Seroprevalence, associated risk factors and clinico-pathological studies of buffalopox disease in various regions of Punjab province, Pakistan.

Buffalopox (BPX) is a highly contagious disease that causes high morbidity and production losses in buffaloes. During this study, seroprevalence, effect of various associated risk factors, and pathological studies of BPX were recorded in the Punjab province. A total of 97 blood samples and 63 scabs were collected from clinically pox suspected buffaloes. Serum was harvested to perform single radial hemolysis to assess the seroprevalence, and scabs were subjected to PCR for BPX virus confirmation. Results revealed that, animal demographics and environmental associated factors showed significant effect (p⟨0.05,1⟨R2⟩0) on BPX occurrence. The overall BPX seroprevalence was recorded 4.18% in the Punjab province. The BPX was recorded 5.48% in Nili Ravi breed during winter (7.42%), aged 5-7 years (7.46%) under loose housing (5.51%) in the Faisalabad region (8.03%). Further, BPX was 5.37% in pregnant, 6.86% pregnant milking buffaloes during the 3rd lactation period (7.28%) in dairy herds (5.20%). The BPX was 5.22% in non-vaccinated buffaloes where multiple animals were reared together (4.99%) in the herds having 21-30 total number of animals. A total of 49 scab samples were found positive for the BPX virus via PCR with C18L gene amplification. Grossly, inflammatory lesions with pits in the center and wart-like nodules were seen on teats and udder of buffaloes. Increased leukocytes, especially neutrophils and lymphocytes, were seen in the blood of the infected animals. These results provide a broader window to understand the effect of associated risk factors, strengthen the diagnostic aid, and to contain the current spread of BPX in Pakistan to safeguard large ruminant-based livelihood.

Essential role of PH domain and leucine-rich repeat protein phosphatase 2 in Nrf2 suppression via modulation of Akt/GSK3ß/Fyn kinase axis during oxidative hepatocellular toxicity.

Instances of sustained oxidative activity have been shown to involve dysregulation of Nrf2-mediated transcriptional induction; however, mechanisms warranting Nrf2-repression remain unclear. In this study, using primary rat hepatocytes, we have attempted to identify factors that may negatively influence Nrf2 survival pathway. Though studies indicate a conspicuous association between Akt and Nrf2, a confirmatory link between the two is unaddressed. On inhibiting PI3K/Akt pathway, we observed compromised activities of antioxidant and detoxification enzymes culminating in oxidative cytotoxicity. This was accompanied by reduced nuclear retention of Nrf2 and its ARE binding affinity, increased Nrf2 ubiquitination and concurrent decline in its downstream targets. Moreover, Akt inhibition enhanced nuclear translocation as well as phosphorylation of Fyn kinase, an enzyme linked to Nrf2 degradation, by relieving GSK3ß from phosphorylation-mediated repression. The involvement of Akt and Fyn kinase in influencing Nrf2 signaling was further confirmed in oxidatively stressed hepatocytes by using tert-butyl hydroperoxide (tBHP). tBHP-induced decrease in Nrf2 levels was associated with enhanced Fyn kinase phosphorylation, Fyn kinase nuclear translocation and decreased levels of phosphorylated GSK3ß(Ser9) in a time-dependent manner. Interestingly, tBHP induced site-specific deactivation of Akt as only Akt(Ser473) phosphorylation was observed to be affected. Further, protein expression as well as nuclear localization of PHLPP2, a phosphatase specific for Akt(Ser473), was found to be significantly enhanced in tBHP-stressed hepatocytes. Silencing of PHLPP2 not only resulted in considerable restoration of Nrf2 signaling, enhanced Nrf2-ARE binding and reduced Nrf2 ubiquitination but also significantly suppressed tBHP-induced ROS generation and alterations in mitochondrial permeability. We infer that cellular PHLPP2 levels may aggravate oxidative toxicity by suppressing Nrf2/ARE transcriptional regulation via Akt(Se473)/GSK3ß/Fyn kinase axis. The study indicates that PHLPP2 could serve as a new target for developing strategies to manage pathological conditions exacerbated due to oxidative stress.

Incidental MRI lesions suggestive of multiple sclerosis in asymptomatic patients in Karachi, Pakistan.

The objective of this study was to identify asymptomatic patients with brain MRI lesions suggestive of multiple sclerosis (MS) in a low-prevalence area of Pakistan. Brain MRIs for 864 patients were reviewed at the Aga Khan University (Karachi, Pakistan) during an 8-month period of 2006 and 2007 to identify patients with lesions suggestive of MS. The lesions were characterised based on modified Barkhof criteria. Six (two females) (0.7%) of 864 patients fulfilled brain MRI criteria suggestive of MS. The mean number of MRI lesions (total lesions on T2) were 9 (range 5-14). Although Pakistan is considered a low-prevalence area for MS, 0.7% of brain MRI scans in patients without clinical MS symptoms showed lesions fulfilling brain MRI criteria of MS.

Safety and efficacy of chimeric yellow Fever-dengue virus tetravalent vaccine formulations in nonhuman primates.

To construct chimeric YF/DEN viruses (ChimeriVax-DEN), the premembrane (prM) and envelope (E) genes of yellow fever (YF) 17D virus were replaced with those of each wild-type (WT) dengue (DEN) virus representing serotypes 1 to 4. ChimeriVax-DEN1-4 vaccine viruses were prepared by electroporation of Vero cells with RNA transcripts prepared from viral cDNA (F. Guirakhoo, J. Arroyo, K. V. Pugachev, C. Miller, Z.-X. Zhang, R. Weltzin, K. Georgakopoulos, J. Catalan, S. Ocran, K. Soike, M. Ratteree, and T. P. Monath, J. Virol. 75:7290-7304, 2001; F. Guirakhoo, K. Pugachev, J. Arroyo, C. Miller, Z.-X. Zhang, R. Weltzin, K. Georgakopoulos, J. Catalan, S. Ocran, K. Draper, and T. P. Monath, Virology 298:146-159, 2002). Progeny viruses were subjected to three rounds of plaque purifications to produce the Pre-Master Seed viruses at passage 7 (P7). Three further passages were carried out using U.S. current Good Manufacturing Practices (cGMP) to produce the Vaccine Lot (P10) viruses. Preclinical studies demonstrated that the vaccine candidates are replication competent and genetically stable and do not become more neurovirulent upon 20 passages in Vero cells. The safety of a tetravalent vaccine was determined and compared to that of YF-VAX in a formal monkey neurovirulence test. Brain lesions produced by the tetravalent ChimeriVax-DEN vaccine were significantly less severe than those observed with YF-VAX. The immunogenicity and protective efficacy of four different tetravalent formulations were evaluated in cynomolgus monkeys following a single-dose subcutaneous vaccination followed by a virulent virus challenge 6 months later. All monkeys developed low levels of viremia postimmunization, and all the monkeys that had received equal concentrations of either a high-dose (5,5,5,5) or a low-dose (3,3,3,3) formulation seroconverted against all four DEN virus serotypes. Twenty-two (92%) of 24 monkeys were protected as determined by lack of viremia post-challenge. This report is the first to demonstrate the safety of a recombinant DEN virus tetravalent vaccine in a formal neurovirulence test, as well as its protective efficacy in a monkey challenge model.

Beneficial effects of fish liver preparations of sea bass (Lates calcarifer) versus gemfibrozil in high fat diet-induced lipid-intolerant rats.

Abnormal lipid metabolism is a main cause of dyslipidemia, which is a major risk factor for coronary heart disease and obesity and is even linked to diabetic-dyslipidemic complications. Fifteen days of high-fat feeding in Charles Foster rats resulted in a significant increase in baseline serum lipid levels accompanied by pronounced dyslipidemia. Treatment with fish liver preparations (FLPs) from sea bass and the standard drug gemfibrozil produced a lowering of serum lipids and glucose levels, along with a fall in very-low-density and low-density lipoprotein and an increase in high-density lipoprotein levels. Simultaneously, reactivation of plasma postheparin lipolytic activity (PHLA) and lecithin:cholesterol acyltransferase (LCAT) activity was also observed. A positive correlation was observed between low-density lipoprotein activity and fecal bile acid excretion, which was enhanced on treatment with FLPs and gemfibrozil, indicating the catabolic process for normal lipids and cholesterol homeostasis. These data suggest that FLPs and gemfibrozil not only lower lipid intolerance but also reduce diabetic-dyslipidemic complications by activating peroxisome proliferator-activated receptors (PPAR).

Antidyslipidemic action of fenofibrate in dyslipidemic-diabetic hamster model.

Fenofibrate is the ligand for PPARalpha subtype that mediates the action of its agonists' in lipid metabolism. How fibrate exerts hypolipidemic effect? The mechanism is studied in a newly developed high-fat fructose enriched diet induced dyslipidemia-diabetic hamster model. Fenofibrate lowered the basal plasma lipids like TC, TG, PL, FFA, glycerol, VLDL, and LDL, but HDL was increased. The activity of lipoprotein lipase in liver, adipose tissue, and small intestine was upregulated. However, that of triglyceride lipase was downregulated in liver. It has also improved the insulin secretion and plasma glucose lowering, caused by impairment in insulin secretion due to high-fat load. The drug was found effective in reducing body weight and diet due to rise in leptin level. Fenofibrate also enhanced the fecal excretion of total lipids, cholic acid, and deoxycholic acid probably by the activation of 7alpha cholesterol hydroxylase enzyme. Thus, causing broad-spectrum lipid lowering along with inhibition of hepatic lipid biosynthesis and maintaining lipid-glucose homeostasis.

Lipid lowering activity of Phyllanthus niruri in hyperlipemic rats.

The lipid lowering activity (LLA) of Phyllanthus niruri has been studied in triton and cholesterol fed hyperlipemic rats. Serum lipids were lowered by P. niruri extract orally fed (250 mg/kg b.w.) to the triton WR-1339 induced hyperlipemic rats. Chronic feeding of this drugs (100 mg/kg b.w.) in animals simultaneously fed with cholesterol (25 mg/kg b.w.) for 30 days caused lowering in the lipids and apoprotein levels of VLDL and LDL in experimental animals. The LLA of this drug is mediated through inhibition of hepatic cholesterol biosynthesis, increased faecal bile acids excretion and enhanced plasma lecithin: cholesterol acyltransferase activity.

Formulations of single or multiple H. pylori antigens with DC Chol adjuvant induce protection by the systemic route in mice. Optimal prophylactic combinations are different from therapeutic ones.

The ability to induce a protective response against Helicobacter pylori infection has been investigated by systemic immunization of mice with urease formulated with the cationic lipid DC Chol. This compound acts both as a formulating agent and as an adjuvant and induces a balanced Th1/Th2 response shown to be more effective for protection in our previous studies. Urease-DC Chol induced a significant protection in prophylaxis but not in therapeutic immunization. The protection level was between 1.5 and 2 log reduction of bacterial density measured by quantitative culture compared to unimmunized-infected mice. In parallel, the protective efficacy of other H. pylori antigens formulated in a similar way and administered with DC Chol was tested. These antigens were tested alone or in combination in prophylactic and therapeutic regimens. Some combinations of antigens induced a better prophylactic or therapeutic activity than urease alone (0.5-1.5 log further reduction in prophylaxis and therapy respectively, P<0.05). The combinations that induced the best protection were different in prophylaxis and therapy. In conclusion, DC Chol provides a convenient and efficient method to formulate different antigens even when they are present in non-compatible buffers initially. Moreover, the results obtained in protection against H. pylori with such formulations should lead the way to future clinical trials.

Helicobacter pylori: isolation from gastric biopsy specimens.

Forty-two gastric biopsies, in two transport media, were homogenized and cultured on three media under micro-aerophilic conditions. Brain-heart infusion agar with a commercial antibiotic supplement (giving 10 mg vancomycin, 5 mg trimethoprim and 2500 i.u. polymixin per litre) yielded the best results. Ordinary chocolate (heated) human blood agar could be used in laboratories with limited resources. Growth was obtained in 4-6 days at 37 degrees C. All isolates were sensitive to metronidazole. The resistance to nalidixic acid and rapid urease production of Helicobacter pylori could be used to differentiate this species from Campylobacter spp. Indicator medium (brain-heart infusion broth with 7% human blood and 40 mg 2,3,5-triphenyletetrazolium chloride agar/litre) also proved useful in identification.

HLA frequencies in Pakistani population.

HLA frequencies on 1231 subjects from within the country, using methods employed by National Institute of Health, USA is reported. Pakistani population appears to be a mixture of an indigenous population with others particularly Orientals and Negroids. Relationship with Caucasians is not convincing.

Trypanosoma cruzi: differential expression and distribution of an 85-kDa polypeptide epitope by in vitro developmental stages.

The expression by Trypanosoma cruzi developmental stages of an 85-kDa polypeptide epitope defined by the 155D3 monoclonal antibody (mAb) has been investigated. Immunoprecipitation revealed the presence of an 85-kDa antigen in the NP-40 soluble extract of parasites freshly released from infected fibroblasts; this antigen was not found in epimastigote and Leishmania infantum promastigote. Indirect immunofluorescence revealed that the mAb 155D3 failed to react with trypomastigotes, whereas extracellular amastigotes were heavily stained. Positive organisms displayed either surface or polar fluorescence. Since the same mAb immunoprecipitated the 85-kDa antigen in both radioactive iodine- and methionine-labeled trypomastigote detergent soluble extracts, the reactive epitope is likely to be hidden in a cryptic site in trypomastigotes. An alternative explanation for the negative immunofluorescence on trypomastigotes and the positive immunoprecipitation is the presence, in the extracts, of a small population of parasites already expressing the 155D3 epitope. Immunoelectron microscopy revealed that the target epitope is heterogenously distributed among the populations of differentiating parasites. Two types of immunogold labeling were observed: (a) mAb revealed a high amount of reactive material associated with the periphery of the parasites and (b) a label was observed on the inner surface of peripheral vacuoles that might correspond to cross sections of inflated flagellar pockets and in association with vesicles which were released by the parasites. The surface expression of the epitope recognized by the 155D3 mAb was followed by fluorescence-activated cell-sorting analysis. The results showed that the epitope is increasingly accessible during trypomastigote differentiation in vitro. Taken together, these results suggest that the epitope reacting with the 155D3 mAb is heavily expressed on extracellular amastigotes after the transformation process and, thus, appears to be developmentally regulated.

Characterization of major surface and excretory-secretory immunogens of Trypanosoma cruzi trypomastigotes and identification of potential protective antigen.

The surface antigens of Trypanosoma cruzi trypomastigotes were identified by immunoprecipitation and were compared with metabolically labelled excretory-secretory products (ES) released by the parasites in vitro. A series of major immunogenic components in the ES antigens were revealed (160 kDa, 130 kDa and 80-110 kDa). The trypomastigote surface also bears the 130 kDa band and the 80-110 kDa complex. Competition experiments demonstrated the common antigenic structure of the ES and the surface antigens. Two-dimensional analysis of ES antigens immunoprecipitated by human Chagasic serum revealed several spots in the 80-110 kDa region with a wide range of isoelectric points (PI between 5.4 and 6.7). This reflects a charge heterogeneity of these polypeptides. The trypomastigote 85 kDa polypeptide was also identified in the ES antigens by using a monoclonal antibody against this antigen. Two-dimensional analysis of the 85 kDa proteins shed from the surface of trypomastigotes and immunoprecipitated by the monoclonal antibody 155D3 showed 2 major spots: a major part of the 85 kDa polypeptide was found at pH 6.5-6.6, whereas a substantial amount of the antigen was found at pH 5.7. An additional component with molecular weight of approximately 58 kDa and isoelectric points of 6.5 and 6.6, was also visualized. Detection of the 85 kDa polypeptide circulating in serum from patients with acute and chronic Chagas' disease was achieved using an enzyme-linked immunosorbent assay. In addition, the data obtained showed that a polyclonal antibody to the 85 kDa polypeptide could be used to passively induce a partial protection of Fischer rats against acute lethal infection. Thus, the antigens recognized by polyclonal antibody appear to play a role in the development of protective immunity against T. cruzi.

IgE receptor on human eosinophils (FcERII). Comparison with B cell CD23 and association with an adhesion molecule.

IgE FcR (FcERII) on human eosinophils was characterized and compared with FcERII present on B cells (CD23). Two mAb, BB10 (anti-eosinophil FcERII) and 135 (anti-CD23), bound to the major component of FcERII at 45,000 to 50,000 Mr, both on purified hypodense eosinophils and on a B cell line (WIL-2WT). The specific ligand, human myeloma IgE, was able to bind to the molecules immunoprecipitated by BB10. A cross-reactivity between BB10 and a mAb anti-Leishmania gp63, which is a "fibronectin (Fn)-like" molecule, containing the L-arginine-L-glycyl-L-aspartyl (RGD) cell attachment domain indicated the presence of such a sequence in the common structure present on eosinophil and B cell FcERII. The synthetic tetrapeptide RGDS as well as its inverted sequence (SDGR) reduced the binding of BB10 and anti-Fn mAb to eosinophils and B cells. Flow microfluorometry analysis revealed a variable binding of BB10 and anti-Fn mAb to eosinophils purified from different patients, results compatible with recent findings on the inducibility of FcERIIb. The significant inhibition of IgE-dependent cytotoxicity against parasite targets by preincubation of eosinophils with BB10, anti-Fn and anti-CD23 mAb, with anti-RGDS polyclonal antibodies or with the SDGR peptide suggested the requirement of this cell adhesion sequence for the function of low affinity FcERII. The presence of such a sequence in the C-terminal domain of B cell FcERII raised the possibility of its role in B cell adhesion or B cell growth.

Anticardiolipin antibodies in Lyme disease.

Sera from 28 patients with Lyme disease were tested for the presence of anticardiolipin antibodies (ACLA). Seven serum samples had elevated levels of IgM ACLA, and 4 had elevated levels of IgG ACLA. Higher IgM ACLA positivity tended to be associated with neurologic disease, and IgM ACLA levels correlated with the specific IgM response to the infecting spirochete (P less than 0.01). Absorption experiments indicated that ACLA and antispirochete antibodies are largely separate populations. Thus, ACLA may occur in patients with Lyme disease, particularly in those with neurologic abnormalities, and the production of these antibodies seems to be linked to the specific IgM response.

The major surface protein of Leishmania promastigotes is a fibronectin-like molecule.

The major surface glycoprotein of Leishmania chagasi promastigotes showed crossreactivity with fibronectin (Fn), a large glycoprotein that is a major constituent of the extracellular matrix of most mononuclear cells. Polyclonal and monoclonal antibodies against Fn precipitated two molecules of 63-58 kDa from the lysates of both 125I and [35S]methionine-labeled promastigotes. In addition, a monoclonal antibody against a 15-kDa fragment of Fn containing the Arg-Gly-Asp-Ser (RGDS) sequence and several polyclonal monospecific mouse antibodies against a synthetic RGDS peptide also recognized the above two molecules. The attachment of Leishmania promastigotes to mouse peritoneal macrophages in vitro was partially inhibited when promastigotes were treated with F(ab')2 fragment of an anti-Fn IgG. Identical results were obtained by saturating the Fn receptors on macrophages using different peptides containing the RGDS sequence. Moreover, antigen preparations rich in glycoprotein 63 could efficiently promote the attachment and spreading of 3T3 mouse fibroblasts to surfaces coated with the antigen. These results clearly suggest that the gp63 of L. chagasi promastigotes is an Fn-like molecule that shares certain biological and molecular characteristics with Fn.

Leishmania chagasi: in vitro differentiation of promastigotes monitored by flow cytometry.

A sequential development from a less infective to an infective stage of Leishmania promastigotes growing in culture has been previously reported. The aim of this work was to investigate whether freeze-fracture electron microscopy and flow cytometry would be able to provide some reliable morphological markers of in vitro differentiation of Leishmania chagasi promastigotes. The flow cytometry technique discriminates between the L. chagasi promastigotes from the different stages of their in vitro differentiation. The "forward scatter" intensity of the parasite, very high 15 hr after seeding when the parasites were very condensed and with a high DNA content per particle, strongly decreased during the culture course. Parallel experiments have shown a striking correlation between forward scatter intensity, growth curves, and infectivity of promastigote populations. By contrast, freeze-fracture techniques showed that in either less infective or infective promastigote plasma membranes, the intramembrane particles density in protoplasmic fracture faces (about 2800/micron 2) and in exoplasmic fracture faces (about 1000/micron 2) was independent of the time of cultivation. The amount of filipin lesions, which reflects the cholesterol content within the plasma membrane, was also constant throughout the culture course. Both data suggest that the architecture of the plasma membrane is an intrinsic characteristic of the promastigote stage. This study shows that whereas freeze-fracture electron microscopy does not provide markers for the differentiation of Leishmania promastigotes, flow cytometry may on the other hand be of value as a screening test for promastigote populations allowing the characterization of their developmental stages in in vitro cultures.

Subspecies-specific surface antigens of promastigotes of the Leishmania donovani complex.

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns of proteins and externally exposed labeled surface constituents were analyzed in promastigotes of three etiological agents of kala azar (Leishmania donovani, HS70 strain from India; L. chagasi, Imperatriz strain from Brazil; L. infantum, ITMPA K263 strain from Morocco and MO strain from France). Coomassie blue-stained gels showed similar protein patterns for L. donovani and L. chagasi and a more distinct one for L. infantum. Surface radioiodination with two different methods, lactoperoxidase and IODO-GEN, gave identical autoradiographic patterns for each parasite. Four major labeled proteins with apparent Mr values of 65,000, 60,000, 50,000, and 26,000 were detected in both L. chagasi and L. donovani. However, the radioiodinated polypeptide pattern of L. infantum only showed two major bands with an apparent Mr of 62,000 and a doublet of 26,000 to 23,000. Immunoprecipitation of detergent extracts of labeled promastigote subspecies with immune sera from rabbits immunized with either L. chagasi or L. infantum and from patients and mice infected with these two parasites, as well as with a monoclonal antibody against the surface of L. donovani promastigotes, demonstrated that the surface antigenic expression of L. infantum is different from that noticed in the two other subspecies, which are similar. Immunofluorescence experiments with some of these antibodies confirmed these results. The present findings should be considered in taxonomic and immunological studies in visceral leishmaniasis.

Infectivity of Leishmania promastigotes is associated with surface antigenic expression.

Differentiation between a non-infective and an infective Leishmania promastigote population was demonstrated. Promastigotes in the stationary phase (day 5) were found to be highly infective in vitro to BALB/c mouse peritoneal macrophages, compared with those of the logarithmic phase (day 3). The infective promastigotes showed surface antigenic determinants different from non-infective ones. Polyclonal anti-3 day and anti-5 day antibodies were bound specifically to the surface of corresponding promastigotes in both SRIA and IFAT; no strong cross-reactions were observed otherwise. Also, polyclonal anti-5 day but not anti-3 day antibodies recognized efficiently the antigenic molecules on the surface of late stage (day 7) sandfly promastigotes. This clearly indicates the appearance of new antigenic molecules on the surface of infective promastigote forms. Intracellular multiplication of Leishmania was significantly inhibited by anti-5 day antibodies compared with anti-3 day antibodies. The presence of new surface molecules on late stage promastigotes may contribute to Leishmania infectivity.