Treatment of bisphosphonate induced osteonecrosis of jaw in rats using an angiogenesis factor (A-Heal) and ABMDO (Autologous Bone Marrow Derived Osteoblasts).

BACKGROUND AND OBJECTIVE: The aims of this study were to create Bisphonates Related Osteonecrosis of the Jaw (BRONJ) in rats and treat them with an angiogenesis factor (A-Heal) and ABMDO (Autologous Bone Marrow Derived Osteoblasts). MATERIALS AND METHODS: Thirty female Wistar rats were procured. Rats were labeled as Group I to III. Group I = Osteoblast group, Group II = A-Heal and Group III Control group. In Groups I-III, BRONJ was created and treated in Group I with ABMDO, Group II with A-Heal and Group III was the control group. At the end of the four weeks post treatment, all the animals were humanely killed. The intact maxillae were removed in total. Histopathological and radiological examinations were carried out with physicians blinded to the groups. RESULTS: Computerized tomography revealed that Groups I and II demonstrated the presence of dense osteosclerosis, intralesional calcifications, and adequate healing of the overlying soft tissues compared to Group III, which showed the presence of bone erosions at the alveolar ridge with a lack of intralesional calcifications and ulceration of the overlying soft tissues. Histologically, H&E staining Group 1 and Group 2 both showed marked reactive bone formation. Group 2 additionally revealed the most prominent vascular proliferation (also highlighted by Factor VIII, an endothelial cell marker) among all groups. Group 3 showed cartilaginous proliferation with less reactive bone formation, implicating decreased endochondral ossification compared to Groups 1 and 2. CONCLUSION: This study shows that angiogenesis factor (A-Heal) and ABMDO were successful in the treatment of experimentally created BRONJ in an animal model.

The clinical and radiological effectiveness of autologous bone marrow derived osteoblasts (ABMDO) in the management of avascular necrosis of femoral head (ANFH) in sickle cell disease (SCD).

PURPOSE: Avascular necrosis of the femoral head is a common issue faced by orthopaedic surgeons that ranges between 10 and 18%, but in patients with SCD, the incidence reaches 30%. There is no definite treatment except joint arthroplasty. Regenerative medicine is an option to cure or delay joint arthroplasty. We report here our experience with the injection of ABMDO to manage ANFH and report our medium-term results, the progression of the ANFH if any and the delay in total hip arthroplasty. (THA). METHODS: Sixty-Three (63) patients with SCD and ANFH were examined and thoroughly investigated, and those who had ANFH < grade II were consented to receive ABMDO. Patients were clinically assessed preoperatively using the Visual analogue scale (VAS), Modified Harris Hips Score (MHHS) and Azam-Sadat Score (ASS) for Quality of Life Score for Chronic Hip Disease. Ten millilitres of bone marrow were aspirated under local anaesthesia and placed in 20 CC of culture media. Osteoblasts were cultured from the aspirated bone marrow. Under anaesthesia, the osteonecrosed lesion was drilled using a 3-mm cannulated drill, and 5 million osteoblasts were injected at the lesion site. Patients were evaluated in the outpatient clinic after 2 weeks. At 4 months, a repeat MRI was done, and patients were followed for a minimum of 2 years. RESULTS: The average age of patients was 25.93 ± 5.48 years. There were 41 (65%) females and 22 (35%) males. The mean hemoglobin S was 83.2 ± 5.1%. The average follow-up was 49.05 ± 12.9 (range: 24-60) months. TheVAS significantly improved from 7.79 ± 1.06 initially to 4.07 ± 1.08 (p < 0.0001) at 2 weeks and continued to improve for the next 24 months, when it was 2.38 ± 0.55 (p < 0.0001). The MHHS improved from 41.77 ± 5.37 initially to 73.19 ± 6.48 at 4 months (p < 0.001), and at 24 months, it was 88.93 ± 3.6 (p < 0.001). The ASS also significantly improved from 2.76 ± 0.49 preoperatively to 7.92 ± 0.09 (p < 0.0001) at 24 months. A comparison of the MRI's from before and after the osteoblast implantation revealed new bone formation and amelioration of the avascular lesions. Three patients were unsatisfied with their outcomes. and one patient suffered a repeat attack of the vaso-occlusive crisis within 6 months of the osteoblast injection. CONCLUSIONS: The results give credence to our earlier short follow-up results showing that osteoblast transplantation has great potential in the healing of avascular lesions. Our study fits the criteria of a Phase II clinical trial, and we believe a larger study equivalent to Phase III numbers should be conducted and include patients with not only SCD but also steroid-induced and idiopathic avascular necrosis. LEVEL OF EVIDENCE: II.

A 20-year retrospective clinical analysis of Candida infections in tertiary centre: Single-center experience.

INTRODUCTION: Fungal infections have risen exponentially in the last decade. In fact, candidiasis has become the most frequent type of hospital acquired infection especially in patients receiving treatment for chronic and terminal illnesses in a hospital. A retrospective analysis for a period of twenty year was undertaken to analyze the incidence rate of candidiasis, especially of Candida species, patients treated in a tertiary care center. MATERIALS AND METHODS: Clinical data was collected from samples of patients who were receiving tertiary care were presenting with clinically suspected fungal infections. Direct microscopy with 10% potassium hydroxide was done to visualize the presence of fungal elements, and Gram staining was done for any suspected yeast infection. The samples were inoculated on Sabouraud's Dextrose Agar and kept at 22 °C. RESULTS: A total of 1256 samples with presumed fungal etiology were included in the study. The maximum number of fungal infections were present in elderly (70-79 years age). Females (53.8%) were more affected (45.5%). 21% isolates were identified as yeast but belonged to non-Candida fungi. Among Candida species, Candida albicans was the most dominant species (58.3%) followed by Candida glabrata (6.4%). The year-round data of fungal cases showed that the highest incident of Candida albicans infection were in January with a mean value of 3.80, while the lowest infections were reported in June, with prevalence of 2.32 of C. albicans. The twenty-year data analysis showed that the years 2001 and 2000 showed the highest incidents of C. albicans, with a mean prevalence of 7.50 and 6.83, respectively. Specimen vs fungal prevalence data showed that 38% of the C. albicans were isolated from body aspirate specimens, followed by 26% from swab specimens. CONCLUSION: The high prevalence of Candida spp. in the present study suggests increased susceptibility of patients with critical or chronic illnesses to fungal infections.

Molecular Epidemiological Surveillance of CTX-M-15-producing Klebsiella pneumoniae from the patients of a teaching hospital in Sindh, Pakistan.

Background  The presence of Extended-spectrum ß-lactamase (ESBL) positive bacteria in hospital setting is an aggravating influential factor for hospitalized patients, and its consequences may be hazardous. Therefore, there is a need for rapid detection methods for newly emerging drug-resistant bacteria. This study was aimed at the molecular characterization of ESBL-positive  Klebsiella pneumoniae isolates recovered from clinical samples.   Methods  A total of 513  K. pneumoniae isolates were obtained from various clinical samples during June 2019 to May 2020. The collected isolates were investigated for antimicrobial susceptibility (antibiogram), and PCR and DNA sequencing were performed to analyse the ESBL genes.   Results  Among the 513 isolates, as many as 359 (69.9%) were ESBL producers and 87.5% were multi-drug resistant, while none had resistance to imipenem. PCR scored 3% blaTEM, 3% blaSHV, and 60% blaCTX-M-15 genes for the tested isolates.   Conclusion  The study showed that CTX-M-15 was the major prevalent ESBL type among the isolates. Additionally, all the isolates were susceptible to carbapenems. Screening and detection of ESBL tests are necessary among all isolates from the enterobacteriaceae family in routine microbiology laboratory to prevent associated nosocomial infections. A larger study is essential to understand molecular epidemiology of ESBL producing organisms to minimize morbidities due to these multidrug resistant organisms.

Autologous Bone Marrow-Derived Chondrocytes for Patients with Knee Osteoarthritis: A Randomized Controlled Trial.

RESULTS: There were a total of 60 patients who were followed up. Three patients in Group II were removed from the analysis as they underwent total knee arthroplasty (TKA). A notably significant improvement was noticed in the ABMDC group on all scores of VAS and MKSSSF with P < 0.0001. The control group continued to be dissatisfied with the treatment they were taking. CONCLUSIONS: This study reveals that a single injection of 5 million of ABMDC was efficient in reducing the symptoms, improving the functional score and betterment of QOL.

Efficacy of autologous bone marrow derived Mesenchymal stem cells (MSCs), osteoblasts and osteoblasts derived exosome in the reversal of ovariectomy (OVX) induced osteoporosis in rabbit model.

Background: A recent study showed that OVX-induced osteoporosis was reversed after injection of osteoblasts cultured from the bone marrow in rats. The present study evaluated the effect of injecting MSCs, osteoblasts, and exosomes isolated from osteoblasts for the treatment of osteoporosis in the rabbit model. Methods: Osteoporosis was created in 40 rabbits by performing ovareictomy at 6 months of age, and 1 mg/kg body weight of methyl prednisolone sodium succinate was injected daily for 8 weeks. Animals were fed twice daily and were given water ad libitum. MSCs and osteoblasts were grown from the bone marrow as per the methodology described earlier. From osteoblasts, exosomes were extracted. After the 15th day, MSCs (Group 2), osteoblasts (Group 3), and exosomes (Group 4) were injected into 5 animals each, and 0.5 ml of normal saline were injected into the control group (Group 1). After 12 weeks (11 months of age), all the animals were euthanized. The whole femur and the lumbar vertebrae 3-5 were dissected out and were subjected to radiological assessment using high-resolution peripheral quantitative computerized tomography (HRpQCT). All parameters of the bone volume, trabecular number, thickness, and spacing were assessed using SPSS (Statistical Package for the Social Sciences), version 21.0, Chicago, Illinois. A p value of <0.05 was considered Statistically significant with a confidence interval (CI) of 95%. Results: Structural indices of the osteoblasts-injected animals were significantly better than the control group for the distal femur. The most significant improvement was seen in the osteoblasts, MSCs, and exosomes group in that order. The p value of all parameters was <0.0001 in the osteoblasts group, whereas the total and bone volume had a lower p value in the MSCs group. In the osteoblasts group, the positive changes were similar in the distal femur and lumbar vertebrae, but with MSCs and exosomes, the changes were more pronounced in the vertebral spine than the distal femur. Conclusions: This study shows that autologous bone marrow-derived osteoblasts have the robust influence of reversing OVX-induced osteoporosis in rabbits.

Direct DNA Sequencing-Based Analysis of Microbiota Associated with Hematological Malignancies in the Eastern Province of Saudi Arabia.

INTRODUCTION: Bloodstream infections (BSI) among patients with hematological malignancies (HM) could predispose them to higher morbidity and mortality for various underlying conditions. Several microorganisms, either pathogenic or opportunistic normal human flora, could cause severe bacteremia and septicemia. While conventional methods have their own limitations, molecular methods such as next-generation sequencing (NGS) can detect these blood infections with more reliability, specificity, and sensitivity, in addition to information on microbial population landscape. Methodology. Blood samples from HM patients (n = 50) and volunteer blood donor control individuals with no HM (n = 50) were subjected to 16S rRNA gene amplification using standard PCR protocols. A metagenomic library was prepared, and NGS was run on a MiSeq (Illumina) sequencer. Sequence reads were analyzed using MiSeq Reporter, and microbial taxa were aligned using the Green Genes library. RESULTS: 82% of the patients showed BSI with Gram-negative bacteria as the most predominant group. E. coli comprised a major chunk of the bacterial population (19.51%), followed by K. pneumoniae (17.07%). The CoNS and Viridans Streptococci groups are 17.07% and 14.63%, respectively. Other major species were S. aureus (9.75%), P. aeruginosa (7.31%), A. baumannii (4.87%), E. cloacae (4.87%), and P. mirabilis (4.87%). 34.14% of the cases among patients showed a Gram-positive infection, while 14.63% showed polymicrobial infections. CONCLUSION: Most of the BSI in patients were characterized by polymicrobial infections, unlike the control samples. Molecular methods like NGS showed robust, fast, and specific identification of infectious agents in BSI in HM, indicating the possibility of its application in routine follow-up of such patients for infections.

Effectiveness of single versus multiple sessions of photodynamic therapy as adjunct to scaling and root planing on periodontopathogenic bacteria in patients with periodontitis.

OBJECTIVE: The aim of the present 6-months' follow-up study was to assess the influence of single versus multiple sessions of photodynamic therapy (PDT) as adjunct to scaling and root planning (SRP) on periodontopathogenic bacteria in patients with periodontitis. METHODS: Forty-five patients with periodontitis were included. The patients were randomly divided into 3 groups. In groups 1 (n = 15), 2 (n = 15) and 3 (n = 15), the patients under went SRP with PDT at (a) baseline; (b) baseline and after 1-month; and (c) baseline and after 1- and 3-months. Peri-implant plaque index (PI), bleeding on probing (BOP), probing depth (PD), clinical attachment loss (CAL) and marginal bone loss (MBL) were measured at baseline and 6-months follow-up. Counts of Porphyromonas gingivalis, Treponema denticola, Prevotella intermedia, Fusobacterium nucleatum and Aggregatibacter Actinomycetemcomitans were determined before SRP and at 4- and 6-months follow-up. RESULTS: All patients had grade-B periodontitis. There was no statistically significant difference in age and gender in all groups. At- 6-months' follow-up, patients that underwent SRP once showed significantly higher counts of periodontopathogenic bacteria in the oral biofilm compared with patients that received photobiomodulation twice or three times (P < 0.05). There was no significant difference in the periodontal parameters and counts of gram-negative bacteria in patients that received photobiomodulation 2 or 3 times. CONCLUSION: At least 2 sessions of PDT following baseline SRP is essential to achieve a significant reduction in the counts of subgingival bacteria in periodontitis patients over a 6-month follow-up period.

Antimicrobial resistance, mechanisms and its clinical significance.

Antimicrobial agents play a key role in controlling and curing infectious disease. Soon after the discovery of the first antibiotic, the challenge of antibiotic resistance commenced. Antimicrobial agents use different mechanisms against bacteria to prevent their pathogenesis and they can be classified as bactericidal or bacteriostatic. Antibiotics are one of the antimicrobial agents which has several classes, each with different targets. Consequently, bacteria are endlessly using methods to overcome the effectivity of the antibiotics by using distinct types of mechanisms. Comprehending the mechanisms of resistance is vital for better understanding and to continue use of current antibiotics. Which also helps to formulate synthetic antimicrobials to overcome the current mechanism of resistance. Also, encourage in prudent use and misuse of antimicrobial agents. Thus, decline in treatment costs and in the rate of morbidity and mortality. This review will be concentrating on the mechanism of actions of several antibiotics and how bacteria develop resistance to them, as well as the method of acquiring the resistance in several bacteria and how can a strain be resistant to several types of antibiotics. This review also analyzes the prevalence, major clinical implications, clinical causes of antibiotic resistance. Further, it evaluates the global burden of antimicrobial resistance, identifies various challenges and strategies in addressing the issue. Finally, put forward certain recommendations to prevent the spread and reduce the rate of resistance growth.

Spinal cord injury regeneration using autologous bone marrow-derived neurocytes and rat embryonic stem cells: A comparative study in rats.

BACKGROUND: Spinal cord injury (SCI) is an important cause of traumatic paralysis and is mainly due to motor vehicle accidents. However, there is no definite treatment for spinal cord damage. AIM: To assess the outcome of rat embryonic stem cells (rESC) and autologous bone marrow-derived neurocytes (ABMDN) treatment in iatrogenic SCI created in rats, and to compare the efficacy of the two different cell types. METHODS: The study comprised 45 male Wistar rats weighing between 250 and 300 g, which were divided into three groups, the control, rESC and ABMDN groups. The anesthetized animals underwent exposure of the thoracic 8th to lumbar 1st vertebrae. A T10-thoracic 12th vertebrae laminectomy was performed to expose the spinal cord. A drop-weight injury using a 10 g weight from a height of 25 cm onto the exposed spinal cord was conducted. The wound was closed in layers. The urinary bladder was manually evacuated twice daily and after each evacuation Ringer lactate 5 mL/100 g was administered, twice daily after each bladder evacuation for the first 7 postoperative days. On the 10th day, the rats underwent nerve conduction studies and behavioral assessment [Basso, Beattie, Brenham (BBB)] to confirm paraplegia. Rat embryonic stem cells, ABMDN and saline were injected on the 10th day. The animals were euthanized after 8 wk and the spinal cord was isolated, removed and placed in 2% formalin for histopathological analysis to assess the healing of neural tissues at the axonal level. RESULTS: All the animals tolerated the procedure well. The BBB scale scoring showed that at the end of the first week no recovery was observed in the groups. Post-injection, there was a strong and significant improvement in rats receiving rESC and ABMDN as compared to the control group based on the BBB scale, and the Train-of-four-Watch SX acceleromyography device exhibited statistically significant (P < 0.0001) regeneration of neural tissue after SCI. Histological evaluation of the spinal cord showed maximum vacuolization and least gliosis in the control group compared to the rESC and ABMDN treated animals. In the ABMDN group, limited vacuolization and more prominent gliosis were observed in all specimens as compared to the control and rESC groups. CONCLUSION: This study provided strong evidence to support that transplantation of rESC and ABMDN can improve functional recovery after iatrogenic SCI. The transplanted cells showed a beneficial therapeutic effect when compared to the control group.

Modulation of host immune status by cryptococcus co-infection during HIV-1 pathogenesis and its impact on CD+4 cell and cytokines environment.

BACKGROUND: Cryptococcus infection is the second most common opportunistic infection in HIV patients with an increased rate of morbidity and mortality. Altered immune system during HIV- Cryptococcus co-infection is yet to be explored by laboratory. This study evaluates pro- and the anti-inflammatory cytokines in HIV patients with Cryptococcus co-infection and correlate them with CD4+T cell counts as well as viral loads before the initiation of drug therapy. This information would enable to understand host immune modulation and cellular environment during co-infection and understand its impact on HIV pathogenesis. METHODOLOGY: The study comprised four categories of patients with cryptococcosis, HIV, HIV-cryptococcosis co-infected and asymptomatic Healthy volunteers. All the patients and healthy individuals were subjected to CD4+T cells count by FACS using monoclonal antibody cocktail CD4+T cell count (counts per mm3) which was counted using multiSET software on FACS caliber. The viral loads were counted in terms of viral RNA copy numbers which was estimated by real-time PCR using by Artus HIV-1 RG. The sensitivity of kit was >70 IU/ml. ELISA was performed for IL-12 p70, IL-12, IL-4, IL-10, IL-6, TNF-α and IFN-Y using commercially kits (BD Biosciences, USA). Significant variations were assayed by Student's t-test and P values ≤ 0.05 were considered statistically significant. RESULTS: Reduction in CD+4 cell counts was highly significant in HIV patients with or without cryptococcosis. CD4+T cell counts were inversely proportional to viral load. TNF-α levels were raised in cryptococcosis patients significantly higher than healthy individuals. TNF-α was more or less not dependent on viral load but it was more related to the cryptococcosis IL-12 levels were increased in patients with infection and was highest in the HIV infected group. Level of IL-4 was similar in healthy and patients with cryptococcosis but it was elevated in HIV-Crypto co-infected patients. HIV infected patients showed a significant increase in IL-4 level and it was elevated higher in co-infected patients. IL-10 and IL-6 were significantly higher in HIV patients. The fungal infection did not influence the levels of IL-10 in HIV group but IL-6 was low in fungal infected patients. CONCLUSION: There are very limited studies related to the immune modulation status of HIV co-infected with Cryptococcus before the initiation of any drug therapy. Such information might through in-depth light to understand the initial state of the immune environment which certainly would play a pivotal role in the outcome of the immune modulation.

Stem-cell therapy for ovariectomy-induced osteoporosis in rats: a comparison of three treatment modalities.

Background: Recent studies have shown that ovariectomy-induced osteoporosis in rats can be reversed by infusion of osteoblasts cultured from mesenchymal stem cells (MSCs). This study compares the influence of MSCs, osteoblasts, and exosomes derived from osteoblasts for the treatment of osteoporosis. Methods: Osteoporosis was induced in 40 female Sprague Dawley rats by performing ovariectomy. After 12 weeks, bone marrow was harvested and MSCs separated from bone-marrow aspirate as described by Piao et al. After 15 days, autologous osteogenically differentiated cells from the MSCs were available. Exosomes were isolated from osteoblasts by modification of the technique described by Ge et al. MSCs and osteoblasts (106 cells in 0.5 mL normal saline) and exosomes (100 µg protein) were injected into the tail veins of the animals. Animals were euthanized after 12 weeks and femurs and lumbar spines dissected and analyzed using high-resolution peripheral quantitative computed tomography. Results: When compared to the control group, osteoblast-treated animals showed significant differences in all parameters compared, with P-values ranging between <0.002 and <0.0001. Comparison among osteoblasts, MSCs, and exosomes, showed that osteoblasts had positive and statistically significant new-bone formation. The comparison for the spine was similar to the distal femur for osteoblasts. Conclusion: This study showed robust positive bone-forming changes after osteoblast injection in the distal femur and the spine when compared to controls, MSCs, and exosomes.

Prevalence of Hepatitis B Virus (HBV) Among Blood Donors in Eastern Saudi Arabia: Results From a Five-Year Retrospective Study of HBV Seromarkers.

BACKGROUND: Transfusion-transmissible hepatitis B virus (HBV) infection is a major problem worldwide. Recently, confirmatory nucleic acid tests (NATs) for HBV DNA have been employed in several countries. We assessed the prevalence and yearly trends of HBV infection in blood donors in the Eastern Province of Saudi Arabia, screening for HBV surface antigen (HBsAg), antibody against HBV core antigen (anti-HBc), and HBV DNA. METHODS: Between 2011 and 2015, a total of 22,842 donors were screenedfor HBsAg, anti-HBc, and HBV DNA using the HBsAg Qualitative II kit (Abbott, Ireland Diagnostics Division, Sligo, Ireland), ARCHITECT Anti-hepatitis B core antigen antibody (HBc) II Assay kit (Abbott GmbH & Co. KG, Wiesbaden, Germany), and NAT Procleix Ultrio Elite Assay kit (Grifols Diagnostic Solutions Inc., Los Angeles, CA, USA), respectively. RESULTS: A total of 739 (3.24%) donors were HbsAg(+), anti-HBc(+), or HBV DNA(+); 63 (0.28%) were HbsAg(+), anti-HBc(+), and HBV DNA(+). Twelve (0.05%) were anti-HBc(+) and HBV DNA(+) but HBsAg(-); they were considered to have occult infection. Further, 664 (2.91%) were HBsAg(-) but anti-HBc(+), indicating chronic or resolving infection. HBV prevalence increased significantly from 2011 to 2012, increased marginally till 2013, and showed a decreasing trend from 2013 (P>0.05). CONCLUSIONS: The five-year prevalence of HBV infection among blood donors in the Eastern Province of Saudi Arabia (3.24%) is lower than that reported for other regions in the country. The occult HBV infection rate of 0.05% emphasizes the importance of NATs in isolating potential infectious blood units.

A simplified colorimetric method for rapid detection of cell viability and toxicity in adherent cell culture systems.

PURPOSE: Evaluation of cell viability and toxicity in adherent culture systems is of critical relevance for a wide range of disciplines of biomedical sciences research, including cancer research, toxicology, pharmacology, cell biology, neurology and nanomedicine. Several well-established cytotoxicity assays are widely used by researchers, including the most-preferred MTT assay. Nevertheless, there are problems associated with them, for example; in terms of the time-factor and solubilization of the formzan crystals before its spectroscopic quantification. In this study, we propose a simple, fast and cost-effective colorimetric assay that is free of these issues. METHODS: Our assay was based upon reductive splitting of blue-green colored supravital safranin derivative dye Janus green B (JG-B) to pink colored diethyl safranin by oxidoreductases of the electron transport chain (ETC) of actively respiring mitochondria. Because this conversion can be easily and reliably followed spectroscopically, measure of diethyl safranin formed from extraneously added JG-B provides a proficient indicator of cellular health and viability. RESULTS: Using MCF-7, a breast cancer cell line, we provide a proof of concept for the suggested assay and compare it with the MTT assay. CONCLUSION: Unlike the MTT assay, our JG-B assay does not require a solubilization/extraction step, and hence follows a much simpler and time-efficient protocol suitable for high-throughput analysis of cell viability in anchorage-dependent cell culture models. Additionally, the JG-B cell viability assay reported here can be suitably applied either independently or in complementation with other assays for the analysis of cellular viability and toxicity in both analytic and therapeutic aspects of research.

Association of Uncoupling Protein 1 (UCP1) gene polymorphism with obesity: a case-control study.

BACKGROUND: Obesity is one of the main causes of morbidity and mortality worldwide. More than 120 genes have been shown to be associated with obesity related phenotypes. The aim of this study was to determine the effect of selected genetic polymorphisms in Uncoupling protein 1 (UCP1) and Niemann-Pick C1 (NPC1) genes in an obese population in Saudi Arabia. METHODS: The genotypes of rs1800592, rs10011540 and rs3811791 (UCP1 gene) and rs1805081 and rs1805082 (NPC1 gene) were determined in a total of 492 subjects using TaqMan chemistry by Real-time PCR. In addition, capillary sequencing assay was performed to identify two specific polymorphisms viz., rs45539933 (exon 2) and rs2270565 (exon 5) of UCP1 gene. RESULTS: A significant association of UCP1 polymorphisms rs1800592 [OR, 1.52 (1.10-2.08); p = 0.009] was observed in the obese cohort after adjusting with age, sex and type 2 diabetes. Further BMI based stratification revealed that this association was inconsistent with both moderate and extreme obese cohort. A significant association of UCP1 polymorphisms rs3811791 was observed only in the moderate-obese cohort [OR = 2.89 (1.33-6.25); p = 0.007] but not in the extreme-obese cohort indicating an overlying genetic complexity between moderate-obesity and extreme-obesity. The risk allele frequencies, which were higher in moderate-obese cohort, had abnormal HDL, LDL and triglyceride levels. CONCLUSION: The rs1800592 and rs3811791 of UCP1 gene are associated with obesity in general and in the moderate-obese group in particular. The associated UCP1 polymorphisms in the moderate-obese group may regulate the impaired energy metabolism which plays a significant role in the initial stages of obesity.

Dysfunction of cortical synapse-specific mitochondria in developing rats exposed to lead and its amelioration by ascorbate supplementation.

BACKGROUND: Lead (Pb) is a widespread environmental neurotoxin and its exposure even in minute quantities can lead to compromised neuronal functions. A developing brain is particularly vulnerable to Pb mediated toxicity and early-life exposure leads to permanent alterations in brain development and neuronal signaling and plasticity, culminating into cognitive and behavioral dysfunctions and elevated risk of neuropsychiatric disorders later in life. Nevertheless, the underlying biochemical mechanisms have not been completely discerned. METHODS: Because of their ability to fulfill high energy needs and to act as calcium buffers in events of high intensity neuronal activity as well as their adaptive regulatory capability to match the requirements of the dynamicity of synaptic signaling, synapse-specific or synaptic mitochondria (SM) are critical for synaptic development, function and plasticity. Our aim for the present study hence was to characterize the effects of early-life Pb exposure on the functions of SM of prepubertal rats. For this purpose, employing a chronic model of Pb neurotoxicity, we exposed rat pups perinatally and postnatally to Pb and used a plethora of colorimetric and fluorometric assays for assessing redox and bioenergetic properties of SM. In addition, taking advantage of its ability as an antioxidant and as a metal chelator, we employed ascorbic acid (vitamin C) supplementation as an ameliorative therapeutic strategy against Pb-induced neurotoxicity and dysfunction of SM. RESULTS: Our results suggest that early-life exposure to Pb leads to elevated oxidative stress in cortical SM with consequent compromises in its energy metabolism activity. Ascorbate supplementation resulted in significant recovery of Pb-induced oxidative stress and functional compromise of SM. CONCLUSION: Alterations in redox status and bioenergetic properties of SM could potentially contribute to the synaptic dysfunction observed in events of Pb neurotoxicity. Additionally, our study provides evidence for suitability of ascorbate as a significant ameliorative agent in tacking Pb neurotoxicity.

Bone Marrow-derived Osteoblasts in the Management of Ovariectomy induced Osteoporosis in Rats.

Background: The objective of this study is to assess if infusion of osteoblasts can temporarily reverse osteoporosis in rats. Methods: Osteoporosis was induced in 20 female Sprague-Dawley rats by performing ovariectomy (OVX) that was carried out at 4 weeks of age. At 3 months a biopsy of the iliac crest was made to assess the bone quality and the same site bone marrow was harvested. From the bone marrow aspirate, MSCs were separated. Osteoblasts were then generated and were characterized using Alizarin red staining. Osteoblasts were injected in the tail vein of 10 rats. Two weeks after the injection of osteoblasts, a second biopsy was done. Animals were euthanized after 8 weeks of osteoblasts infusion by overdose of ketamine mixed with xylazine. The whole femurs and lumbar spine were dissected and the specimens were stored in 2% formalin. The specimens were analyzed using HRpQCT (High-resolution peripheral quantitative computerized tomography (µCT 100, SCANCO Medical AG, Brüttisellen, Switzerland). Results: In all the 10 animals from which bone aspiration was performed, osteoblasts were cultured and transplanted. Analysis showed that there was significant bone formation at bone sites of distal femur and lumbar spine (<0.001), with increased number of trabeculae and thickness (P<0.001). Further analysis revealed that there was robust bone formation in the animals that had osteoblasts injection. Conclusions: This preliminary study indicates that osteoblasts infusion can lead to new bone formation in osteoporosis induced by ovariectomy in rats.

Type 2 diabetes associated variants of KCNQ1 strongly confer the risk of cardiovascular disease among the Saudi Arabian population.

Genome-wide association studies have identified several loci associated with an increased risk for cardiovascular disease (CVD) and type 2 diabetes (T2D). Polymorphisms within the KCNQ1 (potassium voltage-gated channel, KQT-like subfamily, member 1) gene are consistently associated with T2D in a number of populations. The current study was undertaken to evaluate the association of 3 polymorphisms of KCNQ1 (rs2237892, rs151290 and rs2237895) with T2D and/or CVD. Patients diagnosed with either T2D (320 patients), CVD (250 patients) or both (60 patients) and 516 healthy controls were genotyped by TaqMan assay run on a real time PCR thermocycler. A statistically significant association was found for SNPs rs151290 (OR = 1.76; 95%CI = 1.02-3.05; p = 0.0435) and rs2237895 (OR = 2.49; 95%CI = 1.72-3.61; p < 0.0001) with CVD. SNP rs151290 (OR = 7.43; 95%CI = 1.00-55.22; p = 0.0499) showed a strong association in patients with both T2D and CVD. None of the SNPs showed any significant association with T2D. Haploview analysis showed that the ACC (rs151290, rs2237892 and rs2237895) haplotype is the most significant risk allele combination for CVD, while CCA is the most significant risk haplotype for co-morbidity with T2D. KCNQ1 polymorphism at SNPs rs151290 and rs2237895 is strongly associated with CVD in this population, but presented no association with T2D.

Type 2 diabetes associated variants of KCNQ1 strongly confer the risk of cardiovascular disease among the Saudi Arabian population

Abstract Genome-wide association studies have identified several loci associated with an increased risk for cardiovascular disease (CVD) and type 2 diabetes (T2D). Polymorphisms within the KCNQ1 (potassium voltage-gated channel, KQT-like subfamily, member 1) gene are consistently associated with T2D in a number of populations. The current study was undertaken to evaluate the association of 3 polymorphisms of KCNQ1 (rs2237892, rs151290 and rs2237895) with T2D and/or CVD. Patients diagnosed with either T2D (320 patients), CVD (250 patients) or both (60 patients) and 516 healthy controls were genotyped by TaqMan assay run on a real time PCR thermocycler. A statistically significant association was found for SNPs rs151290 (OR = 1.76; 95%CI = 1.02-3.05; p = 0.0435) and rs2237895 (OR = 2.49; 95%CI = 1.72-3.61; p < 0.0001) with CVD. SNP rs151290 (OR = 7.43; 95%CI = 1.00-55.22; p = 0.0499) showed a strong association in patients with both T2D and CVD. None of the SNPs showed any significant association with T2D. Haploview analysis showed that the ACC (rs151290, rs2237892 and rs2237895) haplotype is the most significant risk allele combination for CVD, while CCA is the most significant risk haplotype for co-morbidity with T2D. KCNQ1 polymorphism at SNPs rs151290 and rs2237895 is strongly associated with CVD in this population, but presented no association with T2D.

Stem Cell Therapy for Avascular Necrosis of Femoral Head in Sickle Cell Disease: Report of 11 Cases and Review of Literature.

BACKGROUND AND OBJECTIVE: Sickle cell disease (SCD) is quite common in eastern Saudi Arabia and Avascular necrosis of femoral head (ANFH) occurs in 30% of the young patients leading to early joint arthroplasty. This study was conducted to assess the benefits of injection of osteoblasts in the avascular lesions of the head of femur. PATIENTS AND METHODS: A preset technique was used, 10 CC of bone marrow aspiration was performed under local anesthesia and aseptic technique. Osteoblasts were separated from the bone marrow cells. The avascular area was drilled and 10 million osteoblasts were transplanted at the lesion site. Patients were seen in the out patient clinic after two weeks for removal of the suture and addressed the questionnaire and examined for the range of movement. The follow up MRI was performed at 4 months. RESULTS: The average age was 20.2±3.9 years. The mean hemoglobin S was 81.6±4.8 percent. Quality of Life Score for Chronic Hip Disease was assessed and found at 8.6 (1 being the severe limitation and 10 being normal), whereas Harris hip score improved from 41.7±5.1 to 88.93±3.6 (p < 0.001). MRI of pre and post osteoblast implantation showed robust new bone formation and disappearance of the avascular lesions. CONCLUSIONS: The short term results were good and we believe the injection of osteoblast in the avascular lesion of head of femur is a less invasive procedure devoid of any untoward complications and merits such treatment in large patient group with longer follow up.