Vitamin E protects rat mesenchymal stem cells against hydrogen peroxide-induced oxidative stress in vitro and improves their therapeutic potential in surgically-induced rat model of osteoarthritis.

OBJECTIVE: Oxidative stress is a major obstacle against cartilage repair in osteoarthritis (OA). Anti-oxidant agents can play a vital role in addressing this issue. We evaluated the effect of Vitamin E preconditioning in improving the potential of mesenchymal stem cells (MSCs) to confer resistance against oxidative stress prevailing during OA. METHODS: Vitamin E pretreated MSCs were exposed to oxidative stress in vitro by hydrogen peroxide (H2O2) and also implanted in surgically-induced rat model of OA. Analysis was done in terms of cell proliferation, apoptosis, cytotoxicity, chondrogenesis and repair of cartilage tissue. RESULTS: Vitamin E pretreatment enabled MSCs to counteract H2O2-induced oxidative stress in vitro. Proliferative markers, proliferating cell nuclear antigen (PCNA) and Ki67 were up-regulated, along with the increase in the viability of MSCs. Expression of transforming growth factor-beta (TGFß) was also increased. Reduction of apoptosis, expression of vascular endothelial growth factor (VEGF) and caspase 3 (Casp3) genes, and lactate dehydrogenase (LDH) release were also observed. Transplantation of Vitamin E pretreated MSCs resulted in increased proteoglycan contents of cartilage matrix. Increased expression of chondrogenic markers, Aggrecan (Acan) and collagen type-II alpha (Col2a1) accompanied by decreased expression of collagen type-I alpha (Col1a1) resulted in increased differentiation index that signifies the formation of hyaline cartilage. Further, there was an increased expression of PCNA and TGFß genes along with a decreased expression of Casp3 and VEGF genes with increased histological score. CONCLUSION: Taken together results of this study demonstrated that Vitamin E pretreated MSCs have an improved ability to impede the progression of OA and thus increased potential to treat OA.

Combination of ADMSCs and chondrocytes reduces hypertrophy and improves the functional properties of osteoarthritic cartilage.

OBJECTIVE: To evaluate the therapeutic efficacy of Adipose derived MSCs (ADMSCs) in combination with chondrocytes in counteracting oxidative stress in chondrocytes in vitro and in rat model of osteoarthritis (OA). METHOD: Cultured chondrocytes were exposed to oxidative stress with 200 µM Hydrogen peroxide (H2O2), followed by co-culture with ADMSCs or chondrocytes or combination of both cell types in a transwell culture system for 36 h. The cytoprotective effect was assessed by immunocytochemistry and gene expression analysis. In vivo study evaluated therapeutic effect of the above mentioned three treatments after transplantation in OA rats. RESULTS: The Combination of ADMSCs + Chondrocytes decreased the extent of oxidative stress-induced damage of chondrocytes. Enhanced expression level of Acan and Collagen type-II alpha (Col2a1) with a correspondingly decreased expression of Collagen type-I alpha (Col1a1) and Matrix metallopeptidase 13 (Mmp13) was maximally observed in this group. Moreover, reduced count of annexin-V positive cells, Caspase (Casp3) gene expression and Lactate dehydrogenase (LDH) release with concomitantly enhanced viability and expression of proliferating cell nuclear antigen (PCNA) gene was observed. In vivo study showed that homing of cells and proteoglycan contents of knee joints were significantly better in ADMSCs + Chondrocytes transplanted rats. Increased expression of Acan and Col2a1 along with decreased expression of Col1a1 and Mmp13 indicated formation of hyaline cartilage in this group. These rats also demonstrated significantly reduced expression of Casp3 while increased expression of PCNA genes than the other cell transplanted groups. CONCLUSIONS: Our results demonstrated that a combination of ADMSCs and chondrocytes may be a more effective therapeutic strategy against OA than the use of ADMSCs or chondrocytes separately.

Human platelet antigen polymorphisms (HPA-1, -2, -3, -4, -5 and -15) in major ethnic groups of Pakistan.

OBJECTIVES: Gene frequencies of human platelet antigens (HPA) determine the magnitude of platelet immunological disorders like neonatal alloimmune thrombocytopenia, platelet refractoriness and ease of availability of particular HPA-typed platelet donors in a given community. BACKGROUND: However, the pattern of HPA in Pakistani population is not known. AIM: The aim of present study was to determine the gene frequencies of HPA (HPA-1 to -5 and -15) in individuals belonging to major ethnic groups and castes of Pakistani population. MATERIALS AND METHODS: HPA genotyping was done in 593 individuals belonging to all ethnic groups of Pakistan, by polymerase chain reaction-sequence specific primers with detection on polyacrylamide electrophoresis. RESULTS: The gene frequencies of the 'a' and 'b' alleles of HPA-1 to -5 and -15 in Pakistanis were as follows: HPA-1a/b, 0.885/0.115; HPA-2a/b, 0.92/0.08; HPA-3a/b, 0.69/0.31; HPA-4a/b, 1/0; HPA-5a/b, 0.9/0.1; HPA-15a/b, 0.59/0.41. Except for significant difference regarding gene frequency of HPA-3 between Pathans and Sindhis, there was no significant difference of HPA-1 to -5 and -15 between major ethnic groups of Pakistan. The estimated mismatch probability regarding platelet antigens 1-5 and 15 in Pakistanis, after transfusion of random donor platelets, is from 14 to 37%. The expected incidence of neonatal alloimmune thrombocytopenia due to anti-HPA-1a in Pakistani pregnant females is < 1 of 1000 pregnancies and 8-12 of 1000 in case of anti-HPA-5b. Homozygosity of HPA-1b, -2b and -5b genotypes ranged from 1 to 2% in the Pakistani population, whereas homozygosity of HPA-3b and -15b was 11 and 18%. CONCLUSIONS: There is a need to establish donor registries typed for HPA in the transfusion centres of the country.

Life-supporting pig-to-primate renal xenotransplantation using genetically modified donors.

BACKGROUND: In order to circumvent the complement-mediated hyperacute rejection of discordant xenografts, a colony of pigs transgenic for the human regulator of complement activity, human decay-accelerating factor (hDAF), has been produced. METHODS: Seven kidneys from hDAF transgenic pigs and six kidneys from nontransgenic control pigs were transplanted into cynomolgus monkeys; both native kidneys were removed during the same operation. The recipient animals were immunosuppressed with cyclosporine, steroids, and cyclophosphamide. RESULTS: In the transgenic group, the median survival time was 13 days (range, 6-35 days); the median survival time in the control group was 6.5 days (range, 0.3-30 days). There were no cases of hyperacute rejection in the transgenic group, and the two longest-surviving kidneys in this group showed no evidence of rejection on histological examination. In contrast, all control kidneys underwent antibody-mediated rejection, one demonstrating hyperacute rejection and the others acute vascular rejection. CONCLUSION: This study demonstrates that (i) a kidney from an hDAF transgenic pig can support the life of a primate for up to 35 days (and also shows the basic physiological compatibility between the pig and nonhuman primate); (ii) nontransgenic kidneys are not routinely hyperacutely rejected; and (iii) the presence of hDAF on the kidney confers some protection against acute vascular rejection. Improved immunosuppression and immunological monitoring may enable extended survival.

Long-term survival of nonhuman primates receiving life-supporting transgenic porcine kidney xenografts.

BACKGROUND: Recently, there has been a resumed interest in clinical xenotransplantation using pig organs. However, no data are available yet regarding the capacity of porcine organs to sustain the life of a primate beyond the first month. We have attempted to obtain long-term survival of nonhuman primates using human decay-accelerating factor (hDAF) transgenic pig organs and an immunosuppressive strategy particularly aimed at neutralizing the humoral component of the immune response. METHODS: hDAF transgenic or control kidneys were transplanted into 14 bilaterally nephrectomized cynomolgus monkeys (Macaca fascicularis) that underwent splenectomy and were immunosuppressed with cyclosporine A, cyclophosphamide, and steroids. All animals also received recombinant erythropoietin. Postoperatively, the primates were monitored daily. Laboratory evaluations included serum biochemistry, hematology, and measurements of hemolytic antipig antibodies. To assess the role of splenectomy in the control of humoral response, historical data were also used from a group of monkeys (n=7) that received the same immunosuppressive regimen and an hDAF transgenic porcine kidney but did not have splenectomy or receive recombinant erythropoietin. RESULTS: This immunosuppressive approach obtained the longest survival time (78 days) described to date of a primate receiving a life-supporting porcine renal xenograft. Furthermore, four of nine animals in this series survived for 50 days or more. Most biochemical measurements in this study (including plasma urea, creatinine, sodium, and potassium concentrations) remained within normal ranges for several weeks in all of the longest-surviving animals. CONCLUSIONS: Normalization of renal function (urea and creatinine) in primate recipients of porcine renal xenografts suggests that pig kidneys may be suitable for future clinical xenotransplantation. Additional immunosuppressive approaches, specifically designed to prevent humorally mediated immunological damage, should be explored to further prolong survival of primates that have received porcine xenografts.

Orthotopic heart transplantation in a transgenic pig-to-primate model.

BACKGROUND: Previous studies demonstrated that hearts from transgenic pigs expressing human decay-accelerating factor (hDAF) were not hyperacutely rejected when transplanted heterotopically into the abdomen of cynomolgus monkeys. This study examines orthotopic transplantation of hDAF transgenic pig hearts into baboon recipients. METHODS: Orthotopic xenogeneic heart transplantation was performed using piglets, transgenic for hDAF, as donors. Ten baboons were used as recipients and were immunosuppressed with a combination of cyclophosphamide, cyclosporine, and steroids. RESULTS: Five grafts failed within 18 hr without any histological signs of hyperacute rejection. Pulmonary artery thrombosis induced by a size mismatch was observed in two of these animals. The other three recipients died because of failure to produce even a low cardiac output and/or dysrhythmia. The remaining five animals survived between four and nine days. One animal died of bronchopneumonia on day 4. Three xenografts stopped beating on day 5 due to acute vascular rejection. The longest survivor was killed on day 9 with a beating, histologically normal xenograft, because of pancytopenia. CONCLUSIONS: The results reported here demonstrate that hDAF transgenic pig hearts are not hyperacutely rejected when transplanted into baboon recipients. Orthotopically transplanted transgenic pig hearts are capable of maintaining cardiac output in baboons. An optimum immunosuppressive regimen is the subject of ongoing research.

Life supporting function for over one month of a transgenic porcine heart in a baboon.

BACKGROUND: Inhibition of hyperacute rejection (HAR) and sustained graft survival have been demonstrated in a pig-to-primate model of heterotopic cardiac xenotransplantation using pigs transgenic for human Decay Accelerating Factor (hDAF). Building on this work, an orthotopic model has been developed. This case records 39-day cardiac xenograft function in a life-supporting capacity with clinically applicable immunosuppression. METHODS: Using a heart from an hDAF transgenic pig, an orthotopic cardiac transplant was performed on an adult baboon. The immunosuppressive regimen consisted of induction with a short course of cyclophosphamide, followed by maintenance therapy with cyclosporine A, mycophenolate mofetil and a tapering course of corticosteroids. Post-operative monitoring included daily anti-pig hemolytic antibody titer surveillance and endomyocardial biopsy. RESULTS: The animal survived 39 days and was active and energetic throughout its postoperative course, remaining free of signs of cardiopulmonary failure. Endomyocardial biopsy performed on post-operative Day 36 revealed only patches of sub-endocardial fibrosis with no signs of active rejection. The baboon succumbed to an acute cardiopulmonary decompensation immediately following administration of medication via oral gavage. Post-mortem histopathology demonstrated well-preserved myocardial architecture with small foci of mild humoral rejection. CONCLUSIONS: This case documents the longest survival recorded to date of a discordant orthotopic cardiac xenograft and illustrates that the hDAF transgene combined with a clinically acceptable maintenance immunosuppressive regimen enables sustained, life-supporting function of porcine cardiac xenografts in non-human primates. The inhibition of hyperacute rejection and the subsequent control of humoral and cellular rejection for over 1 month demonstrated in this experiment represent significant progress in the development of a viable strategy for clinical xenotransplantation.

Salmonella speticemia resistant to 3rd generation cephalosporins in a child with sickle crisis.

Full text is available as a scanned copy of the original print version.

Prevalence of antibody to hepatitis C virus in Pakistani thalassaemics by particle agglutination test utilizing C 200 and C 22-3 viral antigen coated particles.

Exposure to hepatitis C virus (HCV) and its effect on ALT levels was studied in 35 transfusion dependent cases of thalassaemia major. Twenty-one (60%) cases were anti HCV positive and also showed raised Alanine Transaminase (ALT) levels. Of 14 anti HCV negative, Hepatitis B Surface Antigen (HBs Ag) negative seven showed raised ALT levels, indicating the chances of acute viraemia. Thus there is an urgent need to start anti HCV screening on all blood donations.

Bacterial isolates from neutropenic febrile pediatric patients and their sensitivity patterns to antibiotics.

Patients on cytotoxic therapy often develop neutropenia and fever. Our interest was to identify the common pathogens isolated from such patients and to study the sensitivity patterns of these organisms to the antibiotics used in their treatment. Thus, guidelines can be established by hospitals to identify which antibiotics can be used in the treatment of these patients when the results of cultures and sensitivities are not available. We conducted a retrospective study of neutropenic pediatrics presenting to AKUH from July, 1990 to June, 1996. A total of 153 isolates in 35 different patients were studied. Samples for culture were taken from the sites at risk. The majority of samples consisted of blood, stool, pus and urine. Twenty stool samples were also sent for microscopy. Malignancies were both hematological and non-hematological. Gram negatives were isolated in 52.9%, gram positives in 33.9% and parasites in 13.2%. Salmonella paratyphi B was the most commonly isolated organism, followed by Pseudomonas aeroginosa, Giardia lamblia was the most common parasite. Sensitivity patterns of these organisms to antibiotics studied showed that Escheria coli had the lowest sensitivity rate being only 40% sensitive to Aztreonam and 64% sensitive to Ofloxacillin. A comparison was made between our findings and those reported in literature, as well as the risk factors for developing neutropenia. A guide to management is also discussed.

Seroprevalence of hepatitis B, C and HIV in blood donors in northern Pakistan.

OBJECTIVE: To study the seroprevalence and trends of Hepatitis B, C and HIV sero markers in healthy blood donors of Northern Pakistan. SUBJECTS AND METHODS: Blood donated by healthy donors from both Armed Forces and civilian population, collected from Jan 1996 to Dec 2000 were tested by Enzyme Linked Immunoassay at Armed Forces Institute of Transfusion Rawalpindi, Pakistan. Demographic data of these donors was also studied. RESULTS: Of 103858 blood donors, 3.3% (95% CI 3.20%-3.41%) were HBsAg, 4.0% (95% CI 3.91%-4.11%) were anti HCV and 0.007% anti HIV positive. Their average was 28 years. HBsAg positive donors were a decade younger than anti HCV positive donors. Pattern in Armed Forces and civilians donors was similar but there was significant reduction in the prevalence of HBsAg carriage in all blood donors. CONCLUSION: This study supports the changing trends in HBV/HCV seroprevalence in blood donors and a low prevalence of HIV in Pakistani population.

Complications of in-dwelling venous access devices: a single institution experience.

OBJECTIVE: To determine the complications of venous access devices (VADs) in cancer patients. SETTING: Retrospective study in a tertiary referral center with specialist hematology and oncology services. SUBJECTS: First one hundred consecutive patients who were implanted a VAD. All patients had an underlying cancer and the devices were inserted by the same surgeon. The duration of use of VADs and causes of their premature removal were noted. RESULTS: One hundred VADs (55 port-a-caths and 45 Hickman's lines) were inserted in a total of 89 patients over a 7.5 year period. Majority of patients had acute myeloid leukemia (22) gastrointestinal malignancies (20) breast cancer (19) and genito-urinary cancers (15). The mean duration of use was 110 days; 157 days for the port-a-cath and 53 days for the Hickman's line. Nineteen devices (10 port-a-caths and 9 Hickman's lines) had to be removed prematurely. Two Hickman's lines got removed accidentally. The causes of premature removal included device failure (9), exist site infection (4), luminal infection (3) and tunnel infection (3). CONCLUSION: The mean duration of use and the complication rates are comparable with studies reported in the literature.

The logistic EuroSCORE in cardiac surgery: how well does it predict operative risk?

OBJECTIVES: To study the ability of the logistic EuroSCORE to predict operative risk in contemporary cardiac surgery. DESIGN: Retrospective analysis of prospectively collected data. SETTING: All National Health Service centres undertaking adult cardiac surgery in northwest England. PATIENTS: All patients undergoing cardiac surgery between April 2002 and March 2004. MAIN OUTCOME MEASURES: The predictive ability of the logistic EuroSCORE was assessed by analysing how well it discriminates between patients with differing observed risk by using the area under the receiver operating characteristic (ROC) curve and studying how well it is calibrated against observed in-hospital mortality. The performance of the EuroSCORE was examined in the following surgical subgroups: all cardiac surgery, isolated coronary artery surgery, isolated valve surgery, combined valve and coronary surgery, mitral valve surgery, aortic valve surgery and other surgery. RESULTS: 9995 patients underwent surgery. The discrimination of the logistic EuroSCORE was good with a ROC curve area of 0.79 for all cardiac surgery (range 0.71-0.79 in the subgroups). For all operations, the predicted mortality was 5.7% and observed mortality was 3.3%. The logistic EuroSCORE overpredicted observed mortality for all subgroups but by differing degrees (p = 0.02) CONCLUSIONS: The logistic EuroSCORE is a reasonable overall predictor for contemporary cardiac surgery but overestimates observed mortality. Its accuracy at predicting risk in different surgical subgroups varies. The logistic EuroSCORE should be recalibrated before it is used to gain reassurance about outcomes. Caution should be exercised when using it to compare hospitals or surgeons with a different operative case mix.

Plasma from rheumatoid patients taking low dose methotrexate enhances platelet aggregation.

Methotrexate (MTX) in low doses is commonly used to treat rheumatoid arthritis (RA). At least 36 deaths have been attributed to bone marrow cytotoxicity associated with low dose MTX. The goal was to determine if plasma from arthritis patients taking low dose MTX induces platelet aggregation in platelet rich plasma from healthy volunteers. Plasma from patients on MTX alone caused a 3-fold increase in aggregation vs plasma from controls (P<0.05). Plasma from patients not taking MTX or taking MTX with diclofenac caused aggregation to a lesser extent. Diclofenac, along with several others NSAIDs and cyclooxygenase inhibitors, depressed aggregation produced by arachidonic acid in platelet rich plasma from healthy volunteers. A precise mechanism for amplification of aggregation by MTX plasma and its relationship to MTX toxicity remains unknown. However, a serum factor may be produced by MTX that modulates the activity of cyclooxygenase, thereby influencing aggregation.