Disrupting Development: Unraveling the Interplay of Aryl Hydrocarbon Receptor (AHR) and Wnt/ß-Catenin Pathways in Kidney Development Under the Influence of Environmental Pollutants.

Understanding the intricate molecular mechanisms governing aryl hydrocarbon receptor (AHR) and Wnt/ß-Catenin pathways crosstalk is of paramount importance for elucidating normal development. We investigated the repercussions of aberrant activation of these signaling pathways on kidney development. HEK-293 cells were subjected to AHR and Wnt activators and inhibitors for 3 and 24 h. Subsequently, pregnant adult female BALB/c mice were administered treatments at gestation day 9 (GD-9), and embryos were analyzed at GD-18 using a combination of cellular, molecular, stereological, and histopathological techniques. Our results demonstrated a noteworthy escalation in oxidative stress and gene expression endpoints associated with apoptosis. Moreover, stereological analyses exhibited alterations in cortex, proximal tubule, and kidney tissue vessels volumes. Remarkably, co-treatment with 6-formylindolo [3,2-b] carbazole (FICZ) and cadmium (Cd) resulted in a significant reduction in glomerulus volume, while elevating the volumes of distal tubule, Henle loop, and connective tissue, compared to the control group. Histopathological investigations further confirmed structural changes in the loop of Henle and proximal tubule, alongside a decline in glomerular volume. Additionally, the expression levels of AHR and Ctnnb1 genes significantly increased in the Cd-treated group compared to the control group. Enhanced expression of apoptosis-related genes, including Bcl-x, Bax, and Caspase3, along with alterations in mitochondrial membrane potential and cytochrome C release, was observed. In contrast, Gsk3 gene expression was significantly decreased. Our findings robustly establish that chemical pollutants, such as Cd, disrupt the AHR and Wnt/ß-Catenin physiological roles during developmental stages by inhibiting the metabolic degradation of FICZ.

Reduced Tumor Volume and Increased Necrosis of Human Breast Tumor Xenograft in Mice Pretreated by a Cocktail of Three Specific Anti-HER2 scFvs.

PURPOSE: We aimed to assess the effects of a cocktail comprising three specific antiHER2 scFvs on breast tumor formation in a xenograft mouse model and to evaluate quantitative changes in the tumor using stereological analysis. METHODS: Three specific anti-HER2 phage antibodies were produced from a scFv-library using phage display technology. The cell binding capacities of the antibodies were assessed via FACS analysis. Soluble forms of the antibodies were prepared by infecting HB2151-E. coli cells and purified using a centrifugal ultrafiltration method. The purification process was evaluated by SDSPAGE analysis. Two forms of scFv cocktails were prepared, soluble scFv and phage-scFv cocktail, which contained an equal amount/phage of the three specific anti-HER2 antibodies. Inbred female BALB/c mice were pretreated with 5 and 20 mg/kg of the soluble scFv cocktail and 1011 phagescFv cocktail/kg. The mice were then injected with 2×106 SKBR-3 human breast cancer cells. Total tumor, inflammatory and non-inflammatory volumes were estimated using the Cavalieri principle after preparing photomicrograph slides. RESULTS: The anti-HER2 scFvs showed significantly higher binding to SKBR-3 cells compared to the isotype control. SDS-PAGE analysis confirmed the high purification of the scFvs. Stereological analysis revealed that the group pretreated with 20 mg/kg of the soluble scFv cocktail exhibited the highest reductions in total tumor volume, non-inflammatory volume, and inflammatory volume, with reductions of 73%, 78%, and 72%, respectively, compared to PBS-pretreated mice (P-value < 0.0001). The volumetric ratio of necrotic tissue to total tumor volume increased by 2.2-fold and 2- fold in the 20mg/kg of soluble scFv cocktail and phage-scFv cocktail groups, respectively, compared to the PBS-treated mice (P-value < 0.05). CONCLUSION: Pre-treatment with a 20 mg/kg anti-HER2 scFv cocktail resulted in a significant reduction in tumor volume and increased necrotic area in a human breast cancer xenograft model, indicating the remarkable anti-tumor effect of the cocktail in vivo.

Evaluation of oral and topical bovine colostrum compared to mesalamine in the treatment of animal model of acetic acid-induced ulcerative colitis.

Background: Oxidative activity and inflammatory responses have been shown to play a pivotal role in the pathogenesis of ulcerative colitis (UC). Colostrum is a natural product with anti-inflammatory and antioxidative properties. Methods: UC was induced in 37 Sprague Dawley rats by administration of a 2 mL enema of 3% acetic acid (AA). The control groups received no treatment during the study, while the experimental groups received either oral or rectal administration of 100 mg/kg 5-aminosalicylic acid, or oral or rectal administration of 300 mg/kg of colostrum. Histopathological and serological analyses were performed 7 days following treatment. Results: A significant decrease in weight was seen in all rats except for the test groups receiving colostrum (P<0.001). After treatment, the level of superoxide dismutase increased more significantly in the test groups that received colostrum (P<0.05). All test groups had a reduction in C-reactive protein and white blood cell levels. The colostrum test groups also showed a decrease in inflammation rate, ulceration, destruction, disorganization, and crypt abscess of the colonic mucosa. Conclusions: The findings of this study show that the administration of colostrum can improve the pathological changes of the intestinal mucosa, as well as inflammatory responses, in animal models of UC. Further studies at both preclinical and clinical levels are suggested to confirm these findings.

Bilirubin improves renal function by reversing the endoplasmic reticulum stress and inflammation in the kidneys of type 2 diabetic rats fed high-fat diet.

Diabetic kidney disease (DKD), as a chronic diabetes-induced complication, is considered the most frequent leading cause of end-stage renal disease (ESRD). Regarding the observed protective effects of bilirubin, as a potential endogenous antioxidant/anti-inflammatory compound, against DKD progression, we planned to evaluate the effects of bilirubin administration on endoplasmic reticulum (ER) stress and inflammation in type 2 diabetic (T2D) rats fed high-fat diet (HFD). In this regard, thirty 8-week adult male Sprague Dawley rats were divided into five groups (n = 6). T2D and obesity were induced by streptozotocin (STZ) (35 mg/kg) and HFD (700 kcal/day), respectively. Bilirubin treatment was carried out for 6- and 14-week intervals (10 mg/kg/day), intraperitoneally. Then, the expression levels of ER stress-related genes (i.e. binding immunoglobulin protein (Bip), C/EBP homologous protein (Chop), and spliced x-box-binding protein 1 (sXbp1), as well as nuclear factor-κB (NF-κB) were analyzed using quantitative Real-time PCR experiments. Moreover, histopathological and stereological changes of kidney and its related structures were investigated for the studied rats. Bip, Chop, and NF-κB expression levels were significantly decreased under bilirubin treatment, while sXbp1 was up-regulated following the bilirubin administration. More interestingly, glomerular constructive damages seen in HFD-T2D rats, were considerably improved in the animals received bilirubin. Stereological assessments also revealed that bilirubin could desirably reverse the mitigation of kidney's total volume and its related structures, such as cortex, glomeruli, and convoluted tubules. Taken together, bilirubin has potential protective/ameliorative effects on DKD progression, especially through alleviating the renal ER stress and inflammatory responses in T2D rats with injured kidneys. In this era, clinical benefits of mild hyperbilirubinemia can be considered in human DKD.

Using Data Mining and Association Rules for Early Diagnosis of Esophageal Cancer.

From 17,000 new cases of esophageal cancer worldwide during last year, 16,000 proved to be fatal. Late or incorrect diagnosis of esophageal cancer cases increases its fatality rate. Today, a data-mining technique can predict the course of the disease with the help of an upto-date technology. With this knowledge, we can reduce esophageal cancer mortality. This study aims to find an association between general characteristics, screening tests, and esophageal cancer based on raw data from the Cancer Research Center within-person interviews, using data mining and classification techniques on mortality. The 5-year medical records of 512 esophageal cancer patients and those with problems related to this cancer, with 50 functional characteristics, were included in this model. In order to provide a prognostic and rule discovery model for esophageal cancer suffering, we used preprocessing EM Algorithm. After accurate identification of the data, WEKA Software tools and Java programming language was used to create Association Rule Classifier and Apriori algorithm for the associated rule discovery. We created 6 significant rules of the association for classification generated by rule miner with 95% and 91% confidence based on screening tests and general attributes, respectively. These substantial rules showed significant association between age, history of medication, smoking, gender, carcinoembryonic antigen (CEA), creatinine, WBCs, and Platelets. The findings of this study can be used as a clue for physicians to consider patients with these characteristics as people who are more likely to develop esophageal cancer and help them for early diagnosis of patients. Keywords:Data mining, esophageal cancer, association rule, healthcare.

Controversial Role of Folic Acid on Diabetic Auditory Neuropathy.

OBJECTIVE: Diabetic auditory neuropathy (DAN) is a common complication of diabetes that seriously affects the quality of life in patients. In this study, we investigate the role of folic acid in the treatment of DAN in an experimental rat model. METHODS: Thirty-two Sprague-Dawley rats were equally divided into four groups: group 1, normal; group 2, diabetic rats; and groups 3 and 4, diabetic rats treated with folic acid (40 and 80 mg/kg, respectively). We used some tools to investigate the therapeutic effect of folic acid on DAN. We evaluated auditory brain stem response (ABR), estimated the volume and number of spiral ganglion and the volume of stria vascularis and spiral ligament by the stereological method, and measured the blood levels of homocysteine (HCY), malondialdehyde (MDA), and superoxide dismutase (SOD). RESULTS: Our study showed that folic acid treatment was not significantly effective in improving structural and functional disorders in DAN, even though its effectiveness in reducing HCY (P < 0.001) and MDA (P < 0.05) as oxidative biomarkers was significant. CONCLUSION: Folic acid is not effective in relieving morphological and functional disorders in DAN.

Development of Human Recombinant Antibodies Against ROR1 Tumor Antigen.

Background: Receptor tyrosine kinase-like orphan receptor 1 (ROR1) is an oncofetal antigen expressed on many types of cancer cells, but not normal adult cells. ROR1 antigen contributes to cancer development and progression by several signaling pathways. ROR1 expression has been associated with tumor growth, survival, and metastasis. In this study specific human recombinant antibodies were selected against ROR1 antigen for their use in cancer immunotherapy. Methods: Phage display technology was used to produce phage antibody from a human scFv library. Phage concentration was determined to confirm the phage rescue process. Panning procedure was performed to isolate specific scFv clones against ROR1 epitope. Phage ELISA was done to evaluate the reactivity of the selected scFvs. Results: Two specific human scFvs with frequencies of 20% and 25% were selected against ROR1 peptide. The antibodies showed specific reaction to the corresponding epitopes in phage ELISA. Discussion: Cancer targeted therapy using human specific antibodies is a new strategy, which is used in cancer therapy. The selected specific scFvs that target ROR1 epitope are human antibodies that originated from a human library and have the potential to be used in clinic in cancer immunotherapy of ROR1 positive tumors without induction of human anti mouse antibody (HAMA) response.

A Quantitative Study on the Protective Effects of Resveratrol against Bisphenol A-induced Hepatotoxicity in Rats: A Stereological Study.

Background: Bisphenol A (BPA) is a widely used chemical with toxic effects on the liver. Resveratrol (RES) is an herbal compound with protective properties. This study aimed to investigate the protective effects of RES on the liver in rats exposed to BPA. Methods: This study was conducted in 2018 in Shiraz, Iran. Thirty Sprague-Dawley male rats were divided into five groups: a control group (distilled water), a sham group (olive oil as a BPA solvent), a BPA group (50 mg/kg), an RES group (100 mg/kg), and a RES+BPA group (50 mg/kg+100 mg/kg). Olive oil, BPA, and RES were administered to the animals via gavage for eight weeks. After eight weeks, the animals' livers were removed, and stereological measurements were made to obtain the total liver volume, portal triad volumes, hepatocyte nucleus and cytoplasm volumes, hepatocyte numbers, sinusoidal space volumes and lengths, and Kupffer cell (KC) numbers. The data were analyzed using the one-way analysis of variance test. Results: The hepatocyte number, the total liver volume, and hepatocyte nucleus and cytoplasm volumes in the BPA group decreased by 41% (P<0.001), 18% (P<0.001), 32% (P=0.030), and 37% (P=0.014), respectively. The number of KCs and the length of sinusoids in the BPA group were increased compared with all the other groups (P<0.001). Our histological study revealed vacuolization, sinusoidal space dilatation, and congestion in the BPA group. Conclusion: In this study, the RES group, compared with the BPA group, exhibited a decrease in the total volume and length of sinusoids and the number of KCs. Additionally, the RES group showed an increase in the total liver volume, hepatocyte nucleus and cytoplasm volumes, portal triad volumes, and hepatocyte numbers after oral administration.

Stereological study of the placental structure in abortion-prone mice model (CBA/J×DBA/2J).

Recurrent spontaneous abortion (RSA) is an important reproductive health issue defined as the loss of two or more consecutive pregnancies before the 20th week of gestation, affecting 2-5% of couples. This study aimed to evaluate the volume, number of cells, and length of the vessels in the placenta in normal and abortion-prone (AP) pregnant mice on gestational day (gd) 13.5. Fetal and placental tissues of female CBA/J mated DBA/2J (AP group) and BALB/c (normal pregnant group) were collected and prepared for stereological assessments on gd13.5. The volumes of the placenta and its main layers decidua basalis (Db), junctional zone (Jz), and labyrinth zone (Lz) were investigated. The number of spongiotrophoblast cells, glycogen cells, giant cells, trophoblast cells, lymphocytes, and neutrophils were estimated as well. The AP group showed a reduction in the volume of the placenta (48.7%) and its components. Moreover, the number of spongiotrophoblast cells (66.7%), glycogen cells (76.2%), giant cells (73.3%), and trophoblast cells (81.4%) was decreased in AP compared to normal pregnant (NP) mice. Also, in AP group recognized a 10-fold increase in the number of lymphocytes and a four-fold increase in the number of neutrophils in comparison to the NP group (p < 0.05). Activation of different immune cell types might induce systemic inflammation at the feto-maternal interface, resulting in impaired placenta formation and abortion.

Co-exposure to an Aryl Hydrocarbon Receptor Endogenous Ligand, 6-Formylindolo[3,2-b]carbazole (FICZ), and Cadmium Induces Cardiovascular Developmental Abnormalities in Mice.

6-Formylindolo[3,2-b]carbazole (FICZ) is a signal substance and an endogenous activator of aryl hydrocarbon receptor (AHR). Cadmium (Cd) is an environmental pollutant that can activate both AHR and Wnt/ß-catenin signaling pathways. We aimed to determine how dysregulated signaling through AHR-Wnt/ß-catenin cross-talk can influence mice heart development. Mice fetuses were exposed to Cd alone or in combination with FICZ in gestation day (GD) 0. In GD18, fetuses were harvested and randomly divided into two parts for stereological and molecular studies. Stereological and tessellation results revealed that when fetuses were co-exposed with FICZ and Cd, abnormalities were synergistically raised. In the presence of FICZ, mRNA expression levels of Wnt/ß-catenin target genes significantly enhanced, especially when animals co-treated with FICZ and Cd. Based on these findings, we propose that chemical pollutants can interfere with the normal function of AHR that has a physiological role in regulating Wnt/ß-catenin during cardiogenesis.

Stereological comparison of intraprostatic injection of alcohol and bleomycin with finasteride gavages in rats.

INTRODUCTION: Resection of the prostate is one of the standard surgical treatments for symptomatic Benign Prostatic Hyperplasia (BPH). To evaluate minimally invasive treatments, intraprostatic injections of ethanol and bleomycin were compared with oral finasteride administration in rats with BPH. MATERIAL AND METHODS: The rats were divided into six groups. The control rats received no BPH/no treatment. BPH was induced using injections of testosterone (2 mg/day/rat for 4 weeks) in groups II-VI. After 4 weeks, Group II received no treatment while Group III received oral finasteride (10 mg/kg/day). Moreover, Groups IV-VI received a single injection of ethanol (95%), bleomycin (5 mg/kg) and normal saline 25 mm3 in each ventral lobe of the prostate respectively. Two weeks after the injections, the ventral lobes underwent a quantitative stereological study. RESULTS: The volume of the ventral lobes, glandular epithelium, fibromuscular tissue and microvessles increased by 1.7, 3.1, 2.4 and 1.6 times in BPH rats respectively (P <0.01). Alcohol or bleomycin injection in PBH rats induced drastic recession of the increased volume of the ventral lobe, glandular epithelium and fibromuscular tissue (P <0.01). Regarding the BPH+alcohol group, the glandular epithelium volume restored to the normal values of the control rats (P <0.01). BPH+finasteride also incited an atrophic change in the volume of the whole prostate and glandular epithelium, but not the fibromuscular tissue and microvessels (P <0.01). CONCLUSIONS: Injection of alcohol and bleomycin (approximately 10% of the volume of ventral prostatic) as well as consuming finasteride can induce a reduction of 1/3, 1/4 and 1/5 in the hypertrophied gland respectively.

Induction of oxidative stress, suppression of glucose-induced insulin release, ATP production, glucokinase activity, and histomorphometric changes in pancreatic islets of hypothyroid rat.

Thyroid hormones have important role in metabolism and impairment of glucose metabolism and insulin secretion has been shown in hypothyroid rats but the exact mechanisms for this defect are poorly understood. The aim of this study was to investigate the effect of hypothyroidism on oxidative stress parameters, insulin secretory pathway and histomorphometric changes of pancreas. In the isolated islets of the control and methimazole -treated hypothyroid insulin secretion and content, ATP production, Glucokinase, and hexokinase specific activity and kATP and L-type channels sensitivity were assayed. In order to determine oxidative stress parameters, antioxidant enzymes and lipid peroxidation were measured in pancreatic homogenates. Histomorphometric changes and histochemistry of the islet in both groups were compared. Results showed that plasma glucose and insulin concentration and their area under the curve during IPGTT in hypothyroid group were respectively higher and lower than the controls. In the hypothyroid islets, glucose stimulated insulin secretion, ATP production, hexokinase and glucokinase activities were decreased. Hypothyroid induced a significant increased lipid peroxidation, and decreased the antioxidant enzyme activity. Compared with the control group, insulin antibody positivity, the total volume of the pancreas, islets, and the total number as well as the mean volume of the beta cells were also significantly decreased in the hypothyroid group. These findings indicate that oxidative stress produced under hypothyroidism could have a role in progression of pancreatic ß-cell dysfunction, reduced beta cell mass and decreased glucokinase activity, impairing glucose tolerance and insulin secretion.

Verapamil, a Calcium-Channel Blocker, Improves the Wound Healing Process in Rats with Excisional Full-Thickness Skin Wounds Based on Stereological Parameters.

OBJECTIVE: Calcium can play noticeable roles in the wound-healing process, such as its effects on organization of F-actinin collagen bundles by fibroblasts at the injury site. In addition, calcium-channel blockers such as verapamil have antioxidant activity by increasing nitric oxide production that promotes angiogenesis, proliferation of fibroblasts, and endothelial cells in the skin-regeneration process. Therefore, in this study, the authors' objective was to investigate the effects of verapamil on the process of wound healing in rat models according to stereological parameters. MATERIALS AND METHODS: In this experimental study, 36 male Wistar rats were randomly divided into 3 groups (n = 12): the control group that received no treatment, gel-base-treated group, and the 5% verapamil gel-treated group. Treatments were done every 24 hours for 15 days. Wound closure rate, volume densities of the collagen bundles and the vessels, vessel's length density and mean diameter, and fibroblast populations were estimated using stereological methods and were analyzed by the Kruskal-Wallis and Mann-Whitney U tests; P < .05 was considered statistically significant. RESULTS: The verapamil-treated group showed a faster wound closure rate in comparison with control and gel-base groups (P = .007 and P = .011). The numerical density of fibroblasts, volume density of collagen bundles, mean diameter, and volume densities of the vessels in the verapamil group were significantly higher than those in the control and the base groups (P < .005). CONCLUSIONS: The authors showed that verapamil has the ability to improve wound healing by enhancing fibroblast proliferation, collagen bundle synthesis, and revascularization in skin injuries.

Scrotal Cooling to Protect Against Cisplatin-induced Spermatogenesis Toxicity: Preliminary Outcome of an Experimental Controlled Trial.

OBJECTIVE: To investigate the protective effects of scrotal cooling on cisplatin-induced gonadal toxicity in an animal model. METHODS: Twenty-one male BALB/c mice were divided into 3 groups. The cisplatin group received 2 cycles of cisplatin (2.5 mg/kg/day for 5 days with 16 days of recovery) intraperitoneally, and the cisplatin + cooling group received the same regimen of cisplatin with a cooling protocol: cooling induction for 30 minutes before injection and cooling for 60 minutes after injection. Mice in control group were given an injection of 2 mL normal saline intraperitoneally. After 35 days of recovery (1 cycle of spermatogenesis), the volume of the testes (Cavalieri method), volume density of the tubules and epithelium (point-counting method), and number of cells (optical dissector method) were estimated. RESULTS: The volume of the testes, tubules, and epithelium was reduced between 61% and 66%, and the number of the spermatogonia, spermatocytes, round spermatids, and long spermatids was reduced between 70% and 93% in cisplatin group compared with that of control mice. Cisplatin affected spermatids to a greater extent, and Sertoli cells to a lesser extent than the other cells. The volume and number of the cells were reduced in the cisplatin + cooling group but to a lesser extent compared with those of mice in the cisplatin group. Sertoli cells were more intact in the cisplatin + cooling group compared with those of the control group. CONCLUSION: Scrotal cooling during cisplatin administration seems to have beneficial effects on spermatogenesis. Scrotal cooling may hold promise as a way to protect fertility.

Topical Nicotinamide Improves Tissue Regeneration in Excisional Full-Thickness Skin Wounds: A Stereological and Pathological Study.

BACKGROUND: Nicotinamide (NA), the active form of vitamin-B3, is hypothesized to have positive effects on the process of wound healing; it has anti-inflammatory, antioxidant, and immunomodulatory properties, as well as an epithelization inducing action. OBJECTIVES: In the present study, we aimed to determine the effects of topical administration of NA on skin wounds, based on histomorphometrical and pathological criteria. MATERIALS AND METHODS: In this study, 36 male Sprague-Dawley rats (220 ± 20 g each), with 1 cm(2) circular full-thickness wounds on their backs were divided into three groups (n = 12): NA group, was treated daily with a Nicotinamide 2% gel , untreated group (control), and base group, which were treated with the vehicle (base) of the gel (carboxymethylcellulose). Skin biopsies were prepared for microscopic analyses. Inflammation, granulation tissue formation, ulceration, epithelization, wound closure rate, fibroblast proliferation, collagen synthesis, and vascularization were studied criteria. RESULTS: The results revealed that besides improving the wound healing by its anti-inflammatory, antioxidant, and epithelization inducing effects, NA also improved tissue regeneration through the increment of fibroblast proliferation, collagen synthesis, and vascularization. CONCLUSIONS: In spite of the few reported side effects, NA can be introduced as an effective agent on the wound healing process, an adjuvant therapy and possibly a treatment by itself. However, its chemical characteristics, as well as possible adverse effects warrants further research.

A simple stereological method for estimating the number and the volume of the pancreatic beta cells.

CONTEXT: Number of the beta cells as well as their volume is a fundamental variable in the pancreatic research. OBJECTIVES: This study describes a simple method for stereological estimation of the volume of the pancreas, the total volume of the islets, and the total number, as well as the mean volume, of the beta cells in rat. METHODS: The primary volume of the pancreas was measured using the immersion method. Also, tissue shrinkage was estimated and the final pancreas volume was corrected without the need for serial sectioning. A limited number (i.e., 10-13) of the isotropic uniform random slabs of each pancreas was embedded in the same block. One 5 µm and one 20 µm sections were obtained and stained with a modified aldehyde fuchsin. The point counting, optical disector and point-sampled intercept methods were used to estimate the volume density of the islet, the numerical density of the beta cells, and the mean cell volume, respectively. RESULTS: After calculating the tissue shrinkage, the mean primary volume of the pancreas (628 mm3; CV: 25%) was corrected to obtain the final volume (442 mm3; CV: 39%). The mean islet volume was reported as 3.8 mm3 (CV: 22%). Besides, the total number of the beta cells was estimated as 2.9x10(6) (CV: 20%). Moreover, the mean volume of the beta cells was obtained (1,158 µm3; CV: 9%). CONCLUSIONS: It takes almost one hour to estimate the volume of the islets and two hours to count the cells and estimate the intercepts per animal.