Protective Effects of Crocin Against Methotrexate-Induced Hepatotoxicity in Adult Male Albino Rats: Histological, Immunohistochemical, and Biochemical Study.

BACKGROUND: Among the many known adverse effects of methotrexate (MTX), hepatotoxicity stands out as a major drawback that limits its therapeutic applicability. There is growing evidence that crocin has antioxidant, anti-hyperglycemic, cardioprotective, and anti-inflammatory effects. This study's aim is to evaluate the potential protective effect of crocin against MTX-induced liver damage in rats using biochemical, histological, and immunohistochemical analyses. METHODS: Twenty-four adult male albino rats were split into four groups at random (six rats/group) as follows: normal control (saline, intraperitoneal (i.p.) injections), crocin-treated (100 mg/kg daily for 14 days, i.p.), MTX-treated (20 mg/kg single i.p. injection on day 15), and crocin/MTX-treated groups (crocin 100 mg/kg/day for 14 days, i.p. + MTX 20 mg/kg single i.p. injection on day 15). On day 16 of the experiment, blood and tissue specimens were used to assess the liver functions, oxidative stress markers, transforming growth factor beta 1 (TGF-ß1), caspase-3, BCL-2-associated X protein (BAX), and B-cell lymphoma 2 (BCL-2) expression. RESULTS: The results of the current research revealed the protective actions of crocin against MTX-induced hepatotoxicity. Our results showed that crocin possesses antioxidants (decrease malondialdehyde (MDA), increase glutathione (GSH) levels, and enhance catalase (CAT) and superoxide dismutase (SOD) enzymatic activity), anti-fibrotic (decrease TGF-ß1), and anti-apoptotic (decrease BAX and caspase-3 expression while increase BCL-2) actions in liver. Moreover, crocin administration along with MTX restores the normal histological structure of hepatic tissues. CONCLUSION:  The data presented in the current study using an in vivo animal model support the notion that crocin should be further studied in humans to assess its potential hepatoprotective effects against MTX-induced liver damage.

Identification of a circulating microRNAs biomarker panel for non-invasive diagnosis of coronary artery disease: case-control study.

BACKGROUND: Circulating microRNAs (miRNAs) are considered a hot spot of research that can be employed for monitoring and/or diagnostic purposes in coronary artery disease (CAD). Since different disease features might be reflected on altered profiles or plasma miRNAs concentrations, a combination of miRNAs can provide more reliable non-invasive biomarkers for CAD. SUBJECTS AND METHODS: We investigated a panel of 14-miRNAs selected using bioinformatics databases and current literature searching for miRNAs involved in CAD using quantitative real-time PCR technique in 73 CAD patients compared to 73 controls followed by function and pathway enrichment analysis for the 14-miRNAs. RESULTS: Our results revealed three out of the 14 circulating miRNAs understudy; miRNAs miR133a, miR155 and miR208a were downregulated. While 11 miRNAs were up-regulated in a descending order from highest fold change to lowest: miR-182, miR-145, miR-21, miR-126, miR-200b, miR-146A, miR-205, miR-135b, miR-196b, miR-140b and, miR-223. The ROC curve analysis indicated that miR-145, miR-182, miR-133a and, miR-205 were excellent biomarkers with the highest AUCs as biomarkers in CAD. All miRNAs under study except miR-208 revealed a statistically significant relation with dyslipidemia. MiR-126 and miR-155 showed significance with BMI grade, while only miR-133a showed significance with the obese patients in general. MiR-135b and miR-140b showed a significant correlation with the Wall Motion Severity Index. Pathway enrichment analysis for the miRNAS understudy revealed pathways relevant to the fatty acid biosynthesis, ECM-receptor interaction, proteoglycans in cancer, and adherens junction. CONCLUSION: The results of this study identified a differentially expressed circulating miRNAs signature that can discriminate CAD patients from normal subjects. These results provide new insights into the significant role of miRNAs expression associated with CAD pathogenesis.

PSORS1 Locus Genotyping Profile in Psoriasis: A Pilot Case-Control Study.

(1) Background: The psoriasis susceptibility 1 (PSORS1) locus, located within the major histocompatibility complex, is one of the main genetic determinants for psoriasis, the genotyping profile for three single-nucleotide polymorphisms (SNPs) comprising the PSORS1 locus: rs1062470 within PSORS1C1/CDSN genes, rs887466 within PSORS1C3 gene, rs10484554 within LOC105375015 gene, were investigated and correlated with psoriasis risk and severity. (2) Methods: This pilot case-controlled study involved 100 psoriatic patients and 100 healthy individuals. We investigated three SNPs and assessed the relative gene expression profile for the PSORS1C1 gene. We then correlated the results with both disease risk and severity. (3) Results: The most significantly associated SNP in PSORS1 locus with psoriasis was rs10484554 with its C/T genotype 5.63 times more likely to develop psoriasis under codominant comparison. Furthermore, C/T and T/T genotypes were 5 times more likely to develop psoriasis. The T allele was 3 times more likely to develop psoriasis under allelic comparison. The relative gene expression of PSORS1C1 for psoriatic patients showed to be under-expressed compared to normal controls. (4) Conclusions: Our study revealed the association of the three studied SNPs with psoriasis risk and severity in an Egyptian cohort, indicating that rs10484554 could be the major key player in the PSORS1 locus.

MicroRNA-17-92a-1 Host Gene (MIR17HG) Expression Signature and rs4284505 Variant Association with Alopecia Areata: A Case-Control Study.

Accumulating evidence indicates the implication of microRNAs (miRs) in cutaneous and hair follicle immunobiology. We evaluated, for the first time, the miR-17-92a-1 cluster host gene (MIR17HG) expression in peripheral blood of 248 unrelated alopecia areata (AA) patients compared to 244 matched controls using Real-Time qPCR. We also tested its association with different rs4284505A>G genotypes (based on TaqMan allelic discrimination PCR) and the available clinical data. The adjusted odds ratio (OR) and 95% confidence interval (CI) were calculated for each genetic association model. The upregulation of miR-17 was observed in the serum of patients with alopecia compared to controls (p-value = 0.004). The ROC curve showed high diagnostic performance of miR-17 in differentiating between patients and controls (AUC = 0.85, p-value < 0.001). rs4284505*A/G heterozygotes were more susceptible to the disease (OR = 1.57, 95% CI = 1.01−2.45) under the over-dominant model. Interestingly, patients with the rs4284505*G/G genotype had a higher level of miR-17 than those with the A/A and A/G genotypes. The G/G genotype was associated with the severe phenotype (p-value = 0.038). A/G carriers were the youngest (p-value < 0.001), had more frequent scalp infection (p-value = 0.006), exhibited the worst dermatology life quality index score (p-value = 0.037), and responded less to treatment (p-value = 0.033). In conclusion, MIR17HG expression and the rs4284505 variant were significantly associated with AA and could play a role in pathogenesis and phenotype in the Egyptian population. Further multi-center studies in other ethnicities are warranted to replicate the findings.

Long Non-Coding RNAs Gene Variants as Molecular Markers for Diabetic Retinopathy Risk and Response to Anti-VEGF Therapy.

BACKGROUND: Long non-coding RNAs (lncRNAs) play essential roles in molecular diagnosis and therapeutic response in several diseases. PURPOSE: For the first time, we aimed to evaluate the association of four lncRNAs TUG1 (rs7284767G/A), MIAT (rs1061540T/C), MALAT1 (rs3200401C/T), and SENCR (rs12420823C/T) variants with susceptibility to diabetic retinopathy (DR), disease severity, and early therapeutic response to intravitreous anti-vascular endothelial growth factor aflibercept therapy. PATIENTS AND METHODS: This case-control study enrolled 126 adult patients with type 2 diabetes. TaqMan assays using Real-Time PCR were run for genotyping. Multivariable regression analyses were applied to assess the role of each polymorphism after the adjustment of covariates. RESULTS: Carriers of TUG1 A/G and MIAT T/C and C/C genotypes were more likely to develop DR [OR=3.15 (95% CI=1.15-8.64), and OR=4.31 (95% CI=1.78-10.47)], while MALAT1 T/C conferred protection (OR=0.40, 95% CI=0.16-0.99). For TUG1, MALAT1, MIAT, and SENCR genotype combinations, GTCT and GCCC had a higher disease risk (P=0.012). For disease severity, MIAT T/T homozygosity was associated with higher DR grade [33.3% (T/T) vs 10% (C/C) and 4.2% (C/T) carriers, P=0.012]. Otherwise, patients with the SENCR T variant exhibited better pre-treatment best-corrected visual acuity level (p=0.021). Following aflibercept administration, carrying the TUG1 A or MIAT T/C was associated with a poor therapeutic response (OR=5.02, 95% CI=1.60-15.76, and OR=10.23, 95% CI=1.51-69.15, respectively). CONCLUSION: The lncRNAs TUG1 (rs7284767G/A) and MIAT (rs1061540T/C) were associated with increased DR susceptibility and poor response to aflibercept treatment, while MALAT1 (rs3200401C/T) conferred protection to DR. These genetic determinants could be useful in DR risk stratification and pharmacogenetics after validation in large-scale studies.

Antioxidant and Anti-Inflammatory Effects of Crocin Ameliorate Doxorubicin-Induced Nephrotoxicity in Rats.

Doxorubicin is a drug that belongs to the anthracycline antibiotics. Nephrotoxicity is one of the serious side effects of doxorubicin treatment. Crocin, which is one of the most bioactive components of saffron, has antioxidant, anti-inflammatory, and antitumor effects. The current study was aimed at investigating the possible protective effects of crocin against doxorubicin-induced nephrotoxicity to elucidate the underlying mechanism of this effect. The study included four groups, six rats in each group: normal control, crocin control, doxorubicin, and crocin/doxorubicin. Doxorubicin and crocin/doxorubicin groups received intraperitoneal injections of doxorubicin (3.5 mg/kg twice weekly for 3 weeks). Rats in the crocin control group and the crocin/doxorubicin group were treated with intraperitoneal injections of crocin (100 mg/kg body weight per day) for 3 weeks. Biomarkers of kidney function and oxidative stress as well as the abundance of mRNA for nuclear factor-κß and inducible nitric oxide synthase were evaluated. In addition, the abundance of cyclooxygenase 2 and tumor necrosis factor α immunoreactivity was evaluated. Crocin treatment had renoprotective effects manifested by significant improvement in kidney function as well as a reduction in the abundance of biomarkers of oxidative stress markers and inflammatory mediators. In conclusion, crocin has a protective effect against doxorubicin-induced nephrotoxicity in rats by serving as an antioxidant and attenuating the expression of NF-κB, iNOS, COX2, and TNFα.

Interleukin 17 receptor A haplotype analysis in chronic spontaneous urticaria: A preliminary study.

BACKGROUND: Chronic spontaneous urticaria (CSU) is a distressing skin disease. Family clustering and heterogeneity in the onset and progression indicate that susceptibility to CSU is a complex trait. In this study, we performed haplotype analysis for one of the key player gene, IL17RA, for CSU to test the association with disease susceptibility and severity. METHODOLOGY: The study included 70 CSU patients and 30 healthy controls. The severity of the disease was evaluated by autologous serum skin test (ASST) and urticaria activity score (UAS). ASST test was done and quality of life was assessed using a questionnaire. Allelic discrimination analysis for rs4819554 and rs879577 was performed using real-time polymerase chain reaction technology. RESULTS: Carriers of rs4819554*G were more prone to develop CSU than its counterpart (P = .039), while rs4819554*A allele displayed more severe phenotype in the form of more prolonged disease duration (P = .040), concurrent angioedema (P < .001), higher level of treatment (P < .001), and higher score of quality of life (P < .001). Additionally, homozygote patients with rs879577*CC were associated with angioedema (P < .001). Haplotype analysis revealed that cohorts with both rs4819554*A and rs879577*T conferred protection against developing CSU (OR = 0.07, 95% CI = 0.01-0.32, P = .001). CONCLUSION: Our results showed that IL17RA gene polymorphisms might contribute to the increased susceptibility to CSU.

The prognostic value of microRNA-biogenesis genes Argonaute 1 and 2 variants in breast cancer patients.

MicroRNA machinery genes Argonaute 1 (AGO1) and 2 (AGO2) are associated with several hallmarks of cancer. They play a key role in transcriptomic silencing, regulation of the immune system, cell differentiation, and angiogenesis processes. The present pilot study aims to explore the impact of genetic variants rs636832 and rs2977490 of AGO1 and AGO2, respectively, on breast cancer (BC) risk in a sample of Mediterranean population. TaqMan genotyping assay of 93 consecutive breast cancer female patients and age- as well as ethnicity-matched controls, was done by Real-Time allele discrimination polymerase chain reaction. Association with the available clinical, histopathological and immunohistochemistry assessments was applied. In silico data analysis was also executed. Although allele and genotype frequencies distribution of both study variants were comparable in BC and healthy control cohorts, AGO1*G variant conferred a significant BC risk under recessive model [adjusted odds ratio (95% confidence interval); 4.90 (1.03-23.39), P = 0.024], and was significantly associated with lymph node infiltration (P = 0.037), distant metastasis (P = 0.019), advanced clinical stage (P < 0.001), recurrence (P = 0.032), and shorter overall survival (P = 0.001). Furthermore, AGO2*G/G genotype showed an association with poor pathological grade (P = 0.029). Our results suggested for the first time that rs636832 and rs2977490 variants of the miRNA-machinery genes AGO1 and 2, respectively, may impact susceptibility and/or clinical outcomes of BC patients in the study population.

Vitamin D receptor Fok1 & Bsm 1 Gene Polymorphisms in Systemic Lupus Erythematosus and Osteoarthritis: Autoimmune Inflammatory versus Degenerative Model.

Vitamin D deficiency has been described in SLE and OA. Low vitamin D level is prevalent in Egyptian SLE patients while controversial studies are present regarding its level in OA patients in Egypt. We investigated whether vitamin D receptor (VDR) genes Bsm1 and Fok1 polymorphisms could be used as genetic markers for the susceptibility to SLE and /or OA in a sample of Egyptian population. The study was carried out on 100 SLE patients, 100 osteoarthritic patients and 100 normal controls using polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP). Our results showed a statistically significant difference in Fok1 genotype distribution between SLE and OA patients (p=0.001). In SLE group, the "f" allele was significantly over-represented where 30% had "f" allele compared to 0% in OA (P = 0.03). Fok1 ff genotypes showed a significant association with disease activity in SLE patients. In addition, the fb haplotype frequency was significantly higher in SLE patients than controls (P=0.01). In conclusion Fok1 genotype and f allelic frequencies may be susceptible risk factors for SLE rather than OA in Egyptian patients.

Effect of feeding a high-fat diet independently of caloric intake on reproductive function in diet-induced obese female rats.

INTRODUCTION: Globally, the prevalence of overweight and obesity is increasing, predisposing females to health hazards including compromised reproductive capacity. Our objective was to investigate the effect of ad libitum, isocalorically and hypocalorically restricted high-fat diet (HFD) feeding on reproductive function in diet-induced obese female rats. MATERIAL AND METHODS: Twenty female albino Sprague Dawley rats were used; 5 rats were kept on a standard pellet animal diet to serve as a control group (A) and 15 rats were fed a HFD for 9 weeks to induce obesity. The HFD fed animals were equally divided into three groups: an ad libitum HFD group (B), an isocalorically restricted HFD group (C), and a hypocalorically restricted HFD group (D). Estrous cyclicity, hormonal levels, ovarian histopathology and caspase-3 immunoreactivity were evaluated. RESULTS: The HFD-fed rats in groups B, C and D had significant irregularity in estrous cyclicity Vs group A (p = 0.001, 0.003 and 0.034 respectively). Groups C and D had significant reduction in serum progesterone level (p = 0.006 and 0.018 Vs A). Isocaloric restriction of HFD feeding significantly increased serum LH. Groups B and C had a significant increase in caspase-3 expression in the ovary (p < 0.001). CONCLUSIONS: Ad libitum HFD interfered with the normal estrous cycle and enhanced apoptosis of luteal cells in obese female rats. The HFD restriction interfered with the normal estrous cycle and caused functional insufficiency of the corpus luteum in obese female rats. These results suggest that HFD feeding determinately affects female reproductive function independently of caloric intake.

Antioxidant and angiostatic effect of Spirulina platensis suspension in complete Freund's adjuvant-induced arthritis in rats.

BACKGROUND: Currently, natural products have built a well-recognized role in the management of many degenerative diseases, mainly rheumatoid arthritis. Recent studies suggest that Spirulina, a unicellular blue-green alga, may have a variety of health benefits and curative properties and is also competent of acting as an anti-inflammatory, antioxidant and recently anti-angiogenic agent. In the present study, the antioxidant and the immunomodulatory effect of Spirulina platensis as well as its anti-angiogenic effect against complete Freund's adjuvant-induced arthritis (AIA) in rat model were tested. RESULTS: We found that the development of arthritis was concealed; moreover it successfully inhibited the development of macroscopic as well as microscopic and histopathological lesions in AIA rats when compared to control. Spirulina treated group showed a higher survival rate and moreover, it reduced the clinical score of RA in a dose dependent manner. Furthermore, Spirulina decreased serum levels of COX-2, TNF-α, IL-6, TBARS, VEGF and increased serum levels of GSH compared to the RA non-treated group. CONCLUSIONS: The present study concluded that Spirulina is able to restrain the changes produced through adjuvant-induced arthritis. The suppressing effect of Spirulina could be attributed, at least in part, to anti-inflammatory, antioxidant and anti-angiogenic properties.

Association of Angiotensin-Converting Enzyme ACE Gene Polymorphism with ACE Activity and Susceptibility to Vitiligo in Egyptian Population.

AIM: The insertion/deletion (I/D) polymorphism of the angiotensin-converting enzyme (ACE) gene is associated with vitiligo in the Indians and Koreans, but not in those of English or Turkish background. We investigated the ACE (I/D) polymorphism in vitiligo patients for the first time in Egypt and compared serum ACE levels between vitiligo patients and controls. The present study was carried out in 100 vitiligo patients (40 males and 60 females) and in 100 healthy controls of an Egyptian population using the polymerase chain reaction genotyping method. RESULTS: The ACE genotype and allele frequency was significantly different between vitiligo patients and controls. Our results revealed a significant increase in the frequency of the ACE I allele (p=0.002; odds ratio: 1.99; 95% confidence intervals: 1.207-3.284) with an overrepresentation of I/D genotype in the vitiligo patient group. Furthermore, there was a significant difference between the segmental, nonsegmental, and focal vitiligo in ACE gene genotype distribution. Serum ACE levels were significantly increased in vitiligo patients compared to controls (p=0.034). CONCLUSION: This study suggests that, for the first time, ACE gene polymorphism confers susceptibility to vitiligo in the Egyptian population.

Effects of naturally-arising HIV Nef mutations on cytotoxic T lymphocyte recognition and Nef's functionality in primary macrophages.

BACKGROUND: Although HIV can infect several cellular subsets, such as CD4⁺ T lymphocytes and macrophages, it remains unclear whether an HIV infection in macrophages supports cytotoxic T lymphocyte (CTL) escape. Here, we tested two naturally-arising mutations located in the well-conserved polyproline region of Nef for their effects on CTL recognition, Nef's functionality, and viral replication capacity in macrophages. These mutations were selected because they are known to cause CTL escape in the context of T lymphocytes. FINDINGS: Monocyte-derived macrophages (MDMs) infected with the wild-type virus, but not with variant viruses, were efficiently killed by CTL clones targeting Nef epitopes, VY8 (VPLRPMTY) and RY11 (RPQVPLRPMTY). The CTL-escape mutation, Arg75Thr, or Arg75Thr/Tyr85Phe double mutation, reduced the HLA class I down-regulation activity and, interestingly, increased the susceptibility of virus-infected MDMs to recognition by CTLs targeting a different epitope. The same mutations reduced the CCR5, but not CD4, down-regulation activity. Moreover, the Nef variants were impaired for Hck activation and enhancement of viral replication in MDMs. CONCLUSIONS: These results suggest that HIV-infected MDMs are killed by CTLs targeting Nef epitopes, contributing to selection and adaptation of CTL-escape viral variants.

Dys-regulated activation of a Src tyroine kinase Hck at the Golgi disturbs N-glycosylation of a cytokine receptor Fms.

HIV-1 Nef accelerates the progression to AIDS by binding with and activating a Src kinase Hck, but underlying molecular basis is not understood. We revealed that Nef disturbed N-glycosylation/trafficking of a cytokine receptor Fms in an Hck-dependent manner, a possible trigger to worsen uncontrolled immune system. Here, we provide direct evidence that dys-regulated activation of Hck pre-localized to the Golgi apparatus causes this Fms maturation arrest. A striking change in Hck induced by Nef other than activation was its skewed localization to the Golgi due to predominant Golgi-localization of Nef. Studies with different Nef alleles and their mutants showed a clear correlation among higher Nef-Hck affinity, stronger Hck activation, severe Golgi-localization of Hck and severe Fms maturation arrest. Studies with a newly discovered Nef-Hck binding blocker 2c more clearly showed that skewed Golgi-localization of active Hck was indeed the cause of Fms maturation arrest. 2c blocked Nef-induced skewed Golgi-localization of an active form of Hck (Hck-P2A) and Fms maturation arrest by Nef/Hck-P2A, but showed no inhibition on Hck-P2A kinase activity. Our finding establishes an intriguing link between the pathogenesis of Nef and a newly emerging concept that the Golgi-localized Src kinases regulate the Golgi function.

Interaction between Hck and HIV-1 Nef negatively regulates cell surface expression of M-CSF receptor.

Nef is a multifunctional pathogenetic protein of HIV-1, the interaction of which with Hck, a Src tyrosine kinase highly expressed in macrophages, has been shown to be responsible for the development of AIDS. However, how the Nef-Hck interaction leads to the functional aberration of macrophages is poorly understood. We recently showed that Nef markedly inhibited the activity of macrophage colony-stimulating factor (M-CSF), a primary cytokine for macrophages. Here, we show that the inhibitory effect of Nef is due to the Hck-dependent down-regulation of the cell surface expression of M-CSF receptor Fms. In the presence of Hck, Nef induced the accumulation of an immature under-N-glycosylated Fms at the Golgi, thereby down-regulating Fms. The activation of Hck by the direct interaction with Nef was indispensable for the down-regulation. Unexpectedly, the accumulation of the active Hck at the Golgi where Nef prelocalized was likely to be another critical determinant of the function of Nef, because the expression of the constitutive-active forms of Hck alone did not fully down-regulate Fms. These results suggest that Nef perturbs the intracellular maturation and the trafficking of nascent Fms, through a unique mechanism that required both the activation of Hck and the aberrant spatial regulation of the active Hck.