Microbes responsible for acute exacerbation of COPD.

This study was designed to find out the microbes responsible for acute exacerbation of chronic obstructive pulmonary disease (COPD). This study was carried out in the National Institute of Diseases of the Chest & Hospital (NIDCH), Dhaka during the period of January 2003 to December 2003. The study was a prospective case control study. There were 88 male and 2 female patients. The majority of the study subjects fell within the range of 50-70 years. All were smokers. 30 stable COPD patients were taken as control for comparison of sputum culture results of acute exacerbated COPD patients. A standard proforma with questionnaire was designed and filled to select patient with COPD. The patients were selected according to the predetermined criteria viz FEV1<70% predicted and FEV1/FVC % <70% of predicted. Morning specimen of sputum was collected after appropriate preparation and physical character of the sputum were noted. Sputum was immediately sent to microbiology lab for culture. Out of 30 stable COPD patients 6(20%) showed positive sputum culture for bacteria, Pseudomonas 3, Klebsiella 1, Streptococcus pneumoniae 1 and Haemophilus influenza 1. Majority of them were Gram-negative organism. Out of 60 patients with acute exacerbation of COPD 39 patients (65%) showed positive culture for bacteria. Pseudomonas 15, Klebsiella 8, Acinetobacter 4, Enterobacter 2, Moraxella catarrhalis 2 and mixed organisms like, Pseudomonas + Klebsiella 2 and Pseudononas + Acinobacter 1. Majority were Gram-negative bacilli viz. Pseudomonas and Klebsiella spp. species. From this study it was concluded that the prevalence of lower airway bacterial colonization in outpatients with stable COPD is high and is mainly due to Gram-negative bacilli like Pseudomonas spp. The greater rate of isolation of pathogenic bacteria in exacerbated COPD than in stable COPD in this study, supports the pathogenic role of bacteria in a proportion of acute exacerbations of chronic obstructive pulmonary disease. The organism commonly play pathogenic role in acute exacerbations of COPD are Pseudomonas and Klebsiella. Acinobacter Moraxella catarrhalis and Enterobacter also contributed in exacerbation of COPD.

Seminal plasma non-heparin binding proteins (NHBP) reduce the cryoinjury to buffalo cauda epididymal spermatozoa induced by heparin binding proteins (HBP).

Previous cryopreservation studies with buffalo cauda epididymal spermatozoa have reported a deleterious effect of seminal plasma heparin binding protein (HBP). The amount of HBP used in these studies was meager compared to the normal level of HBP in the buffalo ejaculate, still the damage induced upon the spermatozoa was substantial when compared to that incurred to the spermatozoa during routine freezing of ejaculated semen. Thus there might be some factor(s) in the seminal plasma, which reduce the deleterious effect of HBP on spermatozoa during cryopreservation of ejaculated semen. This study was conducted to investigate for the presence of any such factor in buffalo seminal plasma. Seminal plasma proteins were separated on their heparin binding properties as heparin binding (HBP) and non-heparin binding (NHBP). The separated proteins were added to the extender of buffalo cauda epididymal semen for cryopreservation either alone or in combination. The spermatozoa were assessed for progressive motility, viability, acrosomal integrity and response to hypo-osmotic solution test (HOST) at prefreeze and post-thaw stages of cryopreservation. NHBP was found to provide some degree of protection to buffalo spermatozoa against cryopreservation stress as well as the deleterious effect of HBP during cryopreservation.

Comparison of sputum induction with broncho-alveolar lavage in the diagnosis of smear-negative pulmonary tuberculosis.

Sputum microscopy and AFB-culture being gold standard and a fundamental tool for diagnosis of pulmonary tuberculosis (PTB) has got its limitation of low sensitivity. Fibreoptic bronchoscopy (FOB) has been widely recommended as the diagnostic procedure of choice in smear negative patients. But bronchoscopy is an invasive procedure, costly, not readily available in our country and needs expertise. Several studies abroad have directly compared the yield of sputum induction (SI) with 3% saline (NaCl solution) with Bronchoalveolar lavage (BAL) through FOB in smear-negative suspected PTB patients and showed that SI was a low cost, safe and well tolerated procedure with equal efficacy to BAL through FOB for the diagnosis of PTB in such patients. For the first time a prospective comparison was conducted in Bangladesh to see the yield of sputum induction (SI) and BAL in 52 selected smear- negative patients of suspected PTB. Each of the samples of induced sputum and BAL fluid were examined for AFB by Ziehl-Neelsen's method. Samples of both SI and BAL from 20 patients were cultured for AFB in Lowenstein-Jensen medium for 6 weeks irrespective of their induced sputum smear being positive or negative for AFB. Data were managed and analyzed using computer program SPSS version 10.0. Agreement of SI and BAL was tested using Pearson Chi-square and Kappa test. The results showed that the yield of SI were significantly more than that of BAL (p<0.05).The AFB smear results from specimens obtained by SI and BAL were in agreement in 75% cases (p=0.02).Statistical analysis of the yield of culture results from SI and BAL group with Fishers Exact test showed they were in agreement in 90% cases (p=0.0001) and was measured by Kappa test as significant (p=0.0004). The sensitivity of AFB-smears in samples from SI and BAL were 74% and 58% respectively. The specificity of smear positivity and of culture was assumed to be 100%. SI is a safe procedure with considerable diagnostic yield and a high agreement with the results of BAL through FOB for the diagnosis of PTB. SI offers an alternative or additional approach to the diagnosis of smear-negative suspected PTB patients and would enhance sensitivity for the diagnosis of tuberculosis.

Effect of egg yolk and seminal plasma heparin binding protein interaction on the freezability of buffalo cauda epididymal spermatozoa.

Egg yolk is routinely used in most of the extenders for cryopreservation of semen, but mechanisms of protection of spermatozoa by egg yolk are not very clear. Investigations with buffalo cauda epididymal sperm have shown that seminal plasma heparin binding proteins have detrimental effects during semen cryopreservation. The present study was conducted to investigate the effect of egg yolk on the detrimental effects of heparin binding proteins during cryopreservation of buffalo cauda epididymal spermatozoa. The results indicated that egg yolk was able to reduce the heparin binding proteins mediated cryoinjury in spermatozoa. One of the mechanisms of protection of spermatozoa from cryoinjury by egg yolk may be due to the inhibition of deleterious actions of heparin binding proteins on the spermatozoa.

Pre-silencing of genes involved in the electron transport chain (ETC) pathway is associated with responsiveness to abatacept in rheumatoid arthritis.

BACKGROUND: In the current context of personalized medicine, one of the major challenges in the management of rheumatoid arthritis (RA) is to identify biomarkers that predict drug responsiveness. From the European APPRAISE trial, our main objective was to identify a gene expression profile associated with responsiveness to abatacept (ABA) + methotrexate (MTX) and to understand the involvement of this signature in the pathophysiology of RA. METHODS: Whole human genome microarrays (4 × 44 K) were performed from a first subset of 36 patients with RA. Data validation by quantitative reverse-transcription (qRT)-PCR was performed from a second independent subset of 32 patients with RA. Gene Ontology and WikiPathways database allowed us to highlight the specific biological mechanisms involved in predicting response to ABA/MTX. RESULTS: From the first subset of 36 patients with RA, a combination including 87 transcripts allowed almost perfect separation between responders and non-responders to ABA/MTX. Next, the second subset of patients 32 with RA allowed validation by qRT-PCR of a minimal signature with only four genes. This latter signature categorized 81% of patients with RA with 75% sensitivity, 85% specificity and 85% negative predictive value. This combination showed a significant enrichment of genes involved in electron transport chain (ETC) pathways. Seven transcripts from ETC pathways (NDUFA6, NDUFA4, UQCRQ, ATP5J, COX7A2, COX7B, COX6A1) were significantly downregulated in responders versus non-responders to ABA/MTX. Moreover, dysregulation of these genes was independent of inflammation and was specific to ABA response. CONCLUSION: Pre-silencing of ETC genes is associated with future response to ABA/MTX and might be a crucial key to susceptibility to ABA response.

Effect of buffalo seminal plasma heparin binding protein (HBP) on freezability and in vitro fertility of buffalo cauda spermatozoa.

The study was conducted to assess the effect of heparin binding seminal plasma proteins (HBP) on freezability and in vitro fertilizing ability of buffalo cauda epididymal spermatozoa. Spermatozoal motility, viability and acrosomal integrity at prefreeze and post-thaw stages were studied. The in vitro fertilizing ability of spermatozoa was assessed by the application of two tests, i.e. bovine cervical mucus penetration test (BCMPT) and hypo-osmotic sperm swelling test (HOST). HBP isolated from buffalo seminal plasma and maintained in the laboratory were used for the study. Twelve pairs of epididymis from adult buffaloes slaughtered at the local abattoir were used for the study. The results indicated that HBP addition improved the progressive motility, BCMPT and HOST response at prefreeze level. HBP at a concentration of 40 microg/ml showed better results than HBP at a concentration of 80 microg/ml. However, subjecting the HBP treated spermatozoa to cryopreservation resulted in significant reduction of motility, viability, acrosomal integrity and response to BCMPT and HOST in the HBP treated groups when compared to those in control group. The deleterious effect of HBP was found to be concentration dependent with the higher concentration causing higher post-thaw damage.

Evidence for alpha-1-acid glycoprotein populations of different pI values after concanavalin A affinity chromatography. Study of their evolution during inflammation in man.

Isoelectric focusing in polyacrylamide gel was used to reveal the two alpha 1-acid glycoprotein populations separated by affinity chromatography on Con A-Sepharose from human purified alpha 1-acid glycoprotein, whole normal serum and sera from patients undergoing an acute inflammatory process. The concanavalin A non-reactive peak had a lower isoelectric point than did the concanavalin A reactive peak. Moreover, crossed immunoaffinity electrophoresis with free concanavalin A revealed three alpha 1-acid glycoprotein populations (concanavalin A reactive, weakly and non-reactive). A significant difference was observed between the patterns of normal and of inflammatory sera, since the concanavalin A reactive fraction was enhanced during the inflammatory process. These results suggest a variation in relative ratios of the different types of peripheral oligosaccharide branches in the alpha 1-acid glycoprotein populations.

Effect of cholesterol supplementation on cryosurvival of goat spermatozoa.

AIM: Sperm membrane cholesterol influences cryodamage during cryopreservation. The present study was carried out to evaluate the effect of varying cholesterol levels in Tris based extenders on the freezability of sexually healthy Malabari buck semen. MATERIALS AND METHODS: A total of 48 ejaculates from two adults healthy sexually healthy Malabari bucks were utilized for the study. The collected and pooled ejaculates were divided into four groups with Group I serving as Control - I, Group II and III were treated with 1 mg and 2 mg of cholesterol-loaded-cyclodextrin (CLC)/120 × 10(6) spermatozoa, respectively, and Group IV, treated with 1 mg methyl-ß-cyclodextrin (MßCD) served as Control - II. Manual freezing was carried out to cryopreserve the treated and control spermatozoa. RESULTS: Treatment of semen samples with CLC resulted in improved maintenance of sperm motility at pre-freeze and post-thaw stages of cryopreservation without affecting hypo-osmotic swelling response. Treatment of semen with 1 mg of CLC/120 × 10(6) spermatozoa was observed to be better than treatment with 2 mg of CLC/120 × 10(6) spermatozoa. In general, MßCD treatment was found to result in significantly lower sperm characteristics than those of Control - I and CLC treatment at pre-feeze and post-thaw stages and when incubated up to 4 h. CONCLUSION: Cholesterol treatment of sexually healthy Malabari buck semen was found to hold promise for improving cryopreservability of spermatozoa.

Absence of expression of interleukin-6 (IL-6) mRNA in regenerating rat liver.

The serum level of IL-6 and expression of IL-6 mRNA in hepatocytes from regenerating liver were investigated in the rat. The IL-6 level in the serum was not significantly different from that of a control group of rats submitted to an acute experimental inflammation. IL-6 mRNA expression did not occur in the liver of hepatectomized rats as judged from Northern blotting experiments using an IL-6 riboprobe. These results suggest that if IL-6 is implicated in hepatic regeneration, this cytokine is not produced by the regenerating liver and must be delivered exogenously to the liver to modulate hepatic regeneration.

The synthesis of human alpha-2-HS glycoprotein is down-regulated by cytokines in hepatoma HepG2 cells.

The regulation of the synthesis of alpha-2-HS glycoprotein (AHSG) by inflammatory mediators from activated monocytes was studied on the human hepatoma cell line HepG2 and compared to that of albumin. Monocyte-conditioned medium, recombinant human interleukin-6 (rhIL6) and interleukin-1 beta (rhIL1 beta) all down-regulated the synthesis of AHSG. This decrease was found both at the protein and the mRNA level. The most efficient mediator was the monocyte-conditioned medium, when rhIL1 beta was found to be less efficient than rhIL6. The combination of rhIL6 and rhIL1 beta resulted in an additive down-regulation of the AHSG mRNA levels. Similar results were obtained with albumin. These data indicate that AHSG is a negative acute-phase protein whose synthesis is regulated by cytokines in a manner similar to that of albumin.

Partial hepatectomy and mediators of inflammation decrease the expression of liver alpha 2-HS glycoprotein gene in rats.

Liver mRNA levels of two acute phase reactant (APR) proteins, alpha 2-HS glycoprotein (a major negative APR) and alpha 1-acid glycoprotein (a major positive APR) were measured in male rats at different times after the administration of turpentine, of tumor necrosis factor, or following partial hepatectomy. In every case, a marked decrease in mRNA levels of alpha 2-HS glycoprotein was observed which reached a maximum at 24 h. A concomitant increase of alpha 1-acid glycoprotein mRNA levels was observed under the same conditions. These results indicate that the decreased levels of alpha 2-HS glycoprotein induced by the acute-phase response following inflammatory mediators and partial hepatectomy are due to a down-regulation of the gene expression of this protein in rat liver.

An enzyme-linked immunoassay for the measurement of rat alpha 1-acid glycoprotein synthesized by cultured hepatocytes.

We have developed a sandwich ELISA to quantify rat alpha 1-acid glycoprotein (AGP). The assay correlated well with RID and the minimum detectable concentration was 1 microgram/l. The assay permits high sensitivity determinations of the rate of synthesis of AGP in vitro. The maximum mean rates observed were 1500 and 1800 ng/24 h/10(6) cells for hepatocytes cultured alone and co-cultured hepatocytes respectively and 39 ng/h/10(6) cells for isolated hepatocytes.

Interleukin-6 (IL-6) and acute-phase proteins in rats with biliary sepsis.

We measured serum interleukin-6 (IL-6) and acute-phase proteins, alpha 1-acid glycoprotein (AGP) and alpha 2-macroglobulin (alpha 2M), after a retrograde intrabiliary bacterial infection in rats with biliary obstruction. Maximum serum IL-6 was obtained at 6 h in rats following inoculation of bacteria (10(6) CFU/ml E. Coli) in the bile duct and it was higher than that observed in rats undergoing a bile duct ligation or a laparotomy. There was a strict relationship between the level of IL-6 at 6 h and the modified levels of AGP and alpha 2M at 48 h. AGP and alpha 2M levels were the highest in sera of rats with bile duct infection as compared with those found in sera of rats with bile duct ligation or laparotomy. After inoculation of E. Coli or E. Fecalis, blood IL-6 level was always higher at 6 h in inferior vena cava as compared with that found in the supra hepatic vein. These results indicate that IL-6 is synthesized after a biliary sepsis and that its blood level is higher in the systemic circulation than in the local circulation.

IL-6-induced changes in synthesis of alpha 1-acid glycoprotein in human hepatoma Hep3B cells are distinctively regulated by monoclonal antibodies directed against different epitopes of IL-6 receptor (gp80).

The synthesis of the human acute-phase alpha 1-acid glycoprotein (AGP) is primarily controlled by IL-6 and IL-1 in liver cells. In the present study, monoclonal antibodies against human gp80 interleukin-6 receptor (IL-6R) were utilized to study the role of the IL-6R in the control of the IL-6-induced AGP synthesis in the human hepatoma Hep3B cell line. Two of the 4 MAbs used in this study, M164 and M195, identified 2 different epitopes involved in IL-6 binding and two others, M91 and M182, recognized epitopes not involved in IL-6 binding. Dose-response experiments indicated that up to 55% of AGP synthesis was inhibited by 10(5) ng/ml of MAbs 164 or 195 when Hep3B cells were treated by IL-6 for 48h. Kinetics of the inhibition of AGP synthesis after addition of anti-IL-6R indicated that the decrease of the IL-6-induced AGP synthesis by Hep3B cells was obtained immediately after the addition of the anti-IL-6R MAbs. Of the two MAbs not involved in IL-6 binding, M91 was unable to interfere with the IL-6-induced AGP synthesis whereas, surprisingly, M182 decreased it by about 25%. Since M182 was also able to interfere with the proliferative response of an IL-6 dependent plasma cell line, our results suggested that M182 may be directed to a structure involved in the IL-6/IL-6R gp130 complex formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of alpha 1-acid glycoprotein plasma concentration by sex steroids and adrenal-cortical hormones during experimental inflammation in the rat.

Treatment of adult intact rats with sex steroids (estradiol-17 beta, ethynylestradiol, dihydrotestosterone) raises the concentration of serum acute-phase alpha 1-acid glycoprotein (AGP). Estrogens are more effective than dexamethasone, and experimental inflammation causes an additive effect on AGP synthesis when ethynylestradiol is given simultaneously. Adrenaline is also able to increase the AGP level. Experiments with adrenalectomized and adrenalectomized plus castrated rats result in a 50% reduction in the serum level of AGP as compared with that in normal and hypophysectomized rats. Although ethynylestradiol is the strongest inducer of AGP synthesis in intact animals, it is unable to enhance significantly the AGP level in adrenalectomized rats, contrary to dexamethasone. Adrenalectomized rats are incapable of undergoing a substantial increase in plasma AGP level following experimental inflammation, and ethynylestradiol or adrenaline cannot take the place of dexamethasone in inducing high levels of AGP in these inflamed rats. These results indicate that glucocorticoids play an obligatory role in modulating AGP synthesis either by directly regulating the AGP gene or in modulating AGP synthesis by increasing the stability of AGP mRNA. Finally, it is suggested that glucocorticoids may also act in unmasking receptor binding sites at the AGP gene level for other mediators such as sex steroids and putative inflammatory factors.

Evaluation of a recombinant LigB protein of Leptospira interrogans serovar Canicola in an enzyme-linked immunosorbent assay for the serodiagnosis of bovine leptospirosis.

A recombinant leptospiral lipoprotein, LigB, was evaluated for use in the diagnosis of bovine leptospirosis by enzyme-linked immunosorbent assay (rLigB IgG ELISA). The standard reference test (Microscopic agglutination test, MAT) of 200 serum samples from cattle suspected of leptospirosis showed that 95 (47.5%) samples had positive agglutination titres, which ranged from 100 to 1600. In rLigB IgG ELISA, 49% of the samples were positive. Sensitivity of IgG ELISA for 95 bovine sera, which had MAT titres of greater than or equal to 100, were 100%. ELISA showed a specificity of 97.1% with 105 bovine sera, which were negative at a 1:50 dilution in MAT for Leptospira interrogans serovars. The results of ELISA and MAT correspond very good. When analytical specificity of IgG ELISA was evaluated using bovine serum samples from animals showing the serum antibodies to other pathogens, no cross-reaction was observed. Thus the recombinant LigB IgG ELISA can be used instead of the MAT as an aid to the diagnosis of bovine leptospirosis.

Identification of PDC-109-like protein(s) in buffalo seminal plasma.

The FN-2 family of seminal plasma proteins represents the major protein fraction of bovine seminal plasma. These proteins also constitute the major seminal plasma proteins fraction in horse, goat and bison seminal plasma and are present in pig, rat, mouse, hamster and human seminal plasma. BSP-A1 and BSP-A2, the predominant proteins of the FN-2 family, are collectively termed as PDC-109. Fn-2 proteins play an important role in fertilization, including sperm capacitation and formation of oviductal sperm reservoirs. Significantly, BSP proteins were also shown to have negative effects in the context of sperm storage. No conclusive evidence for the presence of buffalo seminal plasma protein(s) similar to PDC-109 exists. Studies with buffalo seminal plasma indicated that isolation and identification of PDC-109-like protein(s) from buffalo seminal plasma by conventional methods might be difficult. Thus, antibodies raised against PDC-109 isolated, and purified from cattle seminal plasma, were used for investigating the presence of PDC-109-like protein(s) in buffalo seminal plasma. Buffalo seminal plasma proteins were resolved on SDS-PAGE, blotted to nitro cellulose membranes and probed for the presence of PDC-109-like protein(s) using the PDC-109 antisera raised in rabbits. A distinct immunoreactive band well below the 20-kDa regions indicated the presence of PDC-109-like protein(s) in buffalo seminal plasma.

The human hepatoma Hep3B cell line as an experimental model in the study of the long-term regulation of acute-phase proteins by cytokines.

The regulation of the synthesis by the cytokines interleukin-1 (IL-1) and IL-6 of the positive acute-phase protein alpha 1-acid glycoprotein (AGP) and of the negative acute-phase protein alpha 2-HS glycoprotein (AHSG) has been studied in a long-term culture system of the human hepatoma cell line Hep3B. The culture system contained 30 nM-sodium selenite as the only supplement. This allowed maintenance of the synthesis of the proteins under study at a near steady state for over 3 months. An increase in AGP mRNA and a decrease in AHSG mRNA were observed when cells were treated for two successive 48 h-periods with monocyte-conditioned medium. A return to basal levels was obtained after cessation of the cytokine addition. Two further additions of cytokines led to alterations in mRNA levels similar to those observed following the first cytokine treatment. The amounts of AGP and AHSG secreted were altered in accordance with the mRNA modifications. These results suggest that new cytokine receptors were being constantly synthesized during cell culture. When cytokines were present in the culture medium for 10 days, maximum alterations in AGP and AHSG synthesis were obtained following 2 and 4 days of treatment respectively, but further alterations in protein levels could not be observed afterwards. Expression of IL-6 receptor mRNA was not up-regulated by cytokines, but only by 1 microM-dexamethasone. Our results show that, in this long-term culture system, cytokines induce a response in hepatoma cells similar to that observed in vivo during human inflammatory states. This model could be used to evaluate the effects of agonists or antagonists of cytokines responsible for the hepatic acute-phase protein response.

Long-term biosynthesis of complement component C3 and alpha-1 acid glycoprotein by adult rat hepatocytes in a co-culture system with an epithelial liver cell-type.

We used a system of co-culture of adult rat hepatocytes with another epithelial cell type from rat liver to study the synthesis of two acute-phase reactants, alpha-1 acid glycoprotein (alpha 1AGP) and the third component of complement (C3), and we have obtained long-term secretion of these two proteins. After a period of adaptation corresponding to the first 2-4 days of the co-culture, hepatocytes secreted C3 and alpha 1AGP for at least 2 weeks at a mean level higher than that observed in the first days of a pure culture of hepatocytes. When pulse-chase analysis was performed on day 6 of co-culture, kinetics of synthesis of alpha 1AGP and C3 were the same as those observed on day 1 of a conventional culture of pure hepatocytes. Furthermore, intracellular and extracellular alpha 1AGP had Mr values respectively of 39,000 and of 42,000-52,000, identical with those observed in pure cultures of hepatocytes. Similarly, the molecular size and subunit structures of C3 were the same in co-culture and in cultures, indicating an identical processing of this protein. C3 produced in co-culture was also haemolytically active. Therefore, the system of adult hepatocytes co-cultured with this liver epithelial cell provides a physiological system in vitro which permits long-term synthesis of the two acute-phase reactants C3 and alpha 1AGP. This model opens the possibility to study the modulation of the synthesis of these two proteins during a long period by inflammatory agents or by hormones.