and diffuse immunostaining was observed for α-SMA and intense and diffuse immunopositivity for Bcl-2 and CD34. Based on histopathological and immunohistochemical features, a diagnosis of SFT was rendered in both cases. The low occurrence and nonspecific clinical features of oral SFT may make its clinical diagnosis difficult. Also, morphological and immunohistochemical are essential for differential diagnosis with other mesenchymal neoplasms.

FRI394 Anti-inflammatory Protein Neuregulin-1 (NRG1) And ErbB3 Receptor In Human Antral Follicle And Endometrium

Abstract Disclosure: I. Chowdhury: None. S. Banerjee: None. A. Rodriguez: None. Neuregulin 1 (NRG1) is a member of the EGF-like factor family and has recognized pro-survival and anti-inflammatory roles in neurodevelopmental and cardiac function, neurological and psychiatric disorders, cancer, and development. However, the physiological role of NRG1 in the human ovary and uterus has not been well documented. Therefore, we assessed the expression pattern of NRG1 and its receptor ErbB3 in normal human ovary and uterus tissue samples by immunohistochemistry (IHC). ErbB3 is one of the bona fide receptors for NRG1. NRG1 and ErbB3 were colocalized with steroidogenic acute regulatory protein (StAR) in normal human ovarian tissue samples, whereas NRG1 and ErBB3 were colocalized with CD105 (Endoglin) and epithelial cadherin (E-cadherin) in human uterine tissue samples. Our results showed strong immunoreactive signals of NRG1, ErbB3, and StAR in antral follicles, whereas weak or lack of immunoreactive signals in preantral follicles. The strong immunoreactive signals of NRG1 and ErbB3 were observed in granulosa cells and theca interstitial cells of antral follicles. In the uterus, a differential pattern of NRG1, ErbB3, CD105, and E-cadherin expressions were noticed. Interestingly, strong immunoreactive signals of NRG1, ErbB3, CD105, and E-cadherin in the luminal epithelium, and superficial and deep glands of endometrium were observed, whereas weak immunostaining in myometrium was noticed. These findings suggest that NRG1 and ErbB3 are important intracellular autocrine-paracrine anti-inflammatory factors that may govern antral follicular development and uterine implantation. Nothing to Disclose: SB; AR; IC. Sources of Research Support: This study was supported in part by National Institutes of Health Grants 1SC1 GM130544, G12-RR03034, and P50-HD28934. This investigation was conducted in a facility constructed with support from Research Facilities Improvement Grant #C06 RR18386 from NIH/NCRR. Presentation: Friday, June 16, 2023

Celecoxib-Loaded Cubosomal Nanoparticles as a Therapeutic Approach for Staphylococcus aureus In Vivo Infection.

There is a great need for novel approaches to treating bacterial infections, due to the vast dissemination of resistance among pathogenic bacteria. Staphylococcus aureus are ubiquitous Gram-positive pathogenic bacteria and are rapidly acquiring antibiotic resistance. Here, celecoxib was encapsulated into cubosomal nanoparticles, and the particle morphology, size distribution, zeta potential, entrapment efficiency, and celecoxib release were evaluated in vitro. Also, a systemic infection model in mice elucidated the in vivo antibacterial action of the celecoxib cubosomes. Cubosomes are a nanotechnology-based delivery system which can adhere to the external peptidoglycan layers of Gram-positive bacteria and penetrate them. The size distribution investigation revealed that the prepared celecoxib-loaded cubosomes had a mean particle size of 128.15 ± 3.04 nm with a low polydispersity index of 0.235 ± 0.023. The zeta potential measurement showed that the prepared cubosomes had a negative surface charge of -17.50 ± 0.45, indicating a highly stable nanodispersion formation with little susceptibility to particle aggregation. The cubosomal dispersion exhibited an entrapment efficiency of 88.57 ± 2.36%. The transmission electron micrograph for the prepared celecoxib-loaded cubosomes showed a narrow size distribution for the cubosomal nanoparticles, which had a spherical shape and were non-aggregated. The tested cubosomes diminished the inflammation in the treated mice's liver and spleen tissues, as revealed by hematoxylin and eosin stain and Masson's trichrome stain. The immunostained tissues with nuclear factor kappa B and caspase-3 monoclonal antibodies revealed a marked decrease in these markers in the celecoxib-treated group, as it resulted in negative or weak immunostaining in liver and spleen that ranged from 4.54% to 17.43%. This indicates their inhibitory effect on the inflammatory pathway and apoptosis, respectively. Furthermore, they reduced the bacterial burden in the studied tissues. This is alongside a decrease in the inflammatory markers (interleukin-1 beta, interleukin-6, cyclooxygenase-2, and tumor necrosis factor-alpha) determined by ELISA and qRT-PCR. The IL-1β levels were 16.66 ± 0.5 pg/mg and 17 ± 0.9 pg/mg in liver and spleen, respectively. Also, IL-6 levels were 85 ± 3.2 pg/mg and 84 ± 2.4 pg/mg in liver and spleen, respectively. In conclusion, the current study introduced cubosomes as an approach for the formulation of celecoxib to enhance its in vivo antibacterial action by improving its oral bioavailability.

Clinical significance of galectin-3 expression in urinary bladder carcinoma

To uncover the clinical significance of galectin-3 in the evolution of urinary bladder cancer by defining galectin-3 expression and examining the relationship between its expression in a group of urothelial carcinomas versus normal tissues along with clinicopathological factors. This retrospective study included histopathological reports and archival blocks and slides of all patients with urinary bladder cancer treated at King Abdulaziz University Hospital (Jeddah, Saudi Arabia). An anti-galectin-3 monoclonal antibody was used for immunohistochemical staining of tissue microarray slides comprising 128 cases of urothelium carcinoma and 24 specimens of normal bladder mucosa. Galectin-3 was downregulated during transformation, with positive expression found in 50 (39%) urinary bladder neoplasms, of which 33 (66%) showed weak immunostaining. All positively-stained malignant tumor and normal bladder mucosa samples showed cytoplasmic staining; a few samples also showed nuclear staining. No correlation was noted between galectin-3 and histotype, grade, stage, muscularis propria invasion, lymph node invasion, vascular invasion, or metastasis. A Cox proportional hazards model and Kaplan-Meier survival curves did not show differences in survival on the basis of galectin-3 expression. Galectin-3 is down-regulated in bladder cancer but is not a helpful marker for the diagnosis or prognosis of urinary bladder cancer.

MFSD2a, the transporter of the omega‐3 fatty acid DHA, is expressed in the neural retina

AbstractPurpose: The omega‐3 fatty acid, DHA, is enriched in the outer disk and synapse membranes, and it exerts an anti‐inflammatory and anti‐apoptotic effect in the neuron cells. Animal models and patients with retinal dystrophies were reported to have reduced levels of DHA in the retina, so different nutraceuticals were designed for these patients. Nevertheless, it is still unknown how DHA is cached and distributed in the retina. MFSD2a was reported as the putative transporter of omega‐3, but its expression was only described in the endothelial cells. In this study, we assessed the expression of MFSD2a in the different cell populations of the neural retina.Methods: The expression of MFSD2a was studied by immunofluorescence in cryostat sections of the retina from healthy human donors. Double immunolabelling with different cell markers was used to study MFSD2a expression in the different retinal cell populations. Cone arrestin was used for the identification of cone photoreceptor cells. Parvalbumin is a cell marker for all horizontal cells. The transcription factors Chx10 and Pax6 were used for the identification of bipolar and amacrine cells, respectively. KDEL, an endoplasmic retention signal peptide, was used for the visualization of ganglion cells. Müller cells and RPE are positive for CRALBP.Results: We found that MFSD2a was expressed in the outer nuclear layer as well as in the inner segments of the photoreceptor cells. Outer segments showed weak or negative immunostaining. Immunoreactivity with anti‐MFSD2a was also detected in the inner nuclear layer. Double immunostaining with parvalbumin and the transcriptional factors Chx10 and Pax6 confirmed that all populations of horizontal, bipolar, and amacrine cells, respectively, expressed the omega‐3 transporter. Also, ganglion cells expressed MFSD2a. In the double immunolabelling with anti‐MFSD2a and anti‐CRALBP, Müller cells showed punctae of immunolabelling in the tips of the external limiting membrane. Although, RPE did not seem to express MFSD2a.Conclusions: MFSD2a is expressed by the neural cells of the retina. Photoreceptor cells, Müller cells, and all populations of bipolar, amacrine, horizontal, and ganglion cells expressed the transporter of the omega‐3 fatty acid, DHA.

Levels of Genetic Variants Among Symptomatic Blastocystis Subtypes and their Relationship to Mucosal Immune Surveillance in the Precancerous Colons of Experimentally Infected Rats

PurposeThe relationship between the genetic diversity of Blastocystis and immune surveillance in precancerous colons with blastocystosis is still under investigation. This study aimed to identify the genetic Blastocystis variants among 54 symptomatic human isolates and their relationship to mucosal immune surveillance in the precancerous polyps of experimentally infected rats.MethodsPolymerase chain reaction and high-resolution melting (PCR/HRM) curves discriminated human symptomatic Blastocystis isolates into subtypes (STs)/intrasubtypes, which were orally administered to rats to induce experimental infection. Then, the mucosal immune responses of the infected colons were evaluated in relation to polyp formation through immunostaining to identify mucus MUC2 and determine mucosal immune cell (goblet, lymphocyte and mast) counts, secretory IgA levels and parasitic intestinal invasion.ResultsST1, ST3, and ST4 were found in 18.5% (10/54), 54.7% (29/54), and 27.8% (15/54) of the samples, respectively. Then, the HRM curve discriminated ST3 into the wild, mutant, and heterozygous [17/54 (31.5%), 5/54 (9.3%), and 7/54 (12.9%)] intrasubtypes. ST1 and ST4 had no genetic variations. Precancerous polyps were detected in the colons of 40.5% of the infected rats. ST1 constituted 14.7% of these cases, while the wild, mutant, and heterozygous intrasubtypes of ST3 showed polyps in 12.9%, 5.5%, and 5.5% of cases, respectively. Only 1.9% of the polyps were related to ST4. MUC2 showed weak immunostaining in 44.5% of the infected colons, and 38.9% were polyp inducers. Low goblet cell numbers and high interepithelial lymphocyte counts were significantly associated with polyp formation, particularly with ST1 and wild ST3. Among the polyp inducers, high numbers of mast cells were detected in wild ST3 and ST4, while a low number was found with heterozygous ST3. The level of secretory IgA was low in polyp-inducing STs. Most of the results were statistically significant.ConclusionImmunosurveillance showed a potential relationship between ST1 and the ST3 intrasubtypes and precancerous polyps. This relationship may provide insight into the prevention and/or development of new immunotherapeutic strategies to combat colorectal cancer.

PMON104 Sellar Gangliocytoma-Pituitary Neuroendocrine Tumor (PitNET) Presenting As Acromegaly

Abstract Growth hormone (GH)-secreting pituitary neuroendocrine tumors (PitNET) arising from somatotrophs is the most common cause of acromegaly. It may rarely develop in the setting of a growth hormone-secreting PitNET associated with a neuronal tumor composed of neoplastic ganglion cells, the so-called mixed gangliocytoma somatotroph PitNET. We present a case of acromegaly which proved to be due to a mixed gangliocytoma somatotroph PitNET on pathology.A 58-year-old, healthy woman presented to the ER with a 3-day history of left-sided vision loss and worsening headache. MRI revealed 4.5×4.2×3.8 cm heterogeneously enhancing bilobed mass consistent with a pituitary PitNET with suprasellar extension causing compression of the optic chiasm, invasion into the left cavernous sinus and partial encasement of the left internal carotid artery. She had coarse facial features, wide feet, multiple skin tags, and a history of multiple colonic polyps. Biochemical work up revealed an IGF-I of 664 ng/ml (50-317), AM cortisol 19.7 mg/dl, ACTH 30 pg/ml, prolactin 15.5 ng/ml, FSH 35.6 mIU/ml, LH 11.3 mIU/ml, and FT4 0.7 ng/dl (0.70-1.48). She underwent endoscopic transsphenoidal resection of the tumor. Histopathology demonstrated mature ganglion cells with neuropil-like areas, intermixed with small cell components. The neuronal population was immunopositive for NeuN (Neuronal nuclei antibody), synaptophysin, and NF (Neurofilament). PitNET cells were positive for GH, prolactin, Pit- 1 transcription factor and the somatostatin 5 receptor, histological features consistent with mixed gangliocytoma and sparsely granulated somatotroph pitNET. Some ganglion cells also showed weak immunostaining with Pit-1. A 3-month follow-up MRI showed a residual 3 cm lesion. IGF-I improved but was still elevated at 456 ng/ml associated with a GH 2.9 ng/ml. Medical treatment with pasireotide was recommended, but the patient temporarily declined further treatment to explore other options with her Naturopath.Gangliocytomas are a rare, benign, slow-growing, differentiated form of neuroblastic tumors of sympathetic nerve fibers, largely extracranial and rarely present in the sellar region, most frequently in association with a PitNET, forming the so-called mixed gangliocytoma PitNET. Definitive diagnosis is established by histological and immunohistochemical studies. Though PitNET components of mixed gangliocytoma PitNET are known to frequently secrete an excess hormone, the ganglionic components also show immunoreactivity for hypothalamic releasing hormones along with other neuroendocrine markers. The histological origin of these mixed tumors is controversial, but the small population of ganglionic cells that express the acidophilic lineage transcription factor, Pit-1, favors the theory of transdedifferentiation of neuroendocrine cells into neuronal elements. Treatment of mixed gangliocytoma- PitNET is similar to other PitNETs and the presence of neural components does not modify aggressiveness or risk of recurrence after surgical resection, as gangliocytomas have a low proliferative index. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Lycopene protects against Bisphenol A induced toxicity on the submandibular salivary glands via the upregulation of PPAR-γ and modulation of Wnt/β-catenin signaling

Background and aimThe submandibular salivary glands (SMG) represent a suitable model for studying epithelial cell growth and differentiation. Bisphenol A (BPA) is a xenoestrogen, synthesized to produce polymers such as polycarbonates and epoxy resins. There are concerns about the occurrence of BPA in food, water as well as its appearance in human tissues and body fluids. Lycopene (LYC) is a carotenoid compound that exerts antioxidant and anti-inflammatory properties. This work was performed to study possible protective effect of LYC against BPA toxicity in SMG. Material and Methods40 albino rats were divided into 4 groups; Group I: served as controls. Group II: rats received LYC (4 mg/kg, p.o), Group III: rats received BPA (10 mg/kg, p.o) and Group IV: rats received LYC (4 mg/kg, p.o) and BPA (10 mg/kg, p.o). All drugs were administered for 45 days then under anesthesia, rats were sacrificed. The SMG specimens were taken for histological and biochemical studies. ResultsBPA resulted in a significant rise of malondialdehyde, tumor necrosis factor-α and interleukine-1β. In contrast, the tissue levels of glutathione and PPAR-γ were significantly decreased. BPA activated Wnt/β-catenin pathway evidenced by upregulating WNT3a, β-catenin and c-myc expression. Moreover, SMG of BPA showed degenerative changes that affected the parenchymal and stromal elements of the glands. The immunohistochemical localization of cytokeratin 5,6 and 18 of BPA rats revealed weak immunostaining of the serous secretory cells, myoepithelial cells and ductal cells. Upon treatment with LYC, glutathione and PPAR-γ were restored. ConclusionLYC acted as a protective agent against BPA-induced pathological changes in SMG.

Sex differences in adrenal cortex beta-catenin immunolocalisation of the Saharan gerbil, Libyan jird (Meriones libycus, Lichtenstein, 1823).

The adrenal cortex provides adequate steroidogenic responses to environmental changes. However, in desert rodents, the adrenocortical activity varies according to several factors especially sex, age, and seasonal variations. Herein, we examined the sex differences in the adrenal cortex activity and explored the involvement of sex hormones in the regulation of this function in Libyan jird Meriones libycus. Twenty-four adult male and female animals weighing 109-110 g were captured in the breeding season and equally assigned into control and gonadectomised groups. Animal euthanasia was performed 50 days after the gonadectomy. Adrenal gland was processed for structural and immunohistochemistry study of b-catenin, whereas plasma was used for cortisol assay. The results showed that female adrenal gland weight was heavier than male and gonadectomy reduced this dimorphism. The adrenal cortex thickness was greater in the female than in the male, mainly due to significant development of the zona fasciculata. Females presented higher cell density in fasciculata and reticularis zones. The plasma cortisol was higher in females than in males. The immunolocalisation of beta-catenin showed that the expression was particularly glomerular in both sexes. However, in the female, the immunostaining was present in the zona reticularis while it was absent in the control male. Orchiectomy reduced zona glomerulosa cell density and induced hypertrophy of zona reticularis characterised by strong beta-catenin immunoreactivity. Results indicated that sex hormones had a major role in the regulation of the Saharan gerbil's adrenal homeostasis by modulating beta-catenin signalling. Androgens seem to inhibit the Wnt-beta-catenin pathway and oestrogens are activators of the adrenal inner zones.

Maternal alloxan exposure induces damage in rat offspring lumbar vertebrae and protective role of arachidonic acid.

Vertebral abnormalities in offspring of diabetic mothers make major challenges worldwide and were not sufficiently studied before. To investigate the effects of alloxan-induced diabetes on rats' lumbar vertebrae, and to assess the potential beneficial impact of arachidonic acid. Pregnant rats were randomly equally divided into four groups: control, alloxan-induced diabetes received alloxan injection 150 mg∕kg, alloxan + arachidonic acid group received arachidonic acid 10 μg∕animal then given alloxan injection, and arachidonic acid group received it, until offspring age of three weeks. Six male offspring from each group were included in this study at ages of newborn, three-week-old, two-month-old, and their body measurements were recorded. Lumbar vertebrae and pancreas specimens were examined by light microscopy, morphometry, transmission electron microscopy (TEM), and immunohistochemistry for insulin expression. In alloxan-induced diabetes newborn, three-week-old, and two-month-old rats, body measurements were significantly declined, histomorphometry of 6th lumbar vertebrae revealed disorganized chondrocytes, with vacuolated cytoplasm, empty lacunae, diminished matrix staining, with areas devoid of cells. TEM showed shrunken reserve and proliferative cells, with irregular nuclei, and damaged mitochondria. In contrast, alloxan + arachidonic acid group had cytoarchitecture of lumbar vertebrae that were like control group. Histomorphometry of pancreas in alloxan-induced diabetes group showed significant reduction in pancreatic islets number and surface area, damaged pancreatic islet cells appeared atrophied with apoptotic nuclei, and very weak insulin immunostaining. Whereas alloxan + arachidonic acid group displayed healthy features of pancreatic islets, which resembled control group, with strong insulin immunostaining. Arachidonic acid mitigated alloxan-induced diabetes by its antidiabetic activity.

Claudin 18.2 is a potential therapeutic target for zolbetuximab in pancreatic ductal adenocarcinoma.

BACKGROUNDPancreatic ductal adenocarcinoma (PDAC) is frequently diagnosed and treated in advanced tumor stages with poor prognosis. More effective screening programs and novel therapeutic means are urgently needed. Recent studies have regarded tight junction protein claudin 18.2 (CLDN18.2) as a candidate target for cancer treatment, and zolbetuximab (formerly known as IMAB362) has been developed against CLDN18.2. However, there are few data reported thus far related to the clinicopathological characteristics of CLDN18.2 expression for PDAC.AIMTo investigate the expression of CLDN18.2 in PDAC patients and subsequently propose a new target for the treatment of PDAC.METHODSThe Cancer Genome Atlas, Genotype-Tissue Expression, Gene Expression Omnibus, and European Genome-phenome Archive databases were first employed to analyze the CLDN18 gene expression in normal pancreatic tissue compared to that in pancreatic cancer tissue. Second, we analyzed the expression of CLDN18.2 in 93 primary PDACs, 86 para-cancer tissues, and 13 normal pancreatic tissues by immunohistochemistry. Immunostained tissues were assessed applying the histoscore. subsequently, they fell into two groups according to the expression state of CLDN18.2. Furthermore, the correlations between CLDN18.2 expression and diverse clinicopathological characteristics, including survival, were investigated.RESULTSThe gene expression of CLDN18 was statistically higher (P < 0.01) in pancreatic tumors than in normal tissues. However, there was no significant correlation between CLDN18 expression and survival in pancreatic cancer patients. CLDN18.2 was expressed in 88 (94.6%) of the reported PDACs. Among these tumors, 50 (56.8%) cases showed strong immunostaining. The para-cancer tissues were positive in 81 (94.2%) cases, among which 32 (39.5%) of cases were characterized for strong staining intensities. Normal pancreatic tissue was identified solely via weak immunostaining. Finally, CLDN18.2 expression significantly correlated with lymph node metastasis, distant metastasis, nerve invasion, stage, and survival of PDAC patients, while there was no correlation between CLDN18.2 expression and localization, tumor size, patient age and sex, nor any other clinicopathological characteristic. CONCLUSIONCLDN18.2 expression is frequently increased in PDAC patients. Thus, it may act as a potential therapeutic target for zolbetuximab in PDAC.

Effect of Nigella sativa L. Seed on the Kidney of Monosodium Glutamate Challenged Rats.

Monosodium glutamate (MSG) consumption is responsible for a wide spectrum of health hazards including nephrotoxicity. The search for phytochemical strategies having broad safety profile to counter MSG toxicity is worthwhile. Nigella sativa L. seed (NSS) is very promising in this regard owing to its antioxidant and cytoprotective nature. Therefore, we attempted to investigate the potential protective effect of NSS on MSG-induced renal toxicity in rats. To accomplish this objective, fifteen adult Wistar albino rats were randomly and equally divided into three groups for 21 days: the control group received no treatment, MSG group supplemented with MSG at a dose of 30 g/kg feed, and MSG + NSS group supplemented with MSG at the same previous dose in conjugation with NSS at a dose of 30 g/kg feed. MSG and its combination with NSS failed to cause any significant difference in the kidney function parameters in comparison with the control. A significant elevation in lipid peroxides (LPO) level, glutathione-S-transferase activity and total antioxidant capacity (TAC) and a significant reduction in superoxide dismutase activity were found in MSG group. LPO level and TAC in MSG intoxicated rats significantly normalized by NSS ingestion. NO level showed absence of significant difference among all experimental groups. MSG elicited histopathological lesions such as decreased glycoprotein content and fibrosis however, NSS succeeded in enhancing all these features. MSG group showed positive glutathione reductase and superoxide dismutase 2 immuno-expression whereas, MSG + NSS group showed weak immunostaining. A significant increase in the number of apoptotic cells was observed in MSG group compared to the control. On the other hand, MSG + NSS group exhibited a significant decrease in the number of apoptotic cells. NSS mitigated MSG-induced renal impairments by ameliorating oxidative stress and exerting anti-apoptotic effect.

Presence of Protease Inhibitor 9 and Granzyme B in Healthy and Pathological Human Corneas.

Simple SummaryDetailed knowledge of the structure and properties of the human cornea is a prerequisite not only for the treatment of various corneal diseases but also for successful corneal transplantation and its long-term survival after grafting. Using various cell and molecular biology approaches, we found in cornea the protease inhibitor 9. This protein, known to be present in other human tissues but not yet reported in cornea, is directly involved in the immune response after transplantation. Together with its inhibitor (granzyme B), we localized this protein, especially in the superficial and inner cornea layers. This localization indicates that protease inhibitor 9 protein may be involved in protecting the cornea from external damage, but also in protection against immune cells inducing corneal graft rejection. Furthermore, we have shown on pathological corneal samples from corneal melting and herpes virus keratitis that the increased expression of both proteins is linked to these diseases. These experiments and their results represent an important contribution to the basic research of cornea biological properties with direct overlap into clinical practice.The aim of this study was to find out whether protease inhibitor 9 (PI-9) and granzyme B (GrB) molecules that contribute to immune response and the immunological privilege of various tissues are expressed in healthy and pathological human corneas. Using cryosections, cell imprints of control corneoscleral discs, we showed that PI-9 was expressed particularly in the endothelium, the superficial and suprabasal epithelium of healthy corneas, limbus, and conjunctiva. GrB was localized in healthy corneal and conjunctival epithelium, while the endothelium showed weak immunostaining. The expression of PI-6 and GrB was confirmed by qRT-PCR. Increased expression levels of the PI-9 and GrB genes were determined when the corneas were cultured with proinflammatory cytokines. Fluorescent and enzymatic immunohistochemistry of pathological corneal explants (corneal melting and herpes virus keratitis) showed pronounced PI-9, GrB, human leucocyte antigen (HLA)-DR, and leukocyte-common antigen (CD45) signals localized in multicellular stromal infiltrates and inflammatory cells scattered in the corneal stroma. We conclude that increased expression of the PI-9 and GrB proteins under pathological conditions and their upregulation in an inflammatory environment indicate their participation in immune response of the cornea during the inflammatory process.

BRAF mutation testing for patients diagnosed with stage III or stage IV melanoma: practical guidance for the Australian setting.

Targeted therapy (BRAF inhibitor plus MEK inhibitor) is now among the possible treatment options for patients with BRAF mutation-positive stage III or stage IV melanoma. This makes prompt BRAF mutation testing an important step in the management of patients diagnosed with stage III or IV melanoma; one that can help better ensure that the optimal choice of systemic treatment is initiated with minimal delay. This article offers guidance about when and how BRAF mutation testing should be conducted when patients are diagnosed with melanoma in Australia. Notably, it recommends that pathologists reflexively order BRAF mutation testing whenever a patient is found to have American Joint Committee on Cancer (AJCC)/Union for International Cancer Control (UICC) stage III or IV melanoma (i.e., any metastatic spread beyond the primary tumour) and that patient's BRAF mutation status is hitherto unknown, even if BRAF mutation testing has not been specifically requested by the treating clinician (in Australia, Medicare-subsidised BRAFV600 mutation testing does not need to be requested by the treating clinician). When performed in centres with appropriate expertise and experience, immunohistochemistry (IHC) using the anti-BRAF V600E monoclonal antibody (VE1) can be a highly sensitive and specific means of detecting BRAFV600E mutations, and may be used as a rapid and relatively inexpensive initial screening test. However, VE1 immunostaining can be technically challenging and difficult to interpret, particularly in heavily pigmented tumours; melanomas with weak, moderate or focal BRAFV600E immunostaining should be regarded as equivocal. It must also be remembered that other activating BRAFV600 mutations (including BRAFV600K), which account for ∼10-20% of BRAFV600 mutations, are not detected with currently available IHC antibodies. For these reasons, if available and practicable, we recommend that DNA-based BRAF mutation testing always be performed, regardless of whether IHC-based testing is also conducted. Advice about tissue/specimen selection for BRAF mutation testing of patients diagnosed with stage III or IV melanoma is also offered in this article; and potential pitfalls when interpreting BRAF mutation tests are highlighted.

Piezoelectric Microvibration Mitigates Estrogen Loss-Induced Osteoporosis and Promotes Piezo1, MicroRNA-29a, and Wnt3a Signaling in Osteoblasts.

Biophysical stimulation alters bone-forming cell activity, bone formation and remodeling. The effect of piezoelectric microvibration stimulation (PMVS) intervention on osteoporosis development remains uncertain. We investigated whether 60 Hz, 120 Hz, and 180 Hz PMVS (0.05 g, 20 min/stimulation, 3 stimulations/week for 4 consecutive weeks) intervention affected bone integrity in ovariectomized (OVX) mice or osteoblastic activity. PMVS (120 Hz)-treated OVX mice developed fewer osteoporosis conditions, including bone mineral density loss and trabecular microstructure deterioration together with decreased serum resorption marker CTX-1 levels, as compared to control OVX animals. The biomechanical strength of skeletal tissue was improved upon 120 Hz PMVS intervention. This intervention compromised OVX-induced sparse trabecular bone morphology, osteoblast loss, osteoclast overburden, and osteoclast-promoting cytokine RANKL immunostaining and reversed osteoclast inhibitor OPG immunoreactivity. Osteoblasts in OVX mice upon PMVS intervention showed strong Wnt3a immunoreaction and weak Wnt inhibitor Dkk1 immunostaining. In vitro, PMVS reversed OVX-induced loss in von Kossa-stained mineralized nodule formation, Runx2, and osteocalcin expression in primary bone-marrow stromal cells. PMVS also promoted mechanoreceptor Piezo1 expression together with increased microRNA-29a and Wnt3a expression, whereas Dkk1 rather than SOST expression was repressed in MC3T3-E1 osteoblasts. Taken together, PMVS intervention promoted Piezo1, miR-29a, and Wnt signaling to upregulate osteogenic activity and repressed osteoclastic bone resorption, delaying estrogen deficiency-induced loss in bone mass and microstructure. This study highlights a new biophysical remedy for osteoporosis.

E-Cadherin expression in human tumors: a tissue microarray study on 10,851 tumors

BackgroundThe E-Cadherin gene (CDH1, Cadherin 1), located at 16q22.1 encodes for a calcium-dependent membranous glycoprotein with an important role in cellular adhesion and polarity maintenance.MethodsTo systematically determine E-Cadherin protein expression in normal and cancerous tissues, 14,637 tumor samples from 112 different tumor types and subtypes as well as 608 samples of 76 different normal tissue types were analyzed by immunohistochemistry in a tissue microarray format.ResultsE-Cadherin was strongly expressed in normal epithelial cells of most organs. From 77 tumor entities derived from cell types normally positive for E-Cadherin, 35 (45.5%) retained at least a weak E-Cadherin immunostaining in ≥99% of cases and 61 (79.2%) in ≥90% of cases. Tumors with the highest rates of E-Cadherin loss included Merkel cell carcinoma, anaplastic thyroid carcinoma, lobular carcinoma of the breast, and sarcomatoid and small cell neuroendocrine carcinomas of the urinary bladder. Reduced E-Cadherin expression was linked to higher grade (p = 0.0009), triple negative receptor status (p = 0.0336), and poor prognosis (p = 0.0466) in invasive breast carcinoma of no special type, triple negative receptor status in lobular carcinoma of the breast (p = 0.0454), advanced pT stage (p = 0.0047) and lymph node metastasis in colorectal cancer (p < 0.0001), and was more common in recurrent than in primary prostate cancer (p < 0.0001). Of 29 tumor entities derived from E-Cadherin negative normal tissues, a weak to strong E-Cadherin staining could be detected in at least 10% of cases in 15 different tumor entities (51.7%). Tumors with the highest frequency of E-Cadherin upregulation included various subtypes of testicular germ cell tumors and renal cell carcinomas (RCC). E-Cadherin upregulation was more commonly seen in malignant than in benign soft tissue tumors (p = 0.0104) and was associated with advanced tumor stage (p = 0.0276) and higher grade (p = 0.0035) in clear cell RCC, and linked to advanced tumor stage (p = 0.0424) and poor prognosis in papillary RCC (p ≤ 0.05).ConclusionE-Cadherin is consistently expressed in various epithelial cancers. Down-regulation or loss of E-Cadherin expression in cancers arising from E-Cadherin positive tissues as well as E-Cadherin neo-expression in cancers arising from E-Cadherin negative tissues is linked to cancer progression and may reflect tumor dedifferentiation.