Uroprotective Potential of Campesterol in Cyclophosphamide Induced Interstitial Cystitis; Molecular Docking Studies.

Cyclophosphamide (CYP) is commonly used to treat cancer of the ovaries, breast, lymph, and blood system and produces interstitial cystitis (IC) via its urotoxic metabolite: i. e., acrolein. The present study was aimed to investigate the uroprotective effect of campesterol (a steroidal phytochemical) in cyclophosphamide induced IC. IC was induced by CYP (150 mg/kg, i. p.) in rats. The Enzyme linked immunosorbent assays for oxidative stress markers and Polymerase Chain Reaction (PCR) for inflammatory cytokines were carried out. The Tissue Organ Bath Technique was used for the evaluation of the spasmolytic effect of campesterol. Different pharmacological antagonists have been used to explore the mechanism of action of campesterol. Treatment with campesterol (70 mg/kg) reduced nociception (55 %), edema (67 %), hemorrhage (67 %), and protein leakage significantly (94 %). The antioxidant activity of campesterol was exhibited by a fall in MDA, NO, and an elevation in SOD, CAT, and GPX levels. Campesterol presented anti-inflammatory potential by decreasing IL-1, TNF-α, and TGF-ß expression levels. Histologically, it preserved urothelium from the deleterious effect of CYP. Campesterol showed a spasmolytic effect by reducing bladder overactivity that was dependent on muscarinic receptors, voltage-gated calcium and KATP channels, and cyclo-oxygenase pathways. In silico studies confirmed the biochemical findings. The findings suggest that campesterol could be valorized as a possible therapeutic agent against cyclophosphamide-induced interstitial cystitis.

Attenuation of CFA-induced chronic inflammation by a bicyclic monoterpene fenchone targeting inducible nitric oxide, prostaglandins, C-reactive protein and urea.

Fenchone (a bicyclic monoterpene) is present in the essential oils of plant species like Foeniculum vulgare and Peumus boldus and is used to treat GIT disorders. Research reports have indicated its strong anti-inflammatory, antioxidant, and anti-nociceptive properties. The present study was designed to investigate fenchone's anti-arthritic effects in a rat model of chronic joint inflammation (Complete Freud's Adjuvant-mediated inflammation [CFA]). Molecular docking analysis revealed a high binding interaction of fenchone with inducible nitric oxide synthase (iNOS), Interleukin-17, Prostaglandin E Receptor EP4, and Cycloxygenase-2 (COX-2), indicating its anti-inflammatory efficacy using computational tests. Fenchone treatment at 100 mg/kg, 200 mg/kg, and 400 mg/kg significantly enhanced the tail-flick latency when compared with the solvent-treated group. Correspondingly, the raised mRNA values of iNOS, IL-17, IL-1ß, IL-6, TNF-α, and COX-2 in solvent-treated group were significantly reduced following treatment with fenchone. Moreover, fenchone significantly lowered spleen and thymus indices, Nitric oxide (NO) and PGE2 values as compared to solvent-treated group. Hence, the results of the present study indicated that fenchone has a potent anti-inflammatory effect by inhibiting pro-inflammatory markers and thus may have therapeutic potential for chronic joint inflammation as well as chronic inflammatory disorders.

SLC11A1 genetic variation and low expression may cause immune response impairment in TB patients.

Tuberculosis (TB) is caused by Mycobacterium tuberculosis. Host genetic factors are important for the detection of TB susceptibility. SLC11A1 is located in monocyte phagolysosomes that help to limit M. tuberculosis growth by transferring divalent cations across the membrane. Genetic variation in SLC11A1 may alter its expression and increase the susceptibility of individuals to TB. The current study aimed to provide insight into host genetic variations and gene expression in TB patients. A total of 164 TB patients and 85 healthy controls were enrolled in this study. SLC11A1 polymorphisms were detected by PCR-RFLP. Real-time qPCR was used for SLC11A1 gene expression, and ELISA was used for protein estimation. GTEx Portal was used for quantitative trait loci analysis, while the STRING (v.11) web platform was used for gene interactive network construction. Data were analyzed using SPSS, GraphPad Prism, Haploview, and SNPstats. SLC11A1 polymorphisms and combinatorial genotypes were strongly associated with TB susceptibility, which may explain the greater prevalence of tuberculosis in the local population. Polymorphisms in SLC11A1 have also been linked to gene expression variation. Furthermore, the expression of SLC11A1 was downregulated in TB patients, which may influence the function of other associated genes and may impair the immunological response to tuberculosis.

Nerolidol: a potential approach in rheumatoid arthritis through reduction of TNF-α, IL-1ß, IL-6, NF-kB, COX-2 and antioxidant effect in CFA-induced arthritic model.

Rheumatoid arthritis is primarily associated with inflammation and increased level of proinflammatory cytokines which are released by immune cells, macrophages or activation of arachidonic acid metabolism. The expression of these cytokines, oxidative free radicals and the activation of COX-2 enzymes are crucial targets for chronic inflammation. On the basis of established anti-inflammatory efficacy of nerolidol, the primary study was further appraised to determine its approach against Freund's complete adjuvant (CFA) rheumatoid model. Arthritis was induced by inoculation of 0.1 mL CFA injection into the left hind footpad of rats. Anti-arthritic potential of nerolidol (at 200, 400 and 800 mg/kg doses) was assessed by measuring the paw volume, body weight, serum analysis, histopathological and radiographs of ankle joints. Expressions of cytokine's panels such as IL-10, IL-4, COX-2, NF-kB, TNF-α, IL-6, PGE-2 and IL-1ß were determined by real-time qPCR. Antioxidant enzyme analyses were conducted by measuring the SOD, POD and catalase activity from serum and equated with arthritic control group. Nerolidol prevented body weight loss, stabilized biochemical and haematological homeostasis and significantly reduced the paw volume. Furthermore, X-ray and histopathological assessment of ankle joints showed an improvement in the joint structure of rats treated with nerolidol. Besides that, overexpression of gene pointers like TNF-α, IL-1ß, IL-6, NF-kB, PGE-2 and COX-2 in CFA-treated control rats were also reversed with nerolidol. This anti-arthritic mechanism was further supported by the increased level of IL-10, IL-4 and serum antioxidant activity. The present findings demonstrate that nerolidol reduced adjuvant arthritis by downregulating the proinflammatory cytokines and upregulating the aforementioned anti-inflammatory cytokines and may be used as a therapeutic substance for the management of human rheumatoid arthritis.

Pharmacological evaluation of anti-arthritic potential of terpinen-4-ol using in vitro and in vivo assays.

Terpinen 4-ol, a phytochemical is a monoterpene which has been reported for its anti-inflammatory effect. Present research was planned to check its effect against arthritis through in vitro and in vivo models. Terpinen 4-ol was evaluated through in-vitro procedures including blocking of protein (BSA and egg albumin) denaturation and human RBC membrane stabilization. In in vivo study, terpinen 4-ol (15, 30 and 60 mg/kg) was evaluated using formaldehyde and CFA arthritic models. Terpinen 4-ol significantly inhibited increase in paw and joint swelling as compared to diseased group. Terpinen 4-ol showed remarkable antioxidant effect (SOD, reducing power) and also improved body weight, haematological, histopathological and radiological parameters in CFA model. Also, moreover, the excess production of IL-1ß, TNF-α, IRAK, and NF-kB were noticeably attenuated in all terpinen 4-ol treated rats, however, IL-17 and IL-10 were distinctly increased compared to arthritic control rats in RT-PCR. Also, terpinen 4-ol showed promising antioxidant effect in DPPH assay. Henceforth, it might be concluded that terpinen 4-ol has anti-arthritic effect which can be attributed to the downregulation of pro-inflammatory cytokines.

Novel acetamide derivatives of 2-aminobenzimidazole prevent inflammatory arthritis in rats via suppression of pro-inflammatory mediators.

Benzimidazole ring system is an important pharmacophore with diverse pharmacological activities. In this study, we explored the anti-arthritic effects of newly synthesized acetamide derivatives of 2-aminobenzimidazole (N1 and N2) in rats. FTIR and NMR spectroscopies were used to characterize these compounds. Carrageenan (CRG) induced paw edema model was used to test the acute anti-inflammatory activity of various doses (10, 20 and 30 mg/kg) of N1 and N2 compounds. Based on acute anti-inflammatory effects, the most potent dose of each compound was selected and investigated in complete freund's adjuvant (CFA) induced inflammatory arthritis (RA) model (n = 4 in each group). Histopathological, hematological, radiographic, and RT-qPCR analyses were performed to assess the progression or resolution of inflammatory arthritis. The tested compounds produced a dose-dependent anti-inflammatory activity against CRG induced paw inflammation and similarly reduced edema in CFA induced inflammatory arthritis model. Histopathological and X-ray analyses of ankle joints revealed minimal inflammation and normal joint structures in N1 and N2 treated groups. The tested compounds also reduced the levels of autoantibodies and restored hematological parameters. Interestingly, the tested compounds did not elevate aspartate aminotransferase and alanine transaminase levels and displayed a better safety profile than methotrexate. N1 and N2 compounds also attenuated the transcript levels of IRAK1, NF-kB1, TNF-α, IL-1ß, IL17 and MMP1. In addition, N1 displayed a greater inhibition of mRNA levels of COX1, COX2, mPGES1 and PTGDS as compared to N2. Our findings demonstrate that N1 and N2 compounds possess strong anti-arthritic activity which can be attributed to the suppression of pro-inflammatory mediators.

Four year trend of antimicrobial susceptibility of methicillin-resistant Staphylococcus aureus in a tertiary care hospital, Lahore.

OBJECTIVE: To determine the susceptibility pattern of methicillin-resistant staphylococcus aureus to different antibiotics. METHODS: The descriptive study was conducted at the Microbiology Department of the University of Health Sciences, Lahore, Pakistan, from August 2016 to July 2019, and comprised staphylococcus aureus samples that were processed and identified using colony morphology on blood agar, gram stain, catalase, coagulase and deoxyribonuclease testing. Screening for methicillin-resistant staphylococcus aureus was done using cefoxitin disc 30µg and oxacillin disc 1mg. Antimicrobial susceptibility was tested using the modified Kirby-Bauer disc diffusion method in line with the Clinical and Laboratory Standards Institute guidelines 2019. Data was analysed using SPSS 24. RESULTS: Of the 2704 strains processed, 402(14.86%) were found to be methicillin-resistant staphylococcus aureus. Of them, 204(50.74%) were recovered from pus, while 10(2.48%) were recovered from urine. All 402(100%) isolates were sensitive to vancomycin and linezolid, and resistant to penicillin, followed by erythromycin 306(76.11%) and sulfamethoxazole/ trimethoprim 295(73.38%). Overall, lower resistance was seen with doxycycline 145(36.06%) and clindamycin 160(39.80%). Inducible clindamycin resistance was seen in 142(35.23%) isolates. CONCLUSIONS: An efficacious susceptibility pattern of methicillin-resistant staphylococcus aureus was seen with vancomycin and linezolid, moderate susceptibility with doxycycline and clindamycin, while high resistance was observed for penicillin, erythromycin and trimethoprim/sulfamethoxazole.

Noscapine hydrochloride (benzyl-isoquinoline alkaloid) effectively prevents protein denaturation through reduction of IL-6, NF-kB, COX-2, Prostaglandin-E2 in rheumatic rats.

Noscapine hydrochloride (benzyl-isoquinoline antitussive alkaloid) is an opium derivative and generally used as a cough suppressant. Numerous studies on noscapine hydrochloride have reported that it has potent anti-inflammatory activity. However, the mechanisms by which it exerts an anti-inflammatory function is not well understood. Protein denaturation is the primary step that leads to the organ destruction and permanent arthritic disability. The above-mentioned facts provided the ground to plan this study using different in-vitro and in-vivo approaches. RT-qPCR and ELISA assays were used to assess the inflammatory markers related to protein denaturation in complete adjuvant persuaded rheumatism in Sprague - Dawley rats. The results were collected as paw volume and body weight changes, arthritic scoring and serum antioxidant enzymes assays. These findings demonstrated that all doses of noscapine hydrochloride (10, 20 and 40 mg/kg) studied in this study, significantly (p < 0.001) decreased the protein denaturation by preventing the increase in levels of interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), nuclear factor-kB (NF-kB), cyclooxygenase-2 (COX-2) and prostaglandin E2. Noscapine hydrochloride significantly reduced the paw volume (p < 0.001), arthritic scoring and reversed the body mass as compared to arthritic control diseased rats.

Potentially inappropriate medications use and its association with health-related quality of life among elderly cardiac patients.

BACKGROUND: Previous studies identified alarming use of potentially inappropriate medications (PIMs) in Pakistani population but its effect on health-related quality of life (HRQoL) is still largely unknown. OBJECTIVE: This study aimed to determine the association between PIMs use and HRQoL among elderly cardiac outpatients. METHOD: A descriptive, non-experimental, cross-sectional study was carried out from June 2018 to September 2018 in two outpatient departments of tertiary-care hospitals in the Punjab Province of Pakistan. The population under study were patients aged ≥ 65 years with at least one cardiovascular condition taking at ≥ 1 prescribed medication. Patients with PIMs were identified by using Beers criteria. HRQoL was assessed using EuroQoL-5 dimension (EQ-5D) and EuroQoL-visual analogue scale (EQ-VAS). The association of PIMs with HRQoL was analyzed using χ2 tests, independent sample t-test, and one-way ANOVA tests. Multiple linear regression analysis was used to determine how HRQoL varied by PIMs use after adjusting for patient-level covariates. RESULTS: Of 386 elderly cardiac patients, 260 (67.4%) patients were receiving at least one PIM. Mean EQ-5D scores were significantly lower among patients with PIMs (0.51) compared to patients without PIMs (0.65) (P < 0.001). In multiple linear regression analysis, increasing numbers of PIMs were significantly associated with lower EQ-5D scores [ß = - 0.040 (- 0.075, - 0.005), P < 0.001] and VAS scores [ß = - 1.686 (- 2.916, - 0.456), P < 0.05]. CONCLUSION: The present study concluded that exposure to PIM was significantly associated with lower HRQoL. This indicates that guidelines recommendations should be followed to improve patient's quality of life.

Polystichum braunii extracts inhibit Complete Freund's adjuvant-induced arthritis via upregulation of I-κB, IL-4, and IL-10, downregulation of COX-2, PGE2, IL-1ß, IL-6, NF-κB, and TNF-α, and subsiding oxidative stress.

Polystichum braunii (Spenn.) Fée is a traditional remedy for rheumatoid arthritis, a chronic inflammatory disorder of polygenetic origin. The current project was intended to demonstrate the role of inflammatory and oxidative stress biomarkers in the anti-arthritic activity of the P. braunii extracts. Methanolic and aqueous extracts of the plant roots were prepared by triple maceration. The phytochemical evaluation of the plant extracts was carried out by high-performance liquid chromatography (HPLC). The plant extracts at 150, 300, and 600 mg/kg/day and piroxicam (10 mg/kg/day) were orally administered to Wistar rats for 21 days that were previously immunized with Complete Freund's adjuvant (150 µl on right hind paw) except normal and arthritic control rats. Both plant extracts mitigated the paw oedema, restored the immune organ and body weights, and ameliorated the level of blood parameters such as haemoglobin, red blood cells, platelets, white blood cells, alkaline phosphatase (ALP), C-reactive proteins, and rheumatoid factor. The evaluation of gene expression using quantitative-real-time polymerase chain reaction (qRT-PCR) revealed the substantial downregulation of tumor necrosis factor (TNF)-α, Interleukin (IL)-6, IL-1ß, cyclo-oxygenase (COX)-2, nuclear factor (NF)-κB, and upregulation of IL-4, IL-10 and I-κB in polyarthritic rats treated with the plant extracts. Methanolic plant extract exhibited the maximum effect on upregulation of IL-4 (79 ± 3%), IL-10 (62.66 ± 4.93%), and I-κB (73.66 ± 3.05%) at 600 mg/kg/day. Treatment with the plant extracts also reduced the level of prostaglandin E2 and TNF-α in the serum of arthritic rats' dose dependently. It was also found that the plant extracts and piroxicam increased (p < 0.05) the activities of superoxide dismutase and catalase in the liver tissue while reduced the level of malondialdehyde in arthritic rats. Histological examination of ankle joints revealed that the plant extracts decreased the pannus formation, inflammation, and synovial hyperplasia in arthritic animals. HPLC analysis depicted that the plant extracts had contained kaempferol, quercetin, gallic acid, and other phenolic acids. It can be elucidated from the results that the extracts of P. braunii roots exhibited anti-arthritic activity in Wistar rats through modulation of inflammatory cytokines and boosting the antioxidant defense mechanism.

Moringa rivae leaf extracts attenuate Complete Freund's adjuvant-induced arthritis in Wistar rats via modulation of inflammatory and oxidative stress biomarkers.

Moringa rivae is widely used as a traditional remedy against arthritis. The present research was designed to evaluate the anti-arthritic potential of Moringa rivae extracts. Treatment of rats with adjuvant-induced arthritis with methanolic and aqueous extracts of M. rivae (150, 300 or 600 mg/kg), and piroxicam (10 mg/kg) was started orally at day 8 post-administration of complete Freund's adjuvant and continued till 28th day. The therapeutic effect of the plant extracts was assessed in arthritic rats by arthritic index, body weight, and haematological and biochemical parameters. Furthermore, the modulatory effect on gene expression (I-κB, IL-4 and IL-10, COX-2, IL-1ß and IL-6, NF-κB, and TNF-α) in the blood was determined using qRT-PCR, while ELISA assay was used to find PGE2 and TNF-α concentrations in the serum. Oxidative stress parameters in the liver and ankle joint histopathology were also evaluated. Moreover, the most effective methanolic extract was further characterized by GC-MS for the presence of phytochemicals. Treatment with the plant extracts significantly restored arthritic index, change in the body weight and immune organ weight, and the histopathological indices. Both extracts significantly (p < 0.05) reduced the serum concentration of rheumatoid factor, C-reactive protein, PGE2, and TNF-α in arthritic rats. The extracts persuasively down-regulated the COX-2, PGE2, IL-1ß, IL-6, NF-κB, and TNF-α, and up-regulated the mRNA expression of I-κB, IL-4, and IL-10. Both extracts increased the activities of CAT and SOD while reducing the formation of MDA in a dose- dependent manner in the liver. Histopathological evaluation showed that treatment with the plant extracts significantly (p < 0.05) reduced joint inflammation, pannus formation, and bone erosion in treatment groups in comparison to arthritic control. Phytochemicals detected by GC-MS in the methanolic extract included esters, alcohols, ketones, fatty acids, and vitamin E. These findings provide evidence of the anti-arthritic potential of M. rivae extracts in chronic polyarthritis model.

Correction to: Moringa oleifera leaf extracts attenuate Complete Freund's adjuvant-induced arthritis in Wistar rats via modulation of inflammatory and oxidative stress biomarkers.

In our article entitled "Moringa rivae leaf extracts attenuate Complete Freund's adjuvant-induced arthritis in Wistar rats via modulation of inflammatory and oxidative stress biomarkers", we described the anti-arthritic effects of the plant leaves from Pakistan that we referred to as Moringa rivae (Saleem et al. 2019). Unfortunately, the identity of the plant material was incorrect. In fact, the plant leaves under study were later identified as belonging to wild type Moringa oleifera Lam. rather than Moringa rivae Chiov.

Assessment of a mass media campaign on giving way to ambulances in five cities of Pakistan.

OBJECTIVE: To determine the contribution of a mass media campaign towards encouraging more vehicles to give way to ambulances, and to identify the factors associated with higher likelihood of giving way to ambulances. METHODS: The three-phase observational study was conducted from December 2017 to March 2018 in Karachi, Lahore, Rawalpindi, Islamabad and Peshawar cities of Pakistan. Six road sites in different areas of each city were selected for observation. The surveys in each city were supervised by academic partners, including APPNA Institute of Public Health, Karachi, University of Health Sciences, Lahore, Al-Nafees Medical College, Rawalpindi and Islamabad, and Khyber Medical University, Peshawar. Three observation surveys were carried out before, during and after the media campaign on right of way for ambulances. Only those ambulances were observed which were rushing through and seeking space. The behaviour of only those vehicles was observed which had the space to change the lane when the space was sought from them. The association of the outcome of vehicles giving way to ambulances immediately or in a few seconds with the campaign was determined using logistic regression analysis. RESULTS: After adjustment for city of observation, timing of the day and type of vehicle, vehicles during and after the campaign were significantly more likely give space to ambulance (p<0.05) compared to cars, buses and vans were significantly less likely to give space (p<0.05). CONCLUSIONS: Media campaign produced better results in encouraging vehicle-owners to give right of way to ambulances to ensure timely medical assistance.

Assessment of Vancomycin MIC Creep Phenomenon in Methicillin-Resistant Staphylococcus aureus isolates in a Tertiary Care Hospital of Lahore.

OBJECTIVE: To assess vancomycin MIC creep phenomenon in methicillin-resistant Staphylococcus aureus isolated from clinical specimens. METHODS: This descriptive study was conducted in Microbiology department of University of Health Sciences, Lahore from January 2016- December 2019. In this study, vancomycin MICs were revealed by E test method for clinical MRSA strains. For the final evaluation, a single isolate from each patient was taken. The reported vancomycin MICs results were used and the values were not rounded up to the next upward value. For every study year, MIC50, MIC90, median and geometrical mean MIC, percentages of susceptible and resistant strains were calculated. RESULTS: A total of 352 MRSA strains were isolated out of 2704 staphylococcal isolates. Our study showed elevated vancomycin MIC among MRSA isolates. The majority of isolates showed MIC values ≥1.5µg/ml. MIC50, MIC 90 was constant throughout four years period. However, geometric mean MIC increased gradually during the study period. The MIC greater than base year median was overall 17.3%. A complete shift can be observed between MIC "1.0" and "2.0" the percent of cases with MIC "1.0" decreased and with MIC "2.0" increased over time crossing each other in 2017. CONCLUSION: Vancomycin MIC creep was identified in clinical isolates of MRSA, during four years of study period. Even though there is an absence of VISA and VRSA strains; this significant increase in vancomycin MIC trend is indeed worrying for the clinicians about the threat of potential failure of treatment in MRSA infections.

Consanguinity: A blessing or menace at population level?

Consanguinity has highly complex and multifaceted aspects with sociocultural as well as biological debates on its pros and cons. The biological upshot of consanguinity includes the increased homozygosity, which results in manifold increased risk of genetic disorders at family and population levels. On the other hand, in addition to social, cultural, political, and economic benefits, consanguineous marriages have biological advantages at the population level. The consequence of consanguineous marriages is an upsurge in the number of homozygous diseased individuals with fewer chances of mating and reduced chances of survival, therefore evolutionarily confining the transmission of disease alleles to future generations and encouraging its elimination from a population. Protective effects of consanguinity have also been observed in a few diseases in different populations. Although attractive for many reasons, nonconsanguineous marriages will cause risk alleles to spread throughout the population, making most individuals carriers, and ultimately will resume the production of recessive diseases in subsequent generations. Although consanguinity, from an evolutionary point of view, is beneficial at the population level, it increases the risk of diseases in the very next generation. Presently, there is no treatment for most of the genetic disorders; we cannot opt for consanguinity for long-term benefits. Nonconsanguineous marriages are a better strategy by which we may delay disease manifestation for some generations until science offers a viable solution.

Molecular Detection of blaIMP Genes in Metallo-Beta-Lactamase Producing Clinical Gram-Negative Isolates.

BACKGROUND: The use of carbapenem antibiotics in the treatment of serious Gram-negative bacterial infections is under threat due to the emergence of the blaIMP gene amongst the bacterial pathogens. METHODS: The present descriptive cross-sectional study aimed to determine the occurrence of the blaIMP gene and minimum inhibitory concentrations (MICs) of the bacterial pathogens. The carbapenem-resistant isolates were screened out for the detection of MBLs by using the modified Hodge test and disk potentiation method. MBL producing strains were tested for the presence of the blaIMP gene by using PCR technique. The MICs of the blaIMP gene positive bacterial isolates were detected on the Vitek 2 system (bioMerieux). RESULTS: The primary source of MBLs and blaIPM gene carrying bacterial pathogens was blood (38.5%). We isolated 104 bacterial isolates in the initial screening of carbapenem resistance. Metallo-beta-lactamases (MBLs) were detected in 76 (73%) of the isolates which predominantly included 27 (26%) Klebsiella pneumoniae, 20 (19.2%) Acinetobacter baumannii, 16 (15.4%) Pseudomonas aeruginosa, and 11 (10.6%) E. coli while the other Gram-nega-tive MBL producing bacteria were few in number. The blaIPM gene was detected in 1 (1.3%) case of Acinetobacter baumannii and 1 (1.3%) case of Stenotrophomonas maltophilia. These strains were found to be multi-drug resistant with high MICs (≥ 8 to ≥ 256 µg/mL) against the majority of the drugs. CONCLUSIONS: The emergence of the blaIPM gene is a matter of serious concern as it left us with limited treatment options of minocycline, tigecycline, and levofloxacin. The horizontal transfer of blaIPM gene in other Gram-negative isolates can lead the epidemics of multidrug resistance.

Inhibitory effects of Clematis orientalis aqueous ethanol extract and fractions on inflammatory markers in complete Freund's adjuvant-induced arthritis in Sprague-Dawley rats.

Clematis orientalis Linn has long been used as ethnopharmacy for the treatment of arthritis. This study is intended to evaluate the curative efficacy of Clematis orientalis in treating polyarthritis in rats. Aqueous ethanolic extract and fractions (hexane, butanol and aqueous) were administered orally at 200 mg/kg for 28 days after CFA immunization. Paw swelling, paw diameter, arthritic score, body weight, hematological parameters, radiographic and histological analysis of ankle joints were evaluated. Moreover, levels of various inflammatory markers through RT-PCR and ELISA were measured. DPPH and reducing power assays were used to appraise antioxidant capacity. Qualitative phytochemical analysis, determination of total phenolic and flavonoid contents were also carried out. Aqueous ethanolic extract and fractions significantly (p < 0.001) reduced paw volume, paw thickness and arthritic score and considerably prevented decrease in body weight along with anomalous alterations in hematological parameters in comparison with arthritic control. X-ray and histological examination revealed no significant structural changes in ankle joints of treated rats. Expression levels of IL-1ß, TNF-α, IL-6, COX-2 and NF-Kß were significantly (p < 0.05-0.001) suppressed as well as noteworthy increase in the levels of IL-4 and IL-10 among treated animals has been detected. Overproduction of TNF-α and PGE2 was substantially prevented in animals given different treatments. Aqueous ethanol extract and its fractions demonstrated significant and concentration-dependent antioxidant potential. In general, among fractions aqueous fraction exhibited a greater anti-arthritic effect. Phytochemical analysis of aqueous fraction confirmed the presence of flavonoids and glycosides, 215.29 mgGAE/ml phenolic content and 633.03 µgQE/ml flavonoid content. Thus, we suggest Clematis orientalis as a potent strategy for the treatment of rheumatoid arthritis.

Analysis of toll-like receptors-9 (TLR9) gene polymorphism (rs5743836) in Pakistani patients with HCV.

Toll-like receptors (TLRs) are innate immune receptors that mediate the inflammatory response during HCV infections. The goal of this study was to evaluate the association of TLR9 gene polymorphism (rs5743836) in Pakistani patients infected with genotype 3a of HCV. Total 500 subjects were recruited, 400 HCV patients and 100 healthy individuals. Genotyping of TLR9 (-1237T/C, rs5743836) was carried out in 400 HCV patients (323 interferon responders and 77 interferon non responder) and control group by applying High resolution melting (HRM) curve assay. No remarkable differences in distribution of genotype between HCV (p<0.0001; OR= 3.21, 95% CI= (2.514.12) and control groups (p<0.0001; OR=0.092, 95%CI= (0.0580.14) were observed. In conclusion TLR9-1237T/C gene polymorphism may not be considered as a molecular risk for patients with HCV in Pakistan.

Interleukin 6 Receptor (IL6-R) Gene Polymorphisms Underlie Susceptibility to Rheumatoid Arthritis.

BACKGROUND: Increased production of interleukin 6 (IL-6) is associated with rheumatoid arthritis that acts through its receptor, IL-6R (interleukin 6 receptor). Various single nucleotide polymorphisms in the IL6R gene conferring susceptibility to rheumatoid arthritis have been identified in various populations yet these associations have not been fully established. The present study was pursued with the aim to evaluate a possible association between three single nucleotide polymorphisms (rs2228145, rs4537545, rs4845617) of the IL6R gene and rheumatoid arthritis in Pakistani patients. METHODS: For this purpose, we recruited 60 patients diagnosed with rheumatoid arthritis and 60 healthy age and gender matched controls. Blood samples were collected and DNA was extracted. Sanger DNA sequencing was performed to evaluate the SNPs in IL6R and the data were statistically evaluated using chi-square test. RESULTS: Results of our study indicated that rs2228145 and rs4845617 were significantly associated with rheumatoid arthritis in Pakistani population. However, no association could be established between IL6R (rs4537545) and rheumatoid arthritis in Pakistani population. CONCLUSIONS: This study reports a possible genetic association of IL6R (rs2228145 and rs4845617) to the genetic susceptibility of rheumatoid arthritis.

Role of different regions of the hepatitis C virus genome in the therapeutic response to interferon-based treatment.

Hepatitis C virus (HCV) is considered a significant risk factor in HCV-induced liver diseases and development of hepatocellular carcinoma (HCC). Nucleotide substitutions in the viral genome result in its diversification into quasispecies, subtypes and distinct genotypes. Different genotypes vary in their infectivity and immune response due to these nucleotide/amino acid variations. The current combination treatment for HCV infection is pegylated interferon α (PEG-IFN-α) with ribavirin, with a highly variable response rate mainly depending upon the HCV genotype. Genotypes 2 and 3 are found to respond better than genotypes 1 and 4, which are more resistant to IFN-based therapies. Different studies have been conducted worldwide to explore the basis of this difference in therapy response, which identified some putative regions in the HCV genome, especially in Core and NS5a, and to some extent in the E2 region, containing specific sequences in different genotypes that act differently with respect to the IFN response. In the review, we try to summarize the role of HCV proteins and their nucleotide sequences in association with treatment outcome in IFN-based therapy.