Losartan attenuates progression of osteoarthritis in the synovial temporomandibular and knee joints of a chondrodysplasia mouse model through inhibition of TGF-ß1 signaling pathway.

OBJECTIVE: Transforming growth factor beta 1 (TGF-ß1) is implicated in osteoarthritis (OA). The purpose of this study was to explore the ability of Losartan to inhibit the inflammatory signaling pathway of TGF-ß1 observed during osteoarthritic progression in the temporomandibular joint (TMJ) and knee joint using a genetic mouse model. METHODS: A murine OA model displaying the heterozygous chondrodysplasia gene (cho/+), a col11a1 mutation, was used to test this hypothesis. Following a 7-month treatment period with Losartan, the synovial joints were analyzed for histopathological improvement comparing two experimental groups. Tissues were fixed in paraformaldehyde, processed to paraffin section, and stained with Safranin O and Fast Green to visualize proteoglycans and collagen proteins in cartilage. Using the Modified Mankin scoring system, the degree of staining and OA progression were evaluated. RESULTS: Results show heterozygous animals receiving Losartan having diminished degeneration of TMJ condylar and knee joint articular cartilage. This was confirmed in the TMJ and knee by a statistically significant decrease in the Mankin histopathology score. Decreased expression of HtrA1, a key regulator to the TGF-ß1 signaling pathway, was demonstrated in vitro as well as in vivo, via Losartan inhibition. CONCLUSION: Using a genetic mouse model of OA, this study demonstrated the utility of Losartan to improve treatment of human OA in the TMJ and knee joint through inhibition of the TGF-ß1 signaling cascade. We further demonstrated inhibition of HtrA1, the lowering of Mankin scores to wild type control levels, and the limiting of OA progressive damage with treatment of Losartan.

A comparative study of Desarda's technique with Lichtenstein mesh repair in treatment of inguinal hernia: A prospective cohort study.

BACKGROUND: The Desarda repair technique of inguinal hernia repair introduced in 2001 is still not considered standard tissue based hernia repair technique. We compared the tissue based Desarda technique with standard Lichtenstein repair in treatment of primary inguinal hernia. METHODS: 187 cases were allocated into 2 groups. Desarda (D Group) had 92 and Lichtenstein (L Group) had 95 patients. Primary outcome factor was early (<1 year) recurrence of inguinal hernia. Secondary outcome factors included operative time measured from skin incision to skin closure. Postoperative pain scores was assessed on day 1, 3, 7, 30 and 90 using Visual analogue scale. Time taken to return to basic and home activities was calculated. Cord oedema, groin discomfort, seroma, fever, surgical site infections, chronic pain, etc. were evaluated as postoperative complications. RESULTS: After a 15-month mean follow up period 1 recurrence is noted in each arm (P = 1). Operative time was 73.89 ± 12.63 min in Lichtenstein and 72.60 ± 13.89 min in desarda repair (P = 0.508). Postoperative pain was significantly less in the first 7 post-operative days in Desarda group (P = 0.09) compared to Lichtenstein group. Time taken to return to basic and home activities was significantly less in Desarda group (P = 0.001). There was no statistical difference in rates of post-operative complications among the two arms of the study. CONCLUSION: The results of inguinal hernia treatment with the Desarda technique are similar to the results after standard Lichtenstein operations. Desarda technique does not use a mesh. Patients after Desarda's operative procedure get ambulatory sooner as compared to the standard Lichtenstein mesh repair. Less Postoperative pain, complications similar to standardised technique. Desarda technique has the potential to enlarge the number of tissue based methods available to treat groin hernias.

Dissipation pattern and risk assessment of flubendiamide on chili at different agro-climatic conditions in India.

Dissipation pattern and risk assessment of flubendiamide and its metabolite (desiodo flubendiamide) on chili were studied at four different agro-climatic locations of India at the standard and double dose at 60 and 120 g a.i. ha(-1) at 10 days interval. Quantification of residues was done on a high-performance liquid chromatograph (HPLC) with a photo diode array detector. The limit of quantification (LOQ) of this method was found to be 0.01 mg kg(-1) while limit of detection (LOD) being 0.003 mg kg(-1). Residues of flubendiamide were found to be below the determination limit in 15 days at both the dosages in all locations. Half-life of flubendiamide when applied at 60 and 120 g a.i. ha(-1) ranged from 0.85 to 1.80 and from 0.95 to 2.79 days, respectively. On the basis of data generated under the All India Network Project on Pesticide Residues, a preharvest interval (PHI) of 1 day has been recommended and the flubendiamide 480 SC has been registered for use on chili in India by the Central Insecticide Board and Registration Committee, Ministry of Agriculture, Government of India. The maximum residue limit (MRL) of flubendiamide on chili has been fixed by the Food Safety Standard Authority of India, Ministry of Health and Family Welfare, Government of India, as 0.02 µg g(-1) after its risk assessment.

Near-infrared excited ultraviolet emitting upconverting phosphors as an internal light source in dry chemistry test strips for glucose sensing.

Upconverting phosphors are inorganic crystals with interesting optical properties, including the ability to convert infrared radiation to emission at shorter wavelengths. In this paper we present the utilization of nanosized ß-NaYF4:Yb(3+),Tm(3+), synthesized in the presence of K(+), emitting at 365 nm under 980 nm excitation as an internal light source in glucose sensing dry chemistry test strips. The feasibility of the nanoparticles as an internal UV light source was compared to the use of an external broadband lamp. The results obtained from glucose measurements using UCNPs were in agreement with the traditional method based on measuring reflectance using an external UV light source. In addition the multiple emission peaks of UCNPs offered the possibility of using them as a control signal to account for various sources of error arising in the assay. The high penetration depth of the NIR-excitation made it also possible to excite the UCNPs through a layer of whole blood, giving more freedom to the design of the optical setup.

Residual behavior and risk assessment of flubendiamide on tomato at different agro-climatic conditions in India.

Supervised field trials were conducted at four different agro-climatic zones in India to evaluate the dissipation pattern and risk assessment of flubendiamide on tomato. Flubendiamide 480 SC was sprayed on tomato at 48 and 96 g active ingredient (a.i.) ha(-1). Samples of tomato fruits were drawn at 0, 1, 3, 5, 7, 10, 15, and 20 days after treatment. Quantification of residues was done on a high-performance liquid chromatography (HPLC) device with a photo diode array detector. The limit of quantification (LOQ) of this method was found to be 0.01 mg kg(-1) while limit of detection (LOD) being 0.003 mg kg(-1). Residues of flubendiamide were found below the determination limit of 0.01 mg kg(-1) in 20 days at both the dosages in all the locations. The half-life of flubendiamide at an application rate of 48 g a.i. ha(-1) varied from 0.33 to 3.28 days and at 48-g a.i. ranged from 1.21 to 3.00 days. On the basis of data generated under the All India Network Project on Pesticide Residues, a preharvest interval (PHI) of 1 day has been recommended, and the flubendiamide 480 SC has been registered for its use on tomato by the Central Insecticide Board and Registration Committee, Ministry of Agriculture, Government of India. The maximum residue limit (MRL) of flubendiamide on tomato has been fixed by the Ministry of Health and Family Welfare, Government of India under Food Safety Standard Authority of India, as 0.07 µg g(-1) after its risk assessment.

Safety evaluation of flubendiamide and its metabolites on cabbage and persistence in soil in different agroclimatic zones of India.

Supervised field trials following good agricultural practices were conducted at the research farms of four agricultural universities located at four different agroclimatic zones of India to evaluate the persistence and dissipation of flubendiamide and its metabolite, des-iodo flubendiamide, on cabbage. Two spray applications of flubendiamide 480 SC of standard and double dose at the rate of 24 and 48 g a.i. ha(-1) were given to the crop at a 15-day interval, and the residues of flubendiamide 2 h after spray were found in the range of 0.107-0.33 and 0.20-0.49 mg kg(-1) at respective doses. Residue of des-iodo flubendiamide was not detected in any cabbage sample during study period. No residues were found in the soil samples collected from all treated fields after 15 days of application. On the basis of data generated under All India Network Project on Pesticide Residues, a preharvest interval (PHI) of 10 days has been recommended, and the flubendiamide 480 SC has been registered for its use on cabbage by Central Insecticide Board and Registration Committee, Ministry of Agriculture, Government of India. The maximum residue limit (MRL) of flubendiamide on cabbage has been fixed by the Ministry of Health and Family Welfare, Government of India, under Food Safety Standard Authority of India as 0.05 µg/g after its risk assessment.

Assessment of flubendiamide residues in pigeon pea in different agro-climatic zones of India.

Supervised field trials were conducted at the research farms of four agricultural universities located at different agro-climatic zones of India to find out the harvest time residues of flubendiamide and its des-iodo metabolite on pigeon pea (Cajanus cajan) during the year 2006-2007. Two spray applications of flubendiamide 20 WDG at 50 g (T(1)) and 100 g (T(2)) a.i./ha were given to the crop at 15-days interval. The foliage samples at different time intervals were drawn at only one location, however, the harvest time samples of pigeon pea grain, shell, and straw were drawn at all the four locations. The residues were estimated by HPLC coupled with UV-VIS variable detector. No residues of flubendiamide and its des-iodo metabolite were found at harvest of the crop at or above the LOQ level of 0.05 µg/g. On the basis of the data generated, a pre-harvest interval (PHI) of 28 days has been recommended and the flubendiamide 20 WDG has been registered for use on pigeon pea by Central Insecticide Board and Registration Committee, Ministry of Agriculture, Government of India and the MRL has been fixed by Ministry of Health and Family Welfare, Government of India under Prevention of Food and Adulteration as 0.05 µg/g on pigeon pea grains.

Clinical response of newly diagnosed HIV seropositive & seronegative pulmonary tuberculosis patients with the RNTCP Short Course regimen in Pune, India.

BACKGROUND & OBJECTIVES: In the Revised National Tuberculosis Control Programme (RNTCP) in India prior to 2005, TB patients were offered standard DOTS regimens without knowledge of HIV status. Consequently such patients did not receive anti-retroviral therapy (ART) and the influence of concomitant HIV infection on the outcome of anti-tuberculosis treatment remained undetermined. This study was conducted to determine the results of treatment of HIV seropositive pulmonary tuberculosis patients with the RNTCP (DOTS) regimens under the programme in comparison with HIV negative patients prior to the availability of free ART in India. METHODS: Between September 2000 and July 2006, 283 newly diagnosed pulmonary TB patients were enrolled in the study at the TB Outpatient Department at the Talera Hospital in the Pimpri Chinchwad Municipal Corporation area at Pune (Maharashtra): they included 121 HIV seropositive and 162 HIV seronegative patients. They were treated for tuberculosis as per the RNTCP in India. This study was predominantly conducted in the period before the free ART become available in Pune. RESULTS: At the end of 6 months of anti-TB treatment, 62 per cent of the HIV seropositive and 92 per cent of the HIV negative smear negative patients completed treatment and were asymptomatic; among smear positive patients, 70 per cent of the HIV-seropositive and 81 per cent of HIV seronegative pulmonary TB patients were cured. Considering the results in the smear positive and smear negative cases together, treatment success rates were substantially lower in HIV positive patients than in HIV negative patients, (66% vs 85%). Further, 29 per cent of HIV seropositive and 1 per cent of the HIV seronegative patients expired during treatment. During the entire period of 30 months, including 6 months of treatment and 24 months of follow up, 61 (51%) of 121 HIV positive patients died; correspondingly there were 6 (4%) deaths among HIV negative patients. INTERPRETATION & CONCLUSIONS: The HIV seropositive TB patients responded poorly to the RNTCP regimens as evidenced by lower success rates with chemotherapy and high mortality rates during treatment and follow up. There is a need to streamline the identification and management of HIV associated TB patients in the programme with provision of ART to achieve high cure rates for TB, reducing mortality rates and ensuring a better quality of life.

Opportunistic parasitic infections in HIV/AIDS patients presenting with diarrhoea by the level of immunesuppression.

BACKGROUND & OBJECTIVE: Enteric parasites are major cause of diarrhoea in HIV infected individuals. The present study was undertaken to detect enteric parasites in HIV infected patients with diarrhoea at different levels of immunity. METHODS: The study was carried out at National AIDS Research Institute, Pune, India, between March 2002 and March 2007 among consecutively enrolled 137 HIV infected patients presenting with diarrhoea. Stool samples were collected and examined for enteric parasites by microscopy and special staining methods. CD4 cell counts were estimated using the FACS count system. RESULTS: Intestinal parasitic pathogens were detected in 35 per cent patients, and the major pathogens included Cryptosporidium parvum (12%) the most common followed by Isospora belli (8%), Entamoeba histolytica/Enatmoeba dispar (7%), Microsporidia (1%) and Cyclospora (0.7%). In HIV infected patients with CD4 count < 200 cells/microl, C. parvum was the most commonly observed (54%) pathogen. Proportion of opportunistic pathogens in patients with CD4 count <200 cells/microl was significantly higher as compared with other two groups of patients with CD4 count >200-499 and >or= 500 cells/microl (P=0.001, P=0.016) respectively. INTERPRETATION & CONCLUSION: Parasitic infections were detected in 35 per cent HIV infected patients and low CD4 count was significantly associated with opportunistic infection. Detection of aetiologic pathogens might help clinicians decide appropriate management strategies.

Molecular mechanisms behind the dose-dependent differential activation of MAPK pathways induced by transforming growth factor-beta1 in hematopoietic cells.

Transforming growth factor-beta (TGF-beta) controls a wide range of cellular responses, including cell proliferation, lineage determination, differentiation, and apoptosis, and figures prominently in animal development. It is considered as a pleiotropic factor because it can exert a positive or negative effect on various cellular processes depending on developmental stage of the target cell, its microenvironment, and also its biochemical make up. It has been shown to have a strong inhibitory effect on hematopoietic stem cell proliferation and differentiation. We have earlier shown that TGF-beta1 exerts a bidirectional effect on hematopoietic cell proliferation as a function of its concentration. Although it acted as an inhibitor at high concentrations, at low concentrations it stimulated the stem/progenitor cells. We also provided evidence that the differential activation of mitogen-activated protein kinase pathways was responsible for the observed bidirectional effect. In the present study, we examined the molecular mechanism behind this phenomenon. We observed that the high inhibitory concentrations of TGF-beta1 induced a strong phosphorylation of SMAD 3 and also activated stress kinase-related transcription factors, namely c-Jun and ATF-2. On the other hand, low stimulatory concentrations acted in a SMAD 3-independent pathway and activated STAT proteins. Our results clearly show that differential activation of signal transduction pathways by TGF-beta1 as a function of its concentration underlies its bidirectional effect on hematopoietic cells.

Differential activation of MAPK signaling pathways by TGF-beta1 forms the molecular mechanism behind its dose-dependent bidirectional effects on hematopoiesis.

We have earlier reported that transforming growth factor-beta1 (TGF-beta1), a well-known inhibitor of hematopoiesis, stimulated colony formation from adult human bone marrow mononuclear cells (BM MNC) when used at low concentrations. We examined the possible molecular mechanism behind this bidirectional effect using CD34+ cells isolated from human BM for clonal assays and the KG1a cell line as a model system for analysis of proteins for signaling pathways by immunoblotting. We found that TGF-beta1 at low doses (picogram levels) stimulated the colony formation from CD34+ cells, indicating that these progenitors form the direct target of stimulatory action of TGF-beta1. CD34+ cells were found to be more sensitive to the TGF-beta1 concentration than the total MNC. We used the KG1a cell line as a model system for identification of mitogen-activated protein kinase (MAPK) and AKT signaling pathways involved in the process. Low doses strongly induced p44/42 MAPK phosphorylation, whereas high doses induced p38 activation. Use of specific p44/42 MAPK inhibitor PD 98059 in the colony assay abrogated the stimulatory effect of low TGF-beta1. On the other hand, use of p38 MAPK inhibitor SB 203580 along with low TGF-beta1 concentrations had a synergistic effect on stimulation of colony formation. Treatment of BM MNC with Anisomycin, which activates stress kinases, resulted in a dose-dependent inhibition of colony formation. This inhibition could not be rescued by stimulatory doses of TGF-beta1. Phosphorylation of AKT was found to occur in a dose-dependent way but declined slightly at the highest concentration used (10 ng/ml). Inhibition of the AKT pathway by LY 294002 strongly suppressed colony formation. These data indicate clearly that sustained activation of p44/42 MAPK perhaps forms the stimulatory signal induced by low TGF-beta1, whereas activation of p38 forms the inhibitory pathway.

Colony-stimulating activity of fetal liver cells: synergistic role of stem cell factor in bone marrow recovery from aplastic anemia.

Previously, we and others have shown that fetal liver infusion (FLI) leads to autologous hematopoietic improvement in 40-54% of patients with aplastic anemia. However, whether this recovery was spontaneous or the effect of the infused liver cells was not clear. To dissect the role of FLI in autologous hematopoietic recovery, the colony-supporting potential of fetal liver-conditioned medium (FLCM) was evaluated in bone marrow (BM) cells of normal adult and aplastic anemia patients. In both cases, each sample of FLCM supported the growth of colony-forming cells in semi solid culture medium. The FLCM was assayed for the presence of four principal colony-stimulating cytokines, namely stem cell factor (SCF), granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-3 (IL-3), and erythropoietin (Epo). While GM-CSF, IL-3, and Epo were present in insignificant amounts or were altogether absent, 50-635 pg/ml of SCF was found in 8 of the 13 FLCM samples tested. Preliminary results of bioneutralization assay indicated the possible role of SCF, secreted by the FL cells, in colony-supporting activity of aplastic anemia and normal BM cells. Overall, our in vitro study implicates the paracrine role of infused FL cells in regenerating autologous hematopoiesis in aplastic anemia patients.

Cryopreservation of human hematopoietic cells with membrane stabilizers and bioantioxidants as additives in the conventional freezing medium.

Cord blood (CB) and fetal liver (FL) cells are two alternative sources of human hematopoietic stem cells. Optimization of cryopreservation protocols is an important aspect in the banking of these tissues. Out of the multiple factors responsible for cryodamage of cells, membrane leakage and oxygen free-radical generation have been shown to contribute substantially toward the process. We have studied the effect of certain additives, like membrane stabilizers and bioantioxidants, to the conventional freezing medium on viability, nucleated cell recovery, and clonogenic potential of frozen cells. Our results show that trehalose, a membrane stabilizer, when used in combination with 10% dimethyl sulfoxide (DMSO) affords better cryoprotection as evidenced by significantly increased colony formation as compared to 10% DMSO alone. The cryoprotection afforded by trehalose persists at least for 1.5 years and there is no bias toward protection of a particular lineage. We also found that this increased cryoprotective effect of trehalose is seen both at -196 degrees C and -80 degrees C storage temperatures. Addition of taurine, an amino acid, another membrane stabilizer, and a natural cryoprotectant to the traditional freezing medium also results in beneficial effect. Of the three bioantioxidants tested, i.e., ascorbic acid, alpha-tocopherol acetate, and catalase, catalase shows maximum cryoprotective effect both at -196 degrees C and at -80 degrees C. Because the mode of cryoprotective action of catalase and trehalose are totally different, we tried a combination of these two compounds along with 10% DMSO. At -196 degrees C the protection afforded by the combination was significantly better than that afforded by individual components. At -80 degrees C, however, the combination did not give any added protection as compared to the individual single additives, although it was significantly better than 10% DMSO alone. These results indicate that the addition of membrane stabilizers and antioxidants to the conventional freezing medium may help to improve post thaw recovery of hematopoietic cells.

Stimulation of adult human bone marrow by factors secreted by fetal liver hematopoietic cells: in vitro evaluation using semisolid clonal assay system.

Fetal liver infusion (FLI) therapy has been used in various disorders, such as aplastic anemia, leukemia, metabolic disorders, etc., and has been shown to result in stimulation of autologous hematopoiesis in many cases. The aim of the present study was to elucidate the mechanism of stimulation of adult hematopoiesis by fetal liver hematopoietic cells (FLHC) and to identify the factors involved in the process using a clonal assay system in vitro. The effect of FLHC on the clonal growth of bone marrow cells was studied using a co-culture system consisting of mitomycin C-treated FLHC with 2 x 10(5) bone marrow (BM) mononuclear cells. It was observed that FLHC induced a two- to four-fold increase in the BM colony formation. A further increase in the number of FLHC did not, however, result in an equivalent fold increase in the colony formation, indicating that the number of cells in the BM population responsive to FLHC was perhaps the limiting factor. When the effect of fetal liver cell conditioned medium (FLCM) was examined in a similar fashion, it was observed that the FLCM showed a 1.5- to 4-fold increase in the colony formation when used at 1%-5% along with limiting amounts of growth factors. Higher concentrations of conditioned medium resulted in inhibitory responses. One of the principal factors responsible for the stimulatory activity of FLCM was shown to be transforming growth factor-beta1 (TGF-beta1), by a variety of experiments such as its quantitation in FLCM by enzyme-linked immunosorbent assay, antibody neutralization, and reconstruction experiments using purified TGF-beta1 and normal medium. In these reconstitution experiments, TGF-beta1 stimulated the colony formation when it was applied at 1-50 pg/ml, but at higher concentration it induced an inhibitory effect, mimicking the behavior earlier seen with FLCM. Our data strongly suggest that one of the mechanisms in stimulation of a recipient's hematopoiesis could be mediated by the action of TGF-beta1 secreted by infused FLHC and could provide a rational framework on which FLI therapy can be further evaluated.

Bacteriological study of meningococcal meningitis.

One hundred and thirty samples of cerebro spinal fluid were collected from patients admitted with suspected signs and symptoms of meningococcal meningitis (M. meningitis) during the period from January 1986 to April 1989 and were processed for gram's staining, cultivation and latex agglutination tests for detection of polysaccharide antigen in the CSF. Totally 41.5% of turbid and hazy spinal fluid were positive for N. meningitidis by smear examination. Only 24.6% were positive by culture but 61.5% of sample were positive by latex agglutination tests. All the strains were sensitive to all antibiotics except one strain which was resistant to penicillin but it was sensitive to rifampicin.