TLR4, IgA and EpCAM Expression in Colorectal Cancer and Their Possible Association with Microbiota as a Pathogenic Factor; An Immunohistochemical and Genetic Study.

BACKGROUND: The pathogenesis of inflammatory bowel disease (IBD) and colorectal cancer (CRC) is thought to be related to immune response against gut microbiota. TLR4, IgA, and EpCAM have a role in intestinal local immune response and their altered expression related to both IBD and CRC. Lipopolysaccharide (LPS) is the main activator of TLR4. The objective of this study is to evaluate the possible role of intestinal microbiota in the pathogenesis of IBD and CRC through expression of TLR4, IgA and EpCAM. METHODS: One hundred five cases were divided into (Group 1/ Control: 10 sections of normal colonic mucosa, Group 2/CRC: 51 cases, Group 3/IBD: 44 cases). Immunohistochemistry for TLR4, IgA, and EpCAM was done. LPS was assessed in all groups. TLR4 gene and protein expression were assessed in colorectal cancer cell line by RT-PCR and immunocytochemistry. RESULTS: There was a significant correlation between TLR4 and tumor grade (P value 0.003 and 0.01 respectively). A significant correlation was found between IgA expression and T stage (P value 0.02) and between EpCAM expression and histologic type (P value 0.02). In comparison of CRC patients to controls; there was a statistically significant different expression of TLR4 positivity, IgA positivity and EpCAM (P value <0.001, 0.004, <0.001 respectively). Patients with CRC were compared to colitis patients and there was a statistically significant different expression of IgA positivity and EpCAM expression (P value <0.001). There was significant higher expression of TLR4 in CRC cell line than the fibroblast by both PCR and immunocytochemistry (P-value: 0.003 and 0.024 respectively). LPS level in CRC patients was significantly higher than the control and IBD groups (P values <0.001 and <0.001 respectively). CONCLUSION: TLR4, IgA, EpCAM expression in both CRC and IBD might be related to the pathogenic role of microbiota and could represent potential prevention modalities and therapeutic targets.

Impact of atorvastatin and mesenchymal stem cells combined with ivermectin on murine trichinellosis.

Trichinellosis is one of the global food-borne parasitic diseases that can cause severe tissue damage. The traditionally used drugs for the treatment of trichinellosis have limited efficacy against the encysted larvae in the muscular phase of the disease. Therefore, this study aimed to evaluate the role of atorvastatin and mesenchymal stem cells combined with ivermectin against different phases of Trichinella in experimentally infected mice. A total of 120 male Swiss albino mice were divided into two major groups (n = 60 of each), intestinal and muscular phases. Then, each group was subdivided into 10 subgroups (n = 6); non-infected control, infected non-treated control, infected ivermectin treated, infected atorvastatin treated, infected mesenchymal stem cells treated, infected combined ivermectin and atorvastatin treated, infected combined mesenchymal stem cells and ivermectin treated, infected combined mesenchymal stem cells and atorvastatin treated, infected combined mesenchymal stem cells and a full dose of (ivermectin and atorvastatin) treated, and infected combined mesenchymal stem cells and half dose of (ivermectin and atorvastatin) treated. Mice were sacrificed at days 5 and 35 post-infection for the intestinal and muscular phases, respectively. The assessment was performed through many parameters, including counting the adult intestinal worms and muscular encysted larvae, besides histopathological examination of the underlying tissues. Moreover, a biochemical assay for the inflammatory and oxidative stress marker levels was conducted. In addition, levels of immunohistochemical CD31 and VEGF gene expression as markers of angiogenesis during the muscular phase were investigated. The combined mesenchymal stem cells and atorvastatin added to ivermectin showed the highest significant reduction in adult worms and encysted larvae counts, the most noticeable improvement of the histopathological changes, the most potent anti-inflammatory (lowest level of IL-17) and anti-angiogenic (lowest expression of CD31 and VEGF) activities, and also revealed the highly effective one to relieve the oxidative stress (lowest level of SOD, GSH, and lipid peroxidase enzymes). These observed outcomes indicate that adding mesenchymal stem cells and atorvastatin to ivermectin synergistically potentiates its therapeutic efficacy and provides a promising candidate against trichinellosis.

Interpretation of Farnesoid X Receptor Immunohistochemical Expression in Discriminating Hepatocellular Carcinoma from Its Non-Neoplastic Mimics as an Adjunct to Glypican 3.

AIMS: Differentiating hepatocellular carcinoma (HCC) and non-neoplastic lesions may be challenging. Immunohistochemistry (IHC) can help in the comparative morphologic evaluation of HCC and its mimics. Farnesoid X receptor (FXR) is a nuclear metabolic receptor essential for bile salts homeostasis and other biological functions of liver cells. Preliminary studies have shown that FXR can be useful for diagnosing HCC. This study aimed to assess the role of Farnesoid X receptor (FXR) combined with Glypican 3 (GPC3) in differentiation between HCC and non-neoplastic hepatic lesions. MATERIAL AND METHODS: Immunohistochemistry of GPC3 and FXR was performed in 38 cases of primary hepatic lesions using an automated immunohistochemical stainer. The study included 17 primary HCC cases and 21 non-neoplastic hepatic lesions (5 cases were focal nodular hyperplasia, 7 were regenerative nodules and 9 were dysplastic nodules). RESULTS: The percentage of positive GPC3 and low or negative FXR expression was significantly higher in HCC cases than non-neoplastic hepatic lesions (P value <0.001). The sensitivity and specificity of GPC3 in differentiating HCC from non-neoplastic hepatic lesions were 70.6% and 85.7%, respectively, while the sensitivity and specificity of FXR were 58.8% and 100%, respectively. CONCLUSION: The present work revealed that FXR could be combined with GPC3 in distinguishing between HCC and non-neoplastic hepatic lesions with improved specificity rather than using an individual marker.

Clinico-Pathological Features and Immunohistochemical Comparison of p16, p53, and Ki-67 Expression in Muscle-Invasive and Non-Muscle-Invasive Conventional Urothelial Bladder Carcinoma.

INTRODUCTION: The identification of bladder detrusor muscle invasion in urothelial cancer is essential for prognosis and management. We studied the clinical, histological, and immunohistochemical expression of p16, p53, and Ki-67 in urothelial detrusor muscle-invasive bladder cancer (MIBC) and urothelial non-detrusor muscle-invasive bladder cancer (NMIBC) in Egyptian patients. METHODS: Sixty-two bladder urothelial cancer cases obtained through TURBT were included and divided into two groups: (MIBC, stage T2) and NMIBC (T1). Tissue blocks were recut and re-examined microscopically; then, the immunostaining of p16, p53, and Ki-67 was performed to compare both groups and evaluate the 13% cut-off for Ki-67, 20% for p53, and p16 intensity in various conditions aided by telepathology technology. RESULTS AND CONCLUSION: Hematuria was the main clinical first presentation, with no significant difference between either group. The mean age was 61.6 years, with male predominance (52 males and 10 females). The absence of papillary histological pattern was associated with a higher stage, including detrusor muscle invasion (p = 0.000). The overall average percent of p53 immunostaining was 12.9%, revealing no significant difference between MIBC and NMIBC when a cut-off of 20% was implicated. The Ki-67 expression was correlated with higher grade and muscle invasion; however, no association was found with the other two markers' expression. The negative immunostaining of p16 was associated with low grade and NMIBC in the case of the preservation of the papillary pattern. We recommend further studies on the cut-off of widely used markers and more immunohistochemical and genetic studies on the p16(INK4A), taking into consideration the histological pattern of conventional carcinomas.

Ultrastructural changes and IgA modulatory effect of commercial prebiotic and probiotic in murine giardiasis.

Giardiasis, a parasitic infection of the gastrointestinal tract, is prevalent worldwide. The integrity of the intestinal epithelial barrier plays an important defensive role in giardiasis, and as Oral supplementation with prebiotics and probiotics is known to reinforce the intestinal barrier in many gastrointestinal diseases, this study assessed the effects of prebiotic and probiotic supplementation in giardiasis and compared the results with those obtained after nitazoxanide therapy. Swiss albino male lab-bred mice (n = 50) were divided into three major groups; Group I (control group), i.e., negative (noninfected nontreated) and positive controls (infected nontreated); Group II (preventive group), in which mice were provided prebiotic, probiotic, or a combination for 7 days before of infection, and Group III (therapy group), in which mice were administered prebiotic, probiotic, combined supplements and nitazoxanide from day 12 post-infection. The assessment was achieved through Giardia cyst count, histopathological examination and ultrastructure study. Also, Serological and immunohistochemical parameters were done to evaluate the modulation of IgA levels. Oral supplementation with prebiotic and probiotic, either before or after infection (in preventive or therapy groups respectively) resulted in a significant reduction in Giardia cyst shedding. Remarkable histological and ultrastructure improvement in the intestinal changes, along with a significant increase in the serological and immunohistochemical IgA levels, were seen in mice provided combined supplements and nitazoxanide (in therapy group). Thus, our results indicate that combined prebiotic and probiotic supplementation has promising anti-Giardia activity and that it can restore intestinal structures and modulate IgA response, apart from providing synergistic effects when added to nitazoxanide.

Apoptotic changes in the intestinal epithelium of Cryptosporidium-infected mice after silver nanoparticles treatment versus nitazoxanide.

Cryptosporidium has been identified as one of the prevalent opportunistic parasites that cause diarrhea, which may be persistent and fatal. Current chemotherapeutic agents, including nitazoxanide (NTZ), are frequently associated with therapeutic failure, and their roles in the induction of apoptosis in cryptosporidiosis remain to be a topic of debate. Thus, this study aimed to assess the apoptotic changes in cryptosporidiosis in immunocompetent (IC) and immunosuppressed (IS) mice after treatment with silver nanoparticles (AgNPs) and NTZ either alone or after loading. In total, 120 laboratory-bred Swiss albino mice were divided into two groups. Group A included IC mice, while Group B included IS mice. Both groups were divided into six subgroups: noninfected nontreated, infected nontreated, infected AgNP-treated, infected NTZ-treated, infected AgNP-loaded NTZ (full-dose)-treated, and infected AgNP-loaded NTZ (half-dose)-treated. The assessment was achieved through parasitological, histopathological, and apoptotic marker expression evaluation. AgNP-loaded NTZ (different doses) treatment showed the highest oocyst shedding reduction and remarkable improvement in histopathological changes, followed by individual treatment with NTZ and then AgNPs in IC and IS mice. Results of apoptotic marker expression revealed that AgNP-loaded NTZ treatment exhibited a promising role in regulating apoptotic changes in cryptosporidiosis through the expression of the lowest levels of cytochrome C and caspase-3 in IC and IS mice at the end of the experiment. Therefore, AgNP-loaded NTZ can be a potential therapeutic agent against cryptosporidiosis for IC and IS mice.

Linagliptin and Vitamin D3 Synergistically Rescue Testicular Steroidogenesis and Spermatogenesis in Cisplatin-Exposed Rats: The Crosstalk of Endoplasmic Reticulum Stress with NF-κB/iNOS Activation.

This study investigated the therapeutic effect of linagliptin and/or vitamin D3 on testicular steroidogenesis and spermatogenesis in cisplatin-exposed rats including their impact on endoplasmic reticulum (ER) stress and NF-κB/iNOS crosstalk. Cisplatin (7 mg/kg, IP) was injected into adult male albino rats which then were orally treated with drug vehicle, linagliptin (3 mg/kg/day), vitamin D3 (10 µg/kg/day) or both drugs for four weeks. Age-matched rats were used as the control group. Serum samples and testes were collected for further analyses. Cisplatin induced testicular weight loss, deteriorated testicular architecture, loss of germ cells and declined serum and intra-testicular testosterone levels, compared to the control group. There was down-regulation of steroidogenic markers including StAR, CYP11A1, HSD3b and HSD17b in cisplatin-exposed rats, compared with controls. Cisplatin-exposed rats showed up-regulation of ER stress markers in testicular tissue along with increased expression of NF-κB and iNOS in spermatogenic and Leydig cells. These perturbations were almost reversed by vitamin D3 or linagliptin. The combined therapy exerted a more remarkable effect on testicular dysfunction than either monotherapy. These findings suggest a novel therapeutic application for linagliptin combined with vitamin D3 to restore testicular architecture, aberrant steroidogenesis and spermatogenesis after cisplatin exposure. These effects may be attributed to suppression of ER stress and NF-kB/iNOS.