Search results for: Immunohistochemistry

Authors: Zahra F. Al-Khateeb, Shenel Shekerzade, Hasna Boumenar, Siân M. Henson, Jordi L. Tremoleda, A. T. Michael-Titus

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Traumatic brain injury (TBI) is the leading cause of disability and death in young adults and also increases the risk ofneurodegeneration. The mechanisms linking moderate to severe TBI to neurodegeneration are not known. It has been proposed that cellular senescence inductionpost-injury could amplify neuroinflammation and induce long-term changes. The impact of these processes after injury to an immature brain has not been characterised yet. We carried out a controlled cortical impact injury (CCI) in juvenile 1 month-old male CD1 mice. Animals were anesthetised and received a unilateral CCI injury. The sham group received anaesthesia and had a craniotomy. A naïve group had no intervention. The brain tissue was analysed at 5 days and 35 days post-injury using immunohistochemistry and markers for microglia, astrocytes, and senescence. Compared tonaïve animals, injured mice showed an increased microglial and astrocytic reaction early post-injury, as reflected in Iba1 and GFAP markers, respectively; the GFAP increase persisted in the later phase. The senescence analysis showed a significant increase inγH2AX-53BP1 nuclear foci, 8-oxoguanine, p19ARF, p16INK4a, and p53 expression in naïve vs. sham groups and naïve vs. CCI groups, at 5 dpi. At 35 days, the difference was no longer statistically significant in all markers. The injury induced a decrease p21 expression vs. the naïve group, at 35 dpi. These results indicate the induction of a complex senescence response after immature brain injury. Some changes occur early and may reflect the activation/proliferation of non-neuronal cells post-injury that had been hindered, whereas changes such as p21 downregulation may reflect a delayed response and pro-repair processes.

Keywords: cellular senescence, traumatic brain injury, brain injury, controlled cortical impact

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Authors: J. Vinay , Kusumbati Besra, Niharika Pattnaik, Shivaram Prasad Singh, Manjusha Dixit

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Gallbladder cancer (GBC) is rare but highly malignant cancer; its prevalence is more in certain geographical regions and ethnic groups, which include the Northern and Eastern states of India. Previous studies in India have reported genetic predisposition as one of the risk factors in GBC pathogenesis. Although the matrix metalloproteinase-14 (MMP14) is a well-known modulator of the tumor microenvironment and tumorigenesis and TCGA data also suggests its upregulation yet, its role in the genetic predisposition for GBC is completely unknown. We elucidated the role of MMP14 promoter variants as genetic risk factors and their implications in expression modulation. We screened MMP14 promoter variants association with GBC using Sanger’s sequencing in approximately 300 GBC and 300 control subjects and 26 GBC tissue samples of Indian ethnicity. The immunohistochemistry was used to check the MMP14 protein expression in GBC tissue samples. The role of promoter variants on expression levels was elucidated using a luciferase reporter assay. The variants rs1004030 (p-value = 0.0001) and rs1003349 (p-value = 0.0008) were significantly associated with gallbladder cancer. The luciferase assay in two different cell lines, HEK-293 (p = 0.0006) and TGBC1TKB (p = 0.0036) showed a significant increase in relative luciferase activity in the presence of risk alleles for both the single nucleotide polymorphisms (SNPs). Similarly, genotype-phenotype correlation in patients samples confirmed that the presence of risk alleles at rs1004030 and rs1003349 increased MMP14 expression. Overall, this study unravels the genetic association of MMP14 promoter variants with gallbladder cancer, which may contribute to pathogenesis by increasing its expression.

Keywords: gallbladder cancer, matrix metalloproteinase-14, single nucleotide polymorphism, case control study, genetic association study

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Authors: Ana Claudia Muniz Rennó, Karina Nogueira

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This study aimed to investigate the in vivo tissue response of the introduction of the bioactive mesh (BM) scaffolds using a model of tibial bone defect implants in rats. Although a previous in vivo study demonstrated a highly positive response of particulate bioactive materials in the morphological and biomechanical properties of the bone callus, the effects of material with superior bioactivity, present in form of meshes have not been studied yet. Eighty male Wistar rats with 3 mm tibial defects were used. Animals were divided into four groups: intact group (IG) – tibia without any injury; bone defect day zero (0dD) – bone defects, sacrificed immediately after injury; bone defect control group (CG) – bone defects without any filler and bone defect filled with BM scaffold. The animals of BM and CG groups were sacrificed 15, 30 and 45 days post-injury to compare the temporal-special effects of the scaffolds on bone healing. The histological analysis revealed an organized newly formed bone at 30 and 45 days post-surgery in the BM. Also, this group presented an increased COX-2 expression on days 15 and 30 post-surgery. Furthermore, the immunohistochemistry analysis revealed that, BM presented a positive immunoexpression of RUNX-2 during all periods evaluated. The biomechanical analysis revealed that at 15 day after surgery, no significant statistically difference was observed between BM and CG and both groups had significantly higher values of maximal load compared to 0dG and significantly lower values than IG. On days 30 and 45 post-surgery, BM presented statistically lower values of maximal load compared to the CG. Nevertheless, at the same periods, BM did not show statistically significant difference compared to the IG maximal load values (p > 0, 05). Our results revealed that the implantation of the BM scaffolds was effective in stimulating newly bone formation.

Keywords: bone, biomaterials, scaffolds, cartilage

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Authors: Chodidjah, Edi Dharmana, Hardhono, Sarjadi

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Breast cancer treatment options including surgery, radiation therapy, chemotherapy, and immunotherapy have not been effective. Besides, they have side effects. Keladi Tikus (Typhonium flagelliforme) has been shown to improve immune system, suppress tumor growth and induce apoptosis. One of the parameters for immune system, tumor growth and apoptosis is IFNγ (Interferon γ), VEGF (Vascular Endothelial Growth Factor) and Caspase 3 respectively. The aim of this study was to examine the effect of the administration of Keladi Tikus tuber extract at the dose of 200 mg/kgBW, 400 mg/KgBW, and 800 mg/kgBW on the level of IFNγ, VEGF and caspase 3 expression. In this experimental study using post test randomized control group design, 24 CH3 mice with tumor were randomly divided into 4 groups including control group and treated groups: Treated with 0.2 cc extract of Keladi Tikus at the dose of 200 mg/kgBW, 400 mg/kgBW, 800 mg/kgBW, respectively for 30 days. On day 31 the lymphatic tissue was taken and evaluated for its level of IFNγ, using ELISA. The tumor tissue was taken and subjected to immunohistochemistry staining for VEGF and caspase 3 expression evaluation. The data on IFNγ, VEGF and Caspase 3 expression were analyzed using One Way Anova with significant level of 0.05. One Way Anova resulted in p<0.05. LSD test showed that the level of IFNγ and Caspase 3 for control group was different from that of treated groups. There was no significant different between the treated group of 400 mg/KgBW and 800mg/KgBW. VEGF expressions for all the treated groups were significant. In conclusion, the oral administration of ethanolic extract of Keladi Tikus (Typhonium flagelliforme) at the dose of 200mg/kgBW, 400 mg/kgBW,800 mg/kgBW increases IFNγ, Caspase 3 and decreases VEGF expression in C3H mice with adenocarsinoma mamma.

Keywords: Typhonium flagelliforme, IFNγ, caspase 3, VEGF

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Authors: Saad Al-Shibli, Nasser Amjad, Muna Al Kubaisi, Norra Harun, Shaikh Mizan

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Leptin is a multifunctional hormone produced mainly by adipocyte. Leptin and its receptor have long been found associated with breast cancer. The main aim of this study is to investigate the correlation between Leptin/Leptin receptor and the clinicopathological features of breast cancer. Blood samples for ELISA, tissue samples from tumors and adjacent breast tissue were taken from 51 women with breast cancer with a control group of 40 women with a negative mammogram. Leptin and Leptin receptor in the tissues were estimated by immunohistochemistry (IHC). They were localized at the subcellular level by immunocytochemistry using transmission electron microscopy (TEM). Our results showed significant difference in serum leptin level between control and the patient group, but no difference between pre and post-operative serum leptin levels in the patient group. By IHC, we found that the majority of the breast cancer cells studied, stained positively for leptin and leptin receptors with co-expression of leptin and its receptors. No significant correlation was found between leptin/leptin receptors expression with the race, menopausal status, lymph node metastasis, estrogen receptor expression, progesterone receptor expression, HER2 expression and tumor size. Majority of the patients with distant metastasis were associated with high leptin and leptin receptor expression. TEM views both Leptin and Leptin receptor were found highly concentrated within and around the nucleus of the cancer breast cells, indicating nucleus is their principal seat of actions while the adjacent breast epithelial cells showed that leptin gold particles are scattered all over the cell with much less than that of the cancerous cells. However, presence of high concentration of leptin does not necessarily prove its over-expression, because it could be internalized from outside by leptin receptor in the cells. In contrast, leptin receptor is definitely over-expressed in the ductal breast cancer cells. We conclude that reducing leptin levels, blocking its downstream tissue specific signal transduction, and/or blocking the upstream leptin receptor pathway might help in prevention and therapy of breast cancer.

Keywords: breast cancer, expression, leptin, leptin receptors

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Authors: Freda Halim

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Endometriosis in bowel is rare condition, about 3-37% of endometriosis cases. Most of bowel endometriosis rising in the rectosigmoid (90% of bowel endometriosis). The incidence of caecal endometriosis is very low ( < 5% of bowel endometriosis) and almost never causing acute small bowel obstruction. The aim of this paper is to show that although bowel obstruction caused by caecal endometriosis is difficult to diagnose as it is rare, and may require laparotomy to make definite diagnosis, but it should be considered in infertile female patient. The case is 37 years old woman infertile woman with intestinal obstruction with pre-operative diagnosis total acute small bowel obstruction caused by right colonic mass, with sepsis as the complication. Before the acute small bowel obstruction, she complained of chronic right lower quadrant pain with chronic constipation alternate with chronic diarrhea, symptoms that happened both in bowel endometriosis and colorectal malignancy. She also complained of chronic pelvic pain and dysmenorrhea. She was married for 10 years with no child. The patient was never diagnosed with endometriosis and never seek medical attention for infertility and the chronic pelvic pain. The patient underwent Abdominal CT Scan, with results: massive small bowel obstruction, and caecal mass that causing acute small bowel obstruction. Diagnosis of acute small bowel obstruction due to right colonic mass was made, and exploratory laparotomy was performed in the patient. During the laparotomy, mass at caecum and ileocaecal that causing massive small bowel obstruction was found and standard right hemicolectomy and temporary ileostomy were performed. The pathology examination showed ectopic endometriosis lesions in caecum and ileocaecal valve. The histopathology also confirmed with the immunohistochemistry, in which positive ER, PR, CD 10 and CD7 was found the ileocaecal and caecal mass. In the second operation, reanastomosis of the ileum was done 3 months after the first operation. The chronic pelvic pain is decreasing dramatically after the first and second operation. In conclusion, although bowel obstruction caused by caecal endometriosis is a rare cause of intestinal obstruction, but it can be considered as a cause in infertile female patient

Keywords: acute, bowel obstruction, caecum, endometriosis

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Authors: S. Subramaniam, J. S. A. Rao, P. Ramdas, K. R. Selvaduray, N. M. Han, M. K. Kutty, A. K. Radhakrishnan

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Immune system is a complex system where the immune cells have the capability to respond against a wide range of immune challenges including cancer progression. However, in the event of cancer development, tumour cells trigger immunosuppressive environment via activation of myeloid-derived suppressor cells and T regulatory (Treg) cells. The Treg cells are subset of CD4+ T lymphocytes, known to have crucial roles in regulating immune homeostasis and promoting the establishment and maintenance of peripheral tolerance. Dysregulation of these mechanisms could lead to cancer progression and immune suppression. Recently, there are many studies reporting on the effects of natural bioactive compounds on immune responses against cancer. It was known that tocotrienol-rich-fraction consisting 70% tocotrienols and 30% α-tocopherol is able to exhibit immunomodulatory as well as anti-cancer properties. Hence, this study was designed to evaluate the effects of gamma-tocotrienol (G-T3) supplementation on T-reg cells in a syngeneic mouse model of breast cancer. In this study, female BALB/c mice were divided into two groups and fed with either soy oil (vehicle) or gamma-tocotrienol (G-T3) for two weeks followed by inoculation with tumour cells. All the mice continued to receive the same supplementation until day 49. The results showed a significant reduction in tumour volume and weight in G-T3 fed mice compared to vehicle-fed mice. Lung and liver histology showed reduced evidence of metastasis in tumour-bearing G-T3 fed mice. Besides that, flow cytometry analysis revealed T-helper cell population was increased, and T-regulatory cell population was suppressed following G-T3 supplementation. Moreover, immunohistochemistry analysis showed that there was a marked decrease in the expression of FOXP3 in the G-T3 fed tumour bearing mice. In conclusion, the G-T3 supplementation showed good prognosis towards breast cancer by enhancing the immune response in tumour-bearing mice. Therefore, gamma-T3 can be used as immunotherapy agent for the treatment of breast cancer.

Keywords: breast cancer, gamma tocotrienol, immune suppression, supplement

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Authors: Xinxiu Li, Chuqian Zheng, Yanyan Qian, Hong Fan

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Objectives: In hepatocellular carcinoma (HCC), oncogenes are continuously and robustly transcribed due to aberrant expression of essential components of the trans-acting super-enhancers (SE) complex. Preclinical and clinical trials are now being conducted on small-molecule inhibitors that target core-transcriptional components, including as transcriptional bromodomain protein 4 (BRD4) and cyclin-dependent kinase 7 (CDK7), in a number of malignant tumors. This study aims to explore whether co-overexpression of BRD4 and CDK7 is a potential marker of worse prognosis and a combined therapeutic target in HCC. Methods: The expression pattern of BRD4 and CDK7 and their correlation with prognosis in HCC were analyzed by RNA sequencing data and survival data of HCC patients from TCGA and GEO datasets. The protein levels of BRD4 and CDK7 were determined by immunohistochemistry (IHC), and survival data of patients were analyzed using the Kaplan-Meier method. The mRNA expression levels of genes in HCC cell lines were evaluated by quantitative PCR (q-PCR). CCK-8 and colony formation assays were conducted to assess cell proliferation of HCC upon treatment with BRD4 inhibitor JQ1 or/and CDK7 inhibitor THZ1. Results: It was shown that BRD4 and CDK7 were often overexpressed in HCCs and were associated with poor prognosis of HCC by analyzing the TCGA and GEO datasets. BRD4 or CDK7 overexpression was related to a lower survival rate. It's interesting to note that co-overexpression of CDK7 and BRD4 was a worse prognostic factor in HCC. Treatment with JQ1 or THZ1 alone had an inhibitory effect on cell proliferation; however, when JQ1 and THZ1 were combined, there was a more notable suppression of cell growth. At the same time, the combined use of JQ1 and THZ1 synergistically suppresses the expression of HCC driver genes. Conclusion: Our research revealed that BRD4 and CDK7 coupled can be a useful biomarker in HCC prognosis and the combination of JQ1 and THZ1 can be a promising therapeutic therapy against HCC.

Keywords: BRD4, CDK7, cell proliferation, combined inhibition

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Authors: Ahmed Atwa, Ashraf Hafez, Mohamed Abdelhameed, Adel Nabeeh, Mohamed Dawaba, Tamer Helmy

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Introduction and Objective: Tumour staging and grading do not usually reflect the future behavior of Wilms' tumor (WT) regarding mortality. Therefore, in this study, P53, Ki67 and cyclin A immunohistochemistry were used in a trial to predict WT cancer-specific survival (CSS). Methods: In this nonconcurrent cohort study, patients' archived data, including age at presentation, gender, history, clinical examination and radiological investigations, were retrieved then the patients were reviewed at the outpatient clinic of a tertiary care center by history-taking, clinical examination and radiological investigations to detect the oncological outcome. Cases that received preoperative chemotherapy or died due to causes other than WT were excluded. Formalin-fixed, paraffin-embedded specimens obtained from the previously preserved blocks at the pathology laboratory were taken on positively charged slides for IHC with p53, Ki67 and cyclin A. All specimens were examined by an experienced histopathologist devoted to the urological practice and blinded to the patient's clinical findings. P53 and cyclin A staining were scored as 0 (no nuclear staining),1 (<10% nuclear staining), 2 (10-50% nuclear staining) and 3 (>50% nuclear staining). Ki67 proliferation index (PI) was graded as low, borderline and high. Results: Of the 75 cases, 40 (53.3%) were males and 35 (46.7%) were females, and the median age was 36 months (2-216). With a mean follow-up of 78.6±31 months, cancer-specific mortality (CSM) occurred in 15 (20%) and 11 (14.7%) patients, respectively. Kaplan-Meier curve was used for survival analysis, and groups were compared using the Log-rank test. Multivariate logistic regression and Cox regression were not used because only one variable (cyclin A) had shown statistical significance (P=.02), whereas the other significant factor (residual tumor) had few cases. Conclusions: Cyclin A IHC should be considered as a marker for the prediction of WT CSS. Prospective studies with a larger sample size are needed.

Keywords: wilms’ tumour, nephroblastoma, urology, survival

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Authors: Aditya Arya, Hairin Taha, Ataul Karim Khan, Nayiar Shahid, Hapipah Mohd Ali, Mustafa Ali Mohd

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This study has investigated the antidiabetic and antioxidant potential of Pseudovaria macrophylla bark extract on streptozotocin–nicotinamide induced type 2 diabetic rats. LCMS-QTOF and NMR experiments were done to determine the chemical composition in the methanolic bark extract. For in vivo experiments, the STZ (60 mg/kg/b.w, 15 min after 120 mg/kg/1 nicotinamide, i.p.) induced diabetic rats were treated with methanolic extract of Pseuduvaria macrophylla (200 and 400 mg/kg∙bw) and glibenclamide (2.5 mg/kg) as positive control respectively. Biochemical parameters were assayed in the blood samples of all groups of rats. The pro-inflammatory cytokines, antioxidant status and plasma transforming growth factor βeta-1 (TGF-β1) were evaluated. The histological study of the pancreas was examined and its expression level of insulin was observed by immunohistochemistry. In addition, the expression of glucose transporters (GLUT 1, 2 and 4) were assessed in pancreas tissue by western blot analysis. The outcomes of the study displayed that the bark methanol extract of Pseuduvaria macrophylla has potentially normalized the elevated blood glucose levels and improved serum insulin and C-peptide levels with significant increase in the antioxidant enzyme, reduced glutathione (GSH) and decrease in the level of lipid peroxidation (LPO). Additionally, the extract has markedly decreased the levels of serum pro-inflammatory cytokines and transforming growth factor beta-1 (TGF-β1). Histopathology analysis demonstrated that Pseuduvaria macrophylla has the potential to protect the pancreas of diabetic rats against peroxidation damage by downregulating oxidative stress and elevated hyperglycaemia. Furthermore, the expression of insulin protein, GLUT-1, GLUT-2 and GLUT-4 in pancreatic cells was enhanced. The findings of this study support the anti-diabetic claims of Pseudovaria macrophylla bark.

Keywords: diabetes mellitus, Pseuduvaria macrophylla, alkaloids, caffeic acid

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Authors: Daniela Proca, Yuan Rong, Salvatore Luceno, Jalil Nasibli

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Introduction: Solitary Fibrous Tumors (SFT) have many histologic mimickers and the only way to diagnose it, particularly in an unusual location, such as peripheral nerve trunks, is to use a comprehensive immunohistochemical staining panel. Monoclonal STAT6 immunostain is highly sensitive and specific for SFTs and particularly useful in the diagnosis of difficult SFT cases. Methods: We describe a solitary fibrous tumor (SFT) involving the right branchial plexus in a 66 yo female with 4-year history of slowly growing chest wall mass with recent dysesthesias in fingers 4th and 5th. MRI showed a well-circumscribed heterogenous mass measuring 5.4 x 3.8 x 4.0 cm and encircling peripheral nerves of the branchial plexus; no involvement of the bone or muscle was noted. A biopsy showed a bland spindled and epithelioid proliferation with no significant mitotic activity, no necrosis, and no atypia; peripheral nerve fascicles were encircled by the lesion. The main clinical and pathologic differential diagnosis included peripheral nerve sheath tumor, particularly schwannoma; HE microscopy didn’t show the classic Antoni A and B areas but showed focal subtle nuclear palisading, as well as prominent vessels with hyalinization. Immunohistochemical stains showed focal, weak cytoplasmic S100 positivity in the lesion; CD 34 and Vimentin were strongly and diffusely positive; the neoplastic cells were negative with AE1/AE3, EMA, CD31, SMA, Desmin, Calretinin, HMB-45, Melan A, PAX-8, NSE. The immunohistochemical and histologic pattern was not typical of peripheral nerve sheath tumor. On additional stains, the tumor was positive with STAT-6 and bcl-2 and focally positive with CD99. Given this profile, the final diagnosis was that of a solitary fibrous tumor. Results: NA Conclusion: Very few SFTs involving peripheral nerves and mimicking a peripheral nerve sheath tumor are described in the literature. Although histologically benign on this biopsy, long-term follow-up is required because of the risk of recurrence of these tumors and their uncertain biological behavior.

Keywords: solitary fibrous tumor, pathology, diagnosis, immunohistochemistry

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Authors: Wan-Ting Kuo, Yi-Wen Liu, Hsiao-Sheng Liu

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Annual incidence of bladder cancer increases in the world and occurs frequently in the male. Most common type is transitional cell carcinoma (TCC) which is treated by transurethral resection followed by intravesical administration of agents. In clinical treatment of bladder cancer, chemotherapeutic drugs-induced apoptosis is always used in patients. However, cancers usually develop resistance to chemotherapeutic drugs and often lead to aggressive tumors with worse clinical outcomes. Approximate 70% TCC recurs and 30% recurrent tumors progress to high-grade invasive tumors, indicating that new therapeutic agents are urgently needed to improve the successful rate of overall treatment. Nonapoptotic program cell death may assist to overcome worse clinical outcomes. Autophagy which is one of the nonapoptotic pathways provides another option for bladder cancer patients. Autophagy is reported as a potent anticancer therapy in some cancers. First of all, we established a mouse orthotopic bladder tumor formation model in order to create a similar tumor microenvironment. IVIS system and micro-ultrasound were utilized to noninvasively monitor tumor formation. In addition, we carried out intravesical treatment in our animal model to be consistent with human clinical treatment. In our study, we carried out intravesical instillation of the autophagy inducer in mouse orthotopic bladder tumor to observe tumor formation by noninvasive IVIS system and micro-ultrasound. Our results showed that bladder tumor formation is suppressed by the autophagy inducer, and there are no significant side effects in the physiology of mice. Furthermore, the autophagy inducer upregulated autophagy in bladder tissues of the treated mice was confirmed by Western blot, immunohistochemistry, and immunofluorescence. In conclusion, we reveal that a novel autophagy inducer with low side effects suppresses bladder tumor formation in our mouse orthotopic bladder tumor model, and it provides another therapeutic approach in bladder cancer patients.

Keywords: bladder cancer, transitional cell carcinoma, orthotopic bladder tumor formation model, autophagy

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Authors: KuiDong Kang, Hye Bin Yim, Su Ah Kim

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Purpose: Tectorigenin is an isoflavone derived from the rhizome of Belamacanda chinensis. In this study, oxygen-induced retinopathy was used to characterize the anti-angiogenic properties of tectorigenin in mice. Methods: ICR neonatal mice were exposed to 75% oxygen from postnatal day P7 until P12 and returned to room air (21% oxygen) for five days (P12 to P17). Mice were subjected to daily intraperitoneal injection of tectorigenin (1 mg/kg, 10 mg/kg) and vehicle from P12 to P17. Retro-orbital injection of FITC-dextran was performed and retinal flat mounts were viewed by fluorescence microscopy. The Central avascular area was quantified from the digital images in a masked fashion using image analysis software (NIH ImageJ). Neovascular tufts were quantified by using SWIFT_NV and neovascular lumens were quantified from a histologic section in a masked fashion. Immunohistochemistry and Western blot analysis were also performed to demonstrate the anti-angiogenic activity of this compound in vivo. Results: In the retina of tectorigenin injected mouse (10mg/kg), the central non-perfusion area was significantly decreased compared to the vehicle injected group (1.76±0.5 mm2 vs 2.85±0.6 mm2, P<0.05). In vehicle-injected group, 33.45 ± 5.51% of the total retinal area was avascular, whereas the retinas of pups treated with high-dose (10 mg/kg) tectorigenin showed avascular retinal areas of 21.25 ±4.34% (P<0.05). High dose of tectorigenin also significantly reduced the number of vascular lumens in the histologic section. Tectorigenin (10 mg/kg) significantly reduced the expression of vascular endothelial growth factor (VEGF), matrix metalloproteinase-2 (MMP-2), MMP-9, and angiotensin II compared to the vehicle injected group. Tectorigenin did not affect CD31 abundance at any tested dose. Conclusions: Our results show that tectorigenin possesses powerful anti-angiogenic properties and can attenuate new vessel formation in the retina after systemic administration. These results imply that this compound can be considered as a candidate substance for therapeutic inhibition of retinal angiogenesis.

Keywords: tectorigenin, anti-angiogenic, retinopathy, Belamacanda chinensis

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Authors: Lubna Azmi, Ila Shukla, Shyam Sundar Gupta, Padam Kant, C. V. Rao

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To investigate the chemoprotective potential of NBRIQU16 chemotype isolated from Argyreia speciosa (Family: Convolvulaceae) on N-methyl-N-nitro-N-nitrosoguanidine (MNNG) and NaCl-induced gastric carcinomas in Wistar rats. Forty-six male 6-week-old Wistar rats were divided into two groups. Thirty rats in group A were fed with a diet supplemented with 8 % NaCl for 20 weeks and simultaneously given N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) in drinking water at a concentration of 100 ug/ml for the first 17 weeks. After administration of the carcinogen, 200 and 400 mg/kg of NBRIQU16 were administered orally once a day throughout the study. From week 18, these rats were given normal water. From week 21, these rats were fed with a normal diet for 15 weeks. Group B containing 16 rats was fed standard diet for thirty-five days. It served as control. Ten rats from group A were sacrificed after 20 weeks. Scarification of remaining animals was conducted after 35 weeks. Entire stomach and some part of the duodenum were incised parallel to the greater curvature, and the samples were collected. After opening the stomach location and size of tumors were recorded. The number of tumors with their locations and sizes were recorded. Expression of survivin was examined by recording the Immunohistochemistry of the specimens. The treatment with NBRIQU16 significantly reduced the nodule incidence and nodule multiplicity in the rats after MNNG administration. Surviving expression in glandular stomachs of normal rats, of rats in middle induction period, in adenocarcinomas and NBRIQU16 treated tissues adjacent to tumor were 0, 42.0 %, 79.3%, and 36.4 %, respectively. Expression of survivin was significantly different as compared to the normal rats. Histological observations of stomach tissues too correlated with the biochemical observations.These finding powerfully supports that NBRIQU16 chemopreventive effect by suppressing the tumor burden and restoring the activities of gastric cancer marker enzymes on MNNG and NaCl-induced gastric carcinomas in Wistar rats.

Keywords: Argyreia speciosa, gastric carcinoma, immunochemistry, NBRIQU16

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Authors: Eryati Darwin, Arina Widya Murni, Adnil Edwin Nurdin

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Background: Functional dyspepsia (FD) is a highly prevalent and heterogeneous disorder. Most patients with FD complain of symptoms related to the intake of meals. Psychological stress may promote peptic ulcer and had an effect on ulcers associated Hp, and may also trigger worsen symptoms in inflammatory disorders of the gastrointestinal. Cells in mucosal gastric stimulate the production of several cytokines, which might associated with Helicobacter pylori infection. The cascade of biological events leading to stress-induced FD remains poorly understood. Aim of Study: To determine the prion-flammatory cytokine IL-6, and Helicobacter pylori activity on mucosal gastric of FD and their association with psychological stress. Methods: The subjects of this study were dyspeptic patients who visited M. Djamil General Hospital and in two Community Health Centers in Padang. On the basis of the stress index scale to identify psychological stress by using Depression Anxiety and Stress Scale (DASS 42), subjects were divided into two groups of 20 each, stress groups and non-stress groups. All diagnoses were confirmed by review of cortisol and esophagogastroduodenoscopy reports. Gastric biopsy samples and peripheral blood were taken during diagnostic procedures. Immunohistochemistry methods were used to determine the expression of IL-6 and Hp in gastric mucosal. The data were statistically analyzed by univariate and bivariate analysis. All procedures of this study were approved by Research Ethics Committee of Medical Faculty Andalas University. Results: In this study, we enrolled 40 FD patients (26 woman and 14 men) in range between 35-56 years old. Cortisol level of blood FD patients as parameter of stress hormone which taken in the morning was significantly higher in stress group than non-stress group. The expression of IL-6 in gastric mucosa was significantly higher in stress group in compared to non-stress group (p<0,05). Helicobacter pylori activity in gastric mucosal in stress group were significantly higher than non-stress group. Conclusion: The present study showed that psychological stress can induce gastric mucosal inflammation and increase of Helicobacter pylori activity.

Keywords: functional dyspepsia, Helicobacter pylori, interleukin-6, psychological stress

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Authors: Shadia Al-Bahlani, Ruqaya Al-Rashdi, Shadia Al-Sinawi, Maya Al-Bahri

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Background: Breast cancer is the most common cancer in women worldwide. Triple-negative breast cancer (TNBC) is an aggressive type of breast cancer, which is defined by the absence of Estrogen (ER), Progesterone (PR) and Human epidermal growth factor (Her-2) receptors. The calpain system plays an important role in many cellular processes including apoptosis, necrosis, cell signaling and proliferation. The role of clapins in pathogenesis and tumor progression has been studied in certain cancer types; however, its definite role is not yet established in breast cancer especially in the TNBC subtype. Objectives: This study aims to measure calpain-1 expression and correlate this measurement with the proliferating/apoptotic index as well with the prognostic factors in TNBC patients’ tissue. Materials and Methods: Thirty nine paraffin blocks from patients diagnosed with TNBC were used to measure the expression of calpain-1 and Ki-67 (proliferating marker) proteins using immunohistochemistry. Apoptosis was assessed morphological and biochemically using conventional Haematoxylin and Eosin (H&E) staining method and terminal deoxynucleotidyl transferase-mediate dUTP nick and labeling (TUNEL) assay respectively. Data was statistically analyzed using Pearson X2 test of association. Results: Calpain-1 content was visualized in the nucleus of the TNBC cells and its expression varied from low to high among the patients tissue. Calpain expression showed no significant correlation with the proliferating/apoptotic index as well with the clinicopathological variables. Apoptotic counts quantified by H&E staining showed significant association with the apoptotic TUNEL assay, validating both approaches. Conclusion: Although calpain-1 expression showed no significant association with the clinical outcome, its variable level of expression might indicate a hidden role in breast cancer tissue. Larger number of samples and different mode of assessments are needed to fully investigate such role. Exploring the involvement of calpain-1 in cancer progression might help in considering it as a biomarker of breast cancer.

Keywords: breast cancer, calpain, apoptosis, prognosis

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Authors: C. Flynn, Q. Yuan, C. Reinhardt

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Alzheimer’s Disease (AD) is a progressive neurodegenerative disorder affecting broad areas of the cerebral cortex and hippocampus. More than 95% of AD cases are representative of sporadic AD, where both genetic and environmental risk factors play a role. The main pathological features of AD include the widespread deposition of amyloid-beta and neurofibrillary tau tangles in the brain. The earliest brain pathology related to AD has been defined as hyperphosphorylated soluble tau in the noradrenergic locus coeruleus (LC) neurons, characterized by Braak. However, the cause of this pathology and the ultimate progression of AD is not understood. Increasing research points to a connection between the gut microbiota and the brain, and mounting evidence has shown that there is a bidirectional interaction between the two, known as the gut-brain axis. This axis can allow for bidirectional movement of neuroinflammatory cytokines and pathogenic misfolded proteins, as seen in AD. Prebiotics and probiotics have been shown to have a beneficial effect on gut health and can strengthen the gut-barrier as well as the blood-brain barrier, preventing the spread of these pathogens across the gut-brain axis. Our laboratory has recently established a pretangle tau rat model, in which we selectively express pseudo-phosphorylated human tau (htauE14) in the LC neurons of TH-Cre rats. LC htauE14 produced pathological changes in rats resembling those of the preclinical AD pathology (reduced olfactory discrimination and LC degeneration). In this work, we will investigate the effects of pre/probiotic ingestion on AD behavioral deficits, blood inflammation/cytokines, and various brain markers in our experimental rat model of AD. Rats will be infused with an adeno-associated viral vector containing a human tau gene pseudophosphorylated at 14 sites (common in LC pretangles) into 2-3 month TH-Cre rats. Fecal and blood samples will be taken at pre-surgery, and various post-surgery time points. A collection of behavioral tests will be performed, and immunohistochemistry/western blotting techniques will be used to observe various biomarkers. This work aims to elucidate the relationship between gut health and AD progression by strengthening gut-brain relationship and aims to observe the overall effect on tau formation and tau pathology in AD brains.

Keywords: alzheimer’s disease, aging, gut microbiome, neurodegeneration

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Authors: Sarocha Vivatvakin, Thanaporn Ratchataswan, Thiratest Leesutipornchai, Komkrit Ruangritchankul, Somboon Keelawat, Virachai Kerekhanjanarong, Patnarin Mahattanasakul, Saknan Bongsebandhu-Phubhakdi

Abstract:

In this globalization era, much attention has been drawn to various molecular biomarkers, which may have the potential to predict the progression of cancer. Epidermal growth factor receptor (EGFR) is the classic member of the ErbB family of membrane-associated intrinsic tyrosine kinase receptors. EGFR expression was found in several organs throughout the body as its roles involve in the regulation of cell proliferation, survival, and differentiation in normal physiologic conditions. However, anomalous expression, whether over- or under-expression is believed to be the underlying mechanism of pathologic conditions, including carcinogenesis. Even though numerous discussions regarding the EGFR as a prognostic tool in head and neck cancer have been established, the consensus has not yet been met. The aims of the present study are to assess the correlation between the level of EGFR expression and demographic data as well as clinicopathological features and to evaluate the ability of EGFR as a reliable prognostic marker. Furthermore, another aim of this study is to investigate the probable pathophysiology that explains the finding results. This retrospective study included 30 squamous cell laryngeal carcinoma patients treated at King Chulalongkorn Memorial Hospital from January 1, 2000, to December 31, 2004. EGFR expression level was observed to be significantly downregulated with the progression of the laryngeal cancer stage. (one way ANOVA, p = 0.001) A statistically significant lower EGFR expression in the late stage of the disease compared to the early stage was recorded. (unpaired t-test, p = 0.041) EGFR overexpression also showed the tendency to increase recurrence of cancer (unpaired t-test, p = 0.128). A significant downregulation of EGFR expression was documented in advanced stage laryngeal cancer. The results indicated that EGFR level correlates to prognosis in term of stage progression. Thus, EGFR expression might be used as a prevailing biomarker for laryngeal squamous cell carcinoma prognostic prediction.

Keywords: downregulation, epidermal growth factor receptor, immunohistochemistry, laryngeal squamous cell carcinoma

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Authors: Nadia Naseem, Farwa Batool, Nasir Mehmood, AbdulHannan Nagi

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Background: The incidence and mortality of breast cancer vary significantly in geographically distinct populations. In Pakistan, breast cancer has shown an increase in incidence in young females and is characterized by more aggressive behavior. The tumor suppressor TP53 gene is a crucial genetic factor that plays a significant role in breast carcinogenesis. This study investigated the TP53 mutations in molecular subtypes of both nodes negative and positive breast cancer in young Pakistani patients. Material and Methods: p53, Estrogen Receptor (ER), Progesterone Receptor (PR), Her-2 neu and Ki 67 expressions were analyzed immunohistochemically in a series of 75 node negative (A) and 75 node positive (B) young (aged: 19-40 years) breast cancer patients diagnosed between 2014 to 2017 at two leading hospitals of Punjab, Pakistan. Tumor tissue specimens and peripheral blood samples were examined for TP53 mutations by direct sequencing of the gene (exons 4-9). The relation of TP53 mutations to these markers and clinicopathological data was investigated. Results: Mean age of the patients was 32.4 + 9.1 SD. Invasive breast carcinoma was the most frequent histological variant (A=92%, B=94.6%). Grade 3 carcinoma was the commonest grade (A=72%, B=81.3%). Triple negative cases (ER-, PR-, Her-2) formed most of the molecular subtypes (A=44%, B=50.6%). A total of 17.2% (A: 6.6%, B: 10.6%) patients showed TP53 mutations. Mutations were significantly more frequent in triple negative cases (A: 74.8%, B: 62.2%) compared to HER2-positive patients (P < 0.0001). In the multivariate analysis of the whole patient group, the independent prognosticator were triple negative cases (P=0.021), TP53 overexpression by IHC (P=0.001) and advanced-stage disease (P=0.007). No statistically significant correlation between TP53 mutations and clinicopathological parameters was found (P < 0.05). Conclusions: It is concluded that TP53 mutations are infrequently present in breast carcinoma of young Pakistani population and there was no significant correlation between p53 mutation and early onset disease. Immunohistochemically detected TP53 expression in our resource-constrained to set up can be beneficial in predicting mutations at the younger age in our population.

Keywords: immunohistochemistry (IHC), invasive breast carcinoma (IBC), Pakistan, TP53

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Authors: Rania Hanafi Mahmoud Said, Rasha Mohamed Taha

Abstract:

Background: The use of nanoparticles for medication delivery offers the possibility of avoiding the negative effects of systemic antibiotic dosing as well as antibiotic resistance in bacteria. Aim of the study: The goal of this study was to see the efficiency of local administration of tetracycline loaded on nano chitosan in the treatment of the induced infection of the albino rats gingiva with Porphyromonas gingivalis through Immunohistochemical localization of Interleukin-1beta (IL-1β) as a proinflammatory cytokine.Material and methods: Fifty adult male albino rats 150 - 180 grams body weight used in this investigation. Any changes in rats’ weights were detected. The male albino rats were divided haphazardly into five groups as Group I involved ten rats; they served as a normal negative control group. Group II involved ten rats; they were infected once with P.gingivalis that was injected into the interdental gingiva. Group III involved ten rats; they were subjected to the same procedure as group II and then to daily injection at the site of infection with diluted tetracycline powder. Group IV involved ten rats; they were subjected to the same procedure as group II and then to daily injection of nano Chitosan at the site of injection. Group V involved ten rats; they were subjected to the same procedure as group II and then to daily injection of tetracycline loaded on nano Chitosan at the site of injection. After rats had been euthanized, the extraction and preparation of their gingiva were carried out in order to examine histologically and immunohistochemically. Results: The light microscopic results of groups II, III, and IV showed degeneration represented by swollen epithelial cells, collagen fibers dissociation of the connective tissue of lamina propria, and areas of basement membrane discontinuation, while groups I and V showed an almost normal histological picture of gingival tissue. Immunohistochemical results showed a significant difference in Group II and III when compared to control. No significant difference appears in group V when compared to the control (group I). Conclusion: Using nanochitosan as a carrier for tetracycline is a new technology to get over the increasing resistance of tetracycline.

Keywords: immunohistochemistry, P.gingivalis, nano-chitosan, tetracycline, periodontitis

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Authors: Mohammad Nasb, Muhammad Rehan, Chen Hong

Abstract:

Background Meniscus defects critically alter knee function and lead to degenerative changes. Regenerative medicine applications including stem cell transplantation have showed a promising efficacy in finding alternatives to overcome traditional treatment limitations. However, stem cell therapy remains limited due to the substantially reduced viability and inhibitory microenvironment. Since tissue growth and repair are under the control of biochemical and mechanical signals, several approaches have recently been investigated (e.g., low intensity pulsed ultrasound [LIPUS]) to promote the regeneration process. This study employed LIPUS to improve growth and osteogenic differentiation of mesenchymal stem cells derived from human embryonic stem cells to improve the regeneration of meniscus tissue. Methodology: The Mesenchymal stromal cells (MSCs) were transplanted into the epicenter of the injured meniscus in rabbits, which were randomized into two main groups: a treatment group (n=32 New Zealand rabbits) including 4 subgroups of 8 rabbits in each subgroup (LIPUS treatment, MSC treatment, LIPUS with MSC and control), and a second group (n=9) to track implanted cells and their progeny using green fluorescence protein (GFP). GFP consists of the MSC and LIPUS-MSC combination subgroups. Rabbits were then subjected to histological, immunohistochemistry, and MRI assessment. Results: The quantity of the newly regenerated tissue in the combination treatment group that had Ultrasound irradiation after mesenchymal stem cells were better at all end points. Likewise, Tissue quality scores were also greater in knees treated with both approaches compared with controls and single treatment at all end points, achieving significance at twelve and twenty-four weeks [p < 0.05], and [p = 0.008] at twelve weeks. Differentiation into type-I and II collagen-expressing cells were higher in the combination group at up to twenty-four weeks. Conclusions: the combination of mesenchymal stem cells and LIPUS showed greater adhering to the sites of meniscus injury, differentiate into cells resembling meniscal fibrochondrocytes, and improve both quality and quantity of meniscal regeneration.

Keywords: stem cells, regenerative medicine, osteoarthritis, knee

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Authors: Toumi Farah, Charallah Salima

Abstract:

In the Algerian Sahara, goat farming is predominant, and it’s associated with other types of breeding, particularly camel and sheep; it also constitutes a significant proportion of breeding exclusively goat. This Saharan goat is a small ruminant with a black dress with white’s spots, hanging ears, and a coat more or less long. It is known for its hardiness and resistance to adverse conditions of arid zones and its perfect ecophysiological adaptation to harsh environmental conditions. However, pregnancy alterations, particularly abortion, degrade its productivity and cause economic losses, having both direct and indirect effects on animal production, like the costs of veterinary interventions and the reconstitution of livestock. The purpose of this work is to study the histological aspect of the liver of goats’ aborted living under nomadic herds in the region of Béni-Abbès (30° 7' N, 2° 10 'O). The organs were collected in physiological serum, rinsed, and then fixed with formaldehyde (37°, diluted at 10%). After that, these samples were processed for a topographic study. The morphometric study of the liver was performed by using an image analysis and processing software "Image J"; the various measurements obtained are intended to specify the supposed stage of development according to the body weight. The histological structure of the liver shows that the hepatic parenchyma consists of vascular conjunctive spaces surrounded by Glisson’s capsule. The sinusoids and hepatic portal vein are full of red blood cells, representing sinusoidal congestion and a thrombosed vein. At high magnification, the blood vessels show the presence of vascular thrombosis and haemorrhage in some areas of the hepatic parenchyma. Morphometric analysis shows that the number of liver parenchymal cells and the diameter of liver vessels vary according to the stage of development. The results obtained will provide details of the anatomical and cellular elements that can be used in the diagnosis of early or late abortion and late embryonic death. It would be interesting to find, by immunohistochemistry, some inflammatory markers useful for monitoring the progress of pregnancy and bioindicators of fetomaternal distress.

Keywords: aborting goat, arid zone, liver, histopathology

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Authors: Sheikha Turki Alketbi

Abstract:

Objectives: 1. Documenting the spontaneous resolution of AML following the initiation of anti-TB therapy. 2. Presenting an uncommon type of relapse in Acute Myeloid Leukemia. 3. Highlighting the role of immune markers in the diagnosis of Leukemia cutis. 4. Exploring and highlighting the possibility of skin relapse as the exclusive manifestation, even when skin involvement is known secondary manifestation in AML. Background: Spontaneous remission of Acute Myeloid Leukemia (AML) is a rare phenomenon that has only been reported in some case reports, usually following severe infections. Some studies have described the occurrence of tuberculosis (TB) infection with AML, usually after starting chemotherapy. Spontaneous resolution of AML after starting anti TB therapy (ATT), without starting chemotherapy has never been described in the literature. Moreover, Leukemia cutis is another rare skin manifestation of Acute Myeloid Leukemia as a result of infiltration of the skin or subcutaneous tissue by leukemic cells, in which can present during, precedes, after or independently of systemic leukemia. Methods: Here, we present a case of a 13-year-old male who presented with fever, weight loss, lethargy, epistaxis, bruising and dry cough and was later diagnosed with AML. Before initiating leukemia treatment, the patient was tested for TB and was found to have active TB infection. His leukemia treatment was postponed to clear the TB infection and he was commenced on ATT. Two months later, repeat blood film and bone marrow biopsy showed resolution of his AML. The patient remained in remission for 1 month, after which he presented with symmetrical blue purple well-defined round indurated plaques on the chest and thighs. Our differentials were leukemia cutis and Kaposi sarcoma. Results: Skin Biopsy with immune markers done, showed a picture of Acute Myeloid Leukemia. Immunohistochemistry (IHC) showed neoplastic cells diffusely and strongly positive for LCA, CD2, CD31, MPO, CD117, Lysozymes and TDT, and moderately positive for CD34, CD99, CD43 and CD6 And patchy for CD68. Ki67 showed 60% proliferation index. They were negative for the remaining markers. This suggested acute myeloid leukemia (AML). Conclusion: In summary, we present a rare case of TB with AML that resolved after treatment of TB with ATT but relapsed later as leukemia cutis. While skin involvement might occur as a secondary manifestation of AML, Skin relapse could be the only one.

Keywords: Leukemia cutis, Leukemia relapse, Acute Myeloid Leukemia, spontaneous resolution of AML

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Authors: Bright Chukwunwike Uzuegbunam, Wojciech Paslawski, Hans Agren, Christer Halldin, Wolfgang Weber, Markus Luster, Thomas Arzberger, Behrooz Hooshyar Yousefi

Abstract:

There is a need to develop a PET tracer that will enable to diagnosis and track the progression of Alpha-synucleinopathies (Parkinson’s disease [PD], dementia with Lewy bodies [DLB], multiple system atrophy [MSA]) in living subjects over time. Alpha-synuclein aggregates (a-syn), which are present in all the stages of disease progression, for instance, in PD, are a suitable target for in vivo PET imaging. For this reason, we have developed some promising a-syn tracers based on a disarylbisthiazole (DABTA) scaffold. The precursors are synthesized via a modified Hantzsch thiazole synthesis. The precursors were then radiolabeled via one- or two-step radiofluorination methods. The ligands were initially screened using a combination of molecular dynamics and quantum/molecular mechanics approaches in order to calculate the binding affinity to a-syn (in silico binding experiments). Experimental in vitro binding assays were also performed. The ligands were further screened in other experiments such as log D, in vitro plasma protein binding & plasma stability, biodistribution & brain metabolite analyses in healthy mice. Radiochemical yields were up to 30% - 72% in some cases. Molecular docking revealed possible binding sites in a-syn and also the free energy of binding to those sites (-28.9 - -66.9 kcal/mol), which correlated to the high binding affinity of the DABTAs to a-syn (Ki as low as 0.5 nM) and selectivity (> 100-fold) over Aβ and tau, which usually co-exist with a-synin some pathologies. The log D values range from 2.88 - 2.34, which correlated with free-protein fraction of 0.28% - 0.5%. Biodistribution experiments revealed that the tracers are taken up (5.6 %ID/g - 7.3 %ID/g) in the brain at 5 min (post-injection) p.i., and cleared out (values as low as 0.39 %ID/g were obtained at 120 min p.i. Analyses of the mice brain 20 min p.i. Revealed almost no radiometabolites in the brain in most cases. It can be concluded that in silico study presents a new venue for the rational development of radioligands with suitable features. The results obtained so far are promising and encourage us to further validate the DABTAs in autoradiography, immunohistochemistry, and in vivo imaging in non-human primates and humans.

Keywords: alpha-synuclein aggregates, alpha-synucleinopathies, PET imaging, tracer development

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Authors: Josline Salib, Sayed El-Toumy, Abeer Salama, Enayat Omara, Emad Hassan

Abstract:

Diabetic nephropathy is an important cause of morbidity and mortality and is now among the most common causes of end-stage renal failure (ESRF) in developed countries. Thus, the aim of the present study was to investigate the phenolic compounds content of Cichorium intybus aerial parts extracts as well as the therapeutic effects on diabetic nephropathy, oxidative stress, and anti-inflammatory by characterizing biochemical, histopathological changes and immunohistochemistry in an experimental diabetic rat model as compared with Amaryl. Ten known compounds of flavonoids, coumarins and phenolic acid derivatives were isolated from the C. intybus aqueous methanolic extract. Structures of the isolated compounds were established by chromatography, UV and 1D⁄2D 1H⁄ 13C spectroscopy. The aqueous methanol extract of C. intybus aerial parts was administered to Streptozotocin diabetes rats at doses (100 and 200 mg/kg) for 21 days. After treatment, blood glucose, serum insulin, urea, creatinine, and TNF-α were evaluated. Enzymatic scavengers including catalase (CAT), glutathione (GSH), malondialdehyde (MDA) and nitric oxide (NO) were determined to evaluate the oxidative status in the renal tissue. Diabetic rats treated with C. intybus extract showed a dose-dependent reduction of fasting blood glucose and kidney antioxidant status in comparison to the diabetic control group. The extract was able to enhance the antioxidant defenses of the kidney by increasing the reduced GSH and CAT content and decreasing MDA content in addition to significantly decreasing kidney nitric oxide content compared to diabetic control rats. Furthermore, the histopathological findings in C. intybus extract administered rats were observed at markedly lesser extent than the diabetic control group. Also, inducible nitric oxide synthase (iNOS) levels were decreased significantly after the administration of high-dose C. intybus extract in diabetic rats. Showing significant antihyperglycemic and antioxidant properties of C. intybus aerial parts extract, which is attributed to its polyphenolic content, may offer a potential source for the treatment of diabetes.

Keywords: antioxidant activity, anti-diabetic nephropathy, cichorium intybus aerial parts, phenolic compounds

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Authors: Akterono Budiyati, Gita Kusumo, Teguh Putra, Fritzie Rexana, Antonius Kurniawan, Aru Sudoyo, Ahmad Utomo, Andi Utama

Abstract:

The presence of the programmed death ligand-1 (PD-L1) has been used in multiple clinical trials and approved as biomarker for selecting patients more likely to respond to immune checkpoint inhibitors. However, the expression of PD-L1 is regulated in different ways, which leads to a different significance of its presence. Positive PD-L1 within tumors may result from two mechanisms, induced PD-L1 expression by T-cell presence or genetic mechanism that lead to constitutive PD-L1 expression. Amplification of PD-L1 genes was found as one of genetic mechanism which causes an increase in PD-L1 expression. In case of colorectal cancer (CRC), targeting immune checkpoint inhibitor has been recommended for patients with microsatellite instable (MSI). Although the correlation between PD-L1 expression and MSI status has been widely studied, so far the precise mechanism of PD-L1 gene activation in CRC patients, particularly in MSI population have yet to be clarified. In this present study we have profiled 61 archived formalin fixed paraffin embedded CRC specimens of patients from Medistra Hospital, Jakarta admitted in 2010 - 2016. Immunohistochemistry was performed to measure expression of PD-L1 in tumor cells as well as MSI status using antibodies against PD-L1 and MMR (MLH1, MSH2, PMS2 and MSH6), respectively. PD-L1 expression was measured on tumor cells with cut off of 1% whereas loss of nuclear MMR protein expressions in tumor cells but not in normal or stromal cells indicated presence of MSI. Subset of PD-L1 positive patients was then assessed for copy number variations (CNVs) using single Tube TaqMan Copy Number Assays Gene CD247PD-L1. We also observed KRAS mutation to profile possible genetic mechanism leading to the presence or absence of PD-L1 expression. Analysis of 61 CRC patients revealed 15 patients (24%) expressed PD-L1 on their tumor cell membranes. The prevalence of surface membrane PD-L1 was significantly higher in patients with MSI (87%; 7/8) compared to patients with microsatellite stable (MSS) (15%; 8/53) (P=0.001). Although amplification of PD-L1 gene was not found among PD-L1 positive patients, low-level amplification of PD-L1 gene was commonly observed in MSS patients (75%; 6/8) than in MSI patients (43%; 3/7). Additionally, we found 26% of CRC patients harbored KRAS mutations (16/61), so far the distribution of KRAS status did not correlate with PD-L1 expression. Our data suggest genetic mechanism through amplification of PD-L1 seems not to be the mechanism underlying upregulation of PD-L1 expression in CRC patients. However, further studies are warranted to confirm the results.

Keywords: colorectal cancer, gene amplification, microsatellite instable, programmed death ligand-1

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Authors: Virag Vass, Ilona Bereczki, Erzsebet Szabo, Nora Debreczeni, Aniko Borbas, Pal Herczegh, Arpad Tosaki

Abstract:

Hydrogen sulfide (H₂S) is a toxic gas, but it is produced by certain tissues in a small quantity. According to earlier studies, ibuprofen and H₂S has a protective effect against damaging heart tissue caused by ischemia-reperfusion. Recently, we have been investigating the effect of a new water-soluble H₂S releasing ibuprofen molecule administered after artificially generated ischemia-reperfusion on isolated rat hearts. The H₂S releasing property of the new ibuprofen derivative was investigated in vitro in medium derived from heart endothelial cell isolation at two concentrations. The ex vivo examinations were carried out on rat hearts. Rats were anesthetized with an intraperitoneal injection of ketamine, xylazine, and heparin. After thoracotomy, hearts were excised and placed into ice-cold perfusion buffer. Perfusion of hearts was conducted in Langendorff mode via the cannulated aorta. In our experiments, we studied the dose-effect of the H₂S releasing molecule in Langendorff-perfused hearts with the application of gradually increasing concentration of the compound (0- 20 µM). The H₂S releasing ibuprofen derivative was applied before the ischemia for 10 minutes. H₂S concentration was measured with an H₂S detecting electrochemical sensor from the coronary effluent solution. The 10 µM concentration was chosen for further experiments when the treatment with this solution was occurred after the ischemia. The release of H₂S is occurred by the hydrolyzing enzymes that are present in the heart endothelial cells. The protective effect of the new H₂S releasing ibuprofen molecule can be confirmed by the infarct sizes of hearts using the Triphenyl-tetrazolium chloride (TTC) staining method. Furthermore, we aimed to define the effect of the H₂S releasing ibuprofen derivative on autophagic and apoptotic processes in damaged hearts after investigating the molecular markers of these events by western blotting and immunohistochemistry techniques. Our further studies will include the examination of LC3I/II, p62, Beclin1, caspase-3, and other apoptotic molecules. We hope that confirming the protective effect of new H₂S releasing ibuprofen molecule will open a new possibility for the development of more effective cardioprotective agents with exerting fewer side effects. Acknowledgment: This study was supported by the grants of NKFIH- K-124719 and the European Union and the State of Hungary co- financed by the European Social Fund in the framework of GINOP- 2.3.2-15-2016-00043.

Keywords: autophagy, hydrogen sulfide, ibuprofen, ischemia, reperfusion

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Authors: Ieva Bruzauskaite, Jovile Raudoniute, Karina Poliakovaite, Danguole Zabulyte, Daiva Bironaite, Ruta Aldonyte

Abstract:

Introduction: Air pollution is a growing global problem which has been shown to be responsible for various adverse health outcomes. Immunotoxicity, such as dysregulated inflammation, has been proposed as one of the main mechanisms in air pollution-associated diseases. Chronic obstructive pulmonary disease (COPD) is among major morbidity and mortality causes worldwide and is characterized by persistent airflow limitation caused by the small airways disease (obstructive bronchiolitis) and irreversible parenchymal destruction (emphysema). Exact pathways explaining the air pollution induced and mediated disease states are still not clear. However, modern societies understand dangers of polluted air, seek to mitigate such effects and are in need for reliable biomarkers of air pollution. We hypothesise that post-translational modifications of structural proteins, e.g. citrullination, might be a good candidate biomarker. Thus, we have designed this study, where mice were exposed to diesel exhaust and the ongoing protein modifications and inflammation in lungs and other tissues were assessed. Materials And Methods: To assess the effects of diesel exhaust a in vivo study was designed. Mice (n=10) were subjected to everyday 2-hour exposure to diesel exhaust for 14 days. Control mice were treated the same way without diesel exhaust. The effects within lung and other tissues were assessed by immunohistochemistry of formalin-fixed and paraffin-embedded tissues. Levels of inflammation and citrullination related markers were investigated. Levels of parenchymal damage were also measured. Results: In vivo study corroborates our own data from in vitro and reveals diesel exhaust initiated inflammatory shift and modulation of lung peptidyl arginine deiminase 4 (PAD4), citrullination associated enzyme, levels. In addition, high levels of citrulline were observed in exposed lung tissue sections co-localising with increased parenchymal destruction. Conclusions: Subacute exposure to diesel exhaust renders mice lungs inflammatory and modifies certain structural proteins. Such structural changes of proteins may pave a pathways to lost/gain function of affected molecules and also propagate autoimmune processes within the lung and systemically.

Keywords: air pollution, citrullination, in vivo, lungs

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Authors: Marin Kanarev, Delyan Stoyanov, Ivanna Popova, Nadezhda Petrova

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Thanks to increased survival rates in patients bearing oncological malignancies due to recent developments in anti-cancer therapies and diagnostic techniques, observation of clinical cases of metachronous cancers is more common and can provide more in-depth knowledge of their development and, as a result, help clinicians apply suitable therapy. This unusual case of three metachronous tumors presented the opportunity to follow their occurrence, progression, and treatment thoroughly. A 77-year-old male presented with carcinoma ventriculi of the pylorus region, which was surgically removed via upper subtotal stomach resection, a lateral antecolical gastro-enteroanastomosis, and a subsequent Braun anastomosis. An EOX chemotherapy regimen followed. A CT scan four months later showed no indication of recurrence or dissemination. The same scan, performed as a part of the follow-up plan two years later, showed an indication of neoplastic growth in the urinary bladder. After the patient had been directed to a urologist, the suspicion was confirmed, and the growth was histologically diagnosed as a carcinoma of the urinary bladder. An immunohistochemistry test showed an expression of PDL1 of less than 5%, which resulted in treatment with GemCis chemotherapy regimen that led to full remission. Two years and seven months after the first surgery, a CT scan showed again that the two carcinomas were gone. However, four months later, elevated tumor markers prompted a PET/CT scan, which showed data indicative of recurring neoplastic growth in the region of the stomach cardia. It was diagnosed as an adenocarcinoma infiltrating the esophagus. Preoperative chemotherapy with the ECF regimen was completed in four courses, and a CT scan showed no progression of the disease. In less than a month after therapy, the patient underwent laparotomy, debridement, gastrectomy, and a subsequent mechanical terminal-lateral esophago-jejunoanasthomosis. It was verified that the tumor originated from metastasis from the carcinoma ventriculi, which was located in the pylorus. In conclusion, this case report highlights the importance of patient follow-up and studying recurring neoplastic growth. Despite the absence of symptoms, clinicians should maintain a high level of suspicion when evaluating the patient data and choosing the most suitable therapy.

Keywords: carcinoma, follow-up, metachronous, neoplastic growth, recurrence

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Authors: Y. Landkocz, P. Gosset, A. Héliot, C. Corbière, C. Vendeville, V. Keravec, S. Billet, A. Verdin, C. Monteil, D. Préterre, J-P. Morin, F. Sichel, T. Douki, P. J. Martin

Abstract:

Air Pollution produced by automobile traffic is one of the main sources of pollutants in urban atmosphere and is largely due to exhausts of the diesel engine powered vehicles. The International Agency for Research on Cancer, which is part of the World Health Organization, classified in 2012 diesel engine exhaust as carcinogenic to humans (Group 1), based on sufficient evidence that exposure is associated with an increased risk for lung cancer. Amongst the strategies aimed at limiting exhausts in order to take into consideration the health impact of automobile pollution, filtration of the emissions and use of biofuels are developed, but their toxicological impact is largely unknown. Diesel exhausts are indeed complex mixtures of toxic substances difficult to study from a toxicological point of view, due to both the necessary characterization of the pollutants, sampling difficulties, potential synergy between the compounds and the wide variety of biological effects. Here, we studied the potential indirect genotoxicity of emission of Diesel engines through on-line exposure of rats in inhalation chambers to a subchronic high but realistic dose. Following exposure to standard gasoil +/- rapeseed methyl ester either upstream or downstream of a particle filter or control treatment, rats have been sacrificed and their lungs collected. The following indirect genotoxic parameters have been measured: (i) telomerase activity and telomeres length associated with rTERT and rTERC gene expression by RT-qPCR on frozen lungs, (ii) γH2AX quantification, representing double-strand DNA breaks, by immunohistochemistry on formalin fixed-paraffin embedded (FFPE) lung samples. These preliminary results will be then associated with global cellular response analyzed by pan-genomic microarrays, monitoring of oxidative stress and the quantification of primary DNA lesions in order to identify biological markers associated with a potential pro-carcinogenic response of diesel or biodiesel, with or without filters, in a relevant system of in vivo exposition.

Keywords: diesel exhaust exposed rats, γH2AX, indirect genotoxicity, lung carcinogenicity, telomerase activity, telomeres length

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