Monocyte Chemoattractant Protein-1 (MCP-1/CCL2) Levels and Its Association with Renal Allograft Rejection

Background: CCL2 is a chemoattractant for monocytes/macrophages, T cells, and natural killer cells. It is shown to be involved in the immunological responses against renal allograft. This study was conducted to access the role of urinary CCL2 expression in predicting the rejection episodes in renal transplant patients.

Method: A total of 409 urine samples included in this study. The samples were consisted of (a) biopsy-proven graft rejection (n = 165); (b) non-rejection (n = 93); (c) non-biopsy stable-graft (n = 42), and (d) healthy renal donors (n = 109). The samples were quantified for the CCL2 using the MCP-1/CCL2 ELISA kit. The data were analyzed using the Statistical Package for Social Sciences (SPSS^®) and MedCalc^® statistical software.

Results: Results showed that the CCL2 levels were significantly increased in rejection group when compared with the non-rejection, stable-graft, and control, P < 0.05. The receiver operating curve’s characteristics illustrated that the urinary CCL2 level is a good predictor for graft rejection, with an area under the curve of 0.81 ± 0.03 with optimum sensitivity and specificity of 87% and 62%, respectively, at a cut-off value of 198 pg/mL. Kaplan–Meier curve also showed better cumulative rejection-free graft survival time in group with less than 198 pg/mL of CCL2 as compared to those with expression levels of more than 198 pg/mL (30 weeks vs. 3 weeks; log-rank test, P < 0.001).

Conclusion: In our study, noninvasive investigation of CCL2 levels in urine has showed potential to predict rejection episodes. It is suggested that the CCL2, with others markers, may help in early detection and monitoring of graft rejection episodes.     

Mutational analysis of the CYP1B1 gene in Pakistani primary congenital glaucoma patients: Identification of four known and a novel causative variant at the 3′ splice acceptor site of intron 2

Primary congenital glaucoma (PCG) causes blindness in early age. It has an autosomal recessive pattern of inheritance, hence is more prevalent in populations with frequent consanguineous marriages that occur in the Pakistani population. Mutations in the CYP1B1 gene are commonly associated with PCG. The aim of the present study was to identify genetic mutations in the CYP1B1 gene in PCG cases belonging to 38 Pakistani families. DNA was extracted using blood samples collected from all enrolled patients, their available unaffected family members and controls. Direct sequencing of the CYP1B1 gene revealed a novel 3' splice acceptor site causative variant segregating in an autosomal recessive manner in a large consanguineous family with four PCG‐affected individuals. The novel variant was not detected in 93 ethnically matched controls. Furthermore, four already reported mutations, including p.G61E, p.R355X, p.R368H, and p.R390H were also detected in patients belonging to nine different families. All identified causative variants were evaluated by computational programs, that is, SIFT, PolyPhen‐2, and MutationTaster. Pathogenicity of the novel splice site variant identified in this study was analyzed by Human Splicing Finder and MaxEntScan. Ten out of 38 families with PCG had the disease due to CYP1B1 mutations, suggesting CYP1B1 was contributing to PCG in these Pakistani patients. Identification of this novel 3' splice acceptor site variant in intron 2 is the first report for the CYP1B1 gene contributing to genetic heterogeneity of disease.     

Association between mass media and body weight concern among Jordanian adolescents’ residents of Amman: the role of gender and obesity

Objectives

Body image in the mass media promotes an unrealistic picture of body shape that leads to body dissatisfaction among adolescentsQuery. Therefore, the study presented in this paper aimed to assess the association between mass media and adolescents’ weight concerns and perceptions of body weight and shape.

Methods

A cross-sectional survey was conducted on school adolescents aged between 15 and 18 years during the academic year 2013–2014. Multistage stratified sampling method was used. The number of participants in the study was 795 students: 400 boys and 395 girls.

Results

All participants have a common behavior in rarely reading magazines, but they spend more than 2 h in watching television or less than 3 h using the internet. However, most of obese/non-obese adolescents, boys or girls, have shown high influence (p < 0.05) of reading magazines on the subject of dieting to lose weight.

Conclusion

While obese students read more magazines on dieting to lose weight, other mass media did not show the same results on weight concerns and body shape among Jordanian adolescents.

     

Synthesis of Novel Pyrimidin‐4‐One Bearing Piperazine Ring‐Based Amides as Glycogen Synthase Kinase‐3β Inhibitors with Antidepressant Activity

Novel pyrimidin‐4‐one derivatives have been synthesized using EDC coupling and evaluated as glycogen synthase kinase‐3β (GSK‐3β) inhibitors. Among all the synthesized compounds, compound 5 (3‐methyl‐6‐phenyl‐2‐(piperazin‐1‐yl)‐3,4‐dihydropyrimidin‐4‐one) exhibited the most potent inhibitory activity against GSK‐3β with IC₅₀ value of 74 nm . The molecular docking studies were performed to elucidate the binding modes of the compounds with the target, and a crucial interaction involving hydrogen bond formation with Val‐135 to the active site of GSK‐3β was observed. Furthermore, the synthesized compounds were subjected to in vivo evaluation of their antidepressant activity, and compound 5 showing highest inhibition of GSK‐3β was also found to significantly reduce the duration of immobility at 50 mg/kg, when compared with fluoxetine, a known antidepressant drug. The results of our study suggest that compound 5 may serve as a valuable template for the design and development of inhibitors of GSK‐3β with antidepressant activity.     

Design and Synthesis of Butenolide‐based Novel Benzyl Pyrrolones: Their TNF‐α based Molecular Docking with In vivo and In vitro Anti‐inflammatory Activity

A focused library of novel benzyl pyrrolones has been synthesized and their in silico molecular docking studies carried out against TNF‐α target. Among all the docked molecules, compound 3f showed best glide score of ?6.89. All the synthesized compounds were evaluated for in vivo anti‐inflammatory activity by carrageenan‐induced paw edema model. Compounds showing significant anti‐inflammatory activity were further tested for their in vitro TNF α expression. Compounds 3b and 2b were found to show significant inhibition of 76.22% and 71.47%, respectively after 5 h in comparison with standard drug indomethacin, which showed 80.98% inhibition of inflammation. Compounds 3b and 2b also suppressed TNF α level by 65.03% and 60.90% as compared indomethacin, which showed 68.84% of inhibition. Compound 3b showed significant analgesic activity of 60.04%, and its activity was comparable with indomethacin (64.04%). Compounds 3b and 2b were also tested for their effect on protein expression of COX‐2 and NF‐κB in the liver tissues. Compounds 3b and 2b were further evaluated for their gastric risk and lipid peroxidation action and showed superior GI safety along with reduction of LPO as compared to indomethacin. Hepatotoxicity study showed that these two compounds did not cause any damage to liver.     

Primary hyperoxaluria: Comprehensive mutation screening of the disease causing genes and spectrum of disease-associated pathogenic variants

The primary hyperoxalurias are rare disorders of glyoxylate metabolism. Accurate diagnosis is essential for therapeutic and management strategies. We conducted a molecular study on patients suffering from recurrent calcium-oxalate stones and nephrocalcinosis and screened primary hyperoxaluria causing genes in a large cohort of early-onset cases. Disease-associated pathogenic-variants were defined as missense, nonsense, frameshift-indels, and splice-site variants with a reported minor allele frequency <1% in controls. We found pathogenic-variants in 34% of the cases. Variants in the AGXT gene causing PH-I were identified in 81% of the mutation positive cases. PH-II-associated variants in the GRHPR gene are found in 15% of the pediatric PH-positive population. Only 3% of the PH-positive cases have pathogenic-variants in the HOGA1 gene, responsible to cause PH-III. A population-specific AGXT gene variant c.1049G>A; p.Gly350Asp accounts for 22% of the PH-I-positive patients. Pathogenicity of the identified variants was evaluated by in-silico tools and ACMG guidelines. We have devised a rapid and low-cost approach for the screening of PH by using targeted-NGS highlighting the importance of an accurate and cost-effective screening platform. This is the largest study in Pakistani pediatric patients from South-Asian region that also expands the mutation spectrum of the three known genes.     

Screening of the FANCA gene mutational hotspots in the Pakistani fanconi anemia patients revealed 19 sequence variations

Fanconi anemia (FA) is a recessive disorder that predispose to bone marrow failure and multiple congenital anomalies in affected individuals worldwide. To date, 22 FA genes are known to harbor sequence variations in disease phenotype. Among these, mutations in the FANCA gene are associated with 60% to 70% of FA cases. The aim of the present study was to screen FA cases belonging to consanguineous Pakistani families for selected exons of FANCA gene which are known mutational hotspots for Asian populations. Blood samples were collected from 20 FA cases and 20 controls. RNA was extracted and cDNA was synthesized from blood samples of cases. DNA was extracted from blood samples of cases and ethnically matched healthy controls. Sanger's sequencing of the nine selected exons of FANCA gene in FA cases revealed 19 genetic alterations of which 15 were single nucleotide variants, three were insertions and one was microdeletion. Of the total 19 sequence changes, 13 were novel and six were previously reported. All identified variants were evaluated by computational programs including SIFT, PolyPhen-2 and Mutation taster. Seven out of 20 analyzed patients were carrying homozygous novel sequence variations, predicted to be associated with FA. These disease associated novel variants were not detected in ethnically matched controls and depict genetic heterogeneity of disease.     

Seroepidemiology of human fascioliasis and its relationship with anti-Fasciola IgG and liver enzymes as biomarkers of pathogenicity

BackgroundFascioliasis has never been considered a public health concern in Pakistan, although the increasing numbers of human cases reported in south Asia need a re-consideration in the country. The current study aimed to find the seroprevalence of human fascioliasis, associated risk factors and its relationship with liver enzymes as biomarkers of pathogenicity.MethodsThe cross-sectional study was conducted in different districts of Punjab region from May 2014 to August 2016. A total of 546 respondents were screened by using enzyme-linked immunosorbent assay (ELISA) and serum biochemical tests.ResultsHigher seroprevalence was recorded in Muzaffargarh (6.2%) and Bhara kahu (5.9%), while low infection rate in Gujranwala (1.1%) and Islamabad (1.5%). The results of multiple logistic regression analysis showed rural inhabitants (OR=7.9, 95%CI: 2.5–24.8), females (OR=3.5, 95%CI: 1.7–7.1), family size 3–7 (OR=1.7, 95%CI: 1.0–2.9) and socioeconomic condition (OR=3.9, 95%CI: 1.5–10.4) were the significantly (p<0.005) associated risk factors with disease. The results of liver enzymes i.e. aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase, gamma-glutamyl transferase and cholesterol levels were significantly (p=0.001) elevated and associated with fascioliasis pathogenicity.ConclusionThe higher prevalence recorded may explain with Fasciola IgG antibodies for both active and past infections and cross reactivity of the assay with other helminthes.