Risk and prognostic significance of tuberculosis in patients from The TREAT Asia HIV Observational Database

10.1186/1471-2334-9-46BMC Infectious Diseases94

Interaction of C-Terminal Truncated Human αA-Crystallins with Target Proteins

Significant portion of alphaA-crystallin in human lenses exists as C-terminal residues cleaved at residues 172, 168, and 162. Chaperone activity, determined with alcohol dehydrogenase (ADH) and betaL-crystallin as target proteins, was increased in alphaA(1-172) and decreased in alphaA(1-168) and alphaA(1-162). The purpose of this study was to show whether the absence of the C-terminal residues influences protein-protein interactions with target proteins.Our hypothesis is that the chaperone-target protein binding kinetics, otherwise termed subunit exchange rates, are expected to reflect the changes in chaperone activity. To study this, we have relied on fluorescence resonance energy transfer (FRET) utilizing amine specific and cysteine specific fluorescent probes. The subunit exchange rate (k) for ADH and alphaA(1-172) was nearly the same as that of ADH and alphaA-wt, alphaA(1-168) had lower and alphaA(1-162) had the lowest k values. When betaL-crystallin was used as the target protein, alphaA(1-172) had slightly higher k value than alphaA-wt and alphaA(1-168) and alphaA(1-162) had lower k values. As expected from earlier studies, the chaperone activity of alphaA(1-172) was slightly better than that of alphaA-wt, the chaperone activity of alphaA(1-168) was similar to that of alphaA-wt and alphaA(1-162) had substantially decreased chaperone activity.Cleavage of eleven C-terminal residues including Arg-163 and the C-terminal flexible arm significantly affects the interaction with target proteins. The predominantly hydrophilic flexible arm appears to be needed to keep the chaperone-target protein complex soluble

Human neutrophil clearance of bacterial pathogens triggers anti-microbial gamma delta T cell responses in early infection

Human blood Vc9/Vd2 T cells, monocytes and neutrophils share a responsiveness toward inflammatory chemokines and are rapidly recruited to sites of infection. Studying their interaction in vitro and relating these findings to in vivo observations in patients may therefore provide crucial insight into inflammatory events. Our present data demonstrate that Vc9/Vd2 T cells provide potent survival signals resulting in neutrophil activation and the release of the neutrophil chemoattractant CXCL8 (IL-8). In turn, Vc9/Vd2 T cells readily respond to neutrophils harboring phagocytosed bacteria, as evidenced by expression of CD69, interferon (IFN)-c and tumor necrosis factor (TNF)-a. This response is dependent on the ability of these bacteria to produce the microbial metabolite (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), requires cell-cell contact of Vc9/Vd2 T cells with accessory monocytes through lymphocyte function-associated antigen-1 (LFA-1), and results in a TNF-a dependent proliferation of Vc9/Vd2 T cells. The antibiotic fosmidomycin, which targets the HMB-PP biosynthesis pathway, not only has a direct antibacterial effect on most HMB-PP producing bacteria but also possesses rapid anti-inflammatory properties by inhibiting cd T cell responses in vitro. Patients with acute peritoneal-dialysis (PD)-associated bacterial peritonitis – characterized by an excessive influx of neutrophils and monocytes into the peritoneal cavity – show a selective activation of local Vc9/Vd2 T cells by HMB-PP producing but not by HMB-PP deficient bacterial pathogens. The cd T celldriven perpetuation of inflammatory responses during acute peritonitis is associated with elevated peritoneal levels of cd T cells and TNF-a and detrimental clinical outcomes in infections caused by HMB-PP positive microorganisms. Taken together, our findings indicate a direct link between invading pathogens, neutrophils, monocytes and microbe-responsive cd T cells in early infection and suggest novel diagnostic and therapeutic approaches.Martin S. Davey, Chan-Yu Lin, Gareth W. Roberts, Sinéad Heuston, Amanda C. Brown, James A. Chess, Mark A. Toleman, Cormac G.M. Gahan, Colin Hill, Tanya Parish, John D. Williams, Simon J. Davies, David W. Johnson, Nicholas Topley, Bernhard Moser and Matthias Eber

Characterization of an IncFII Plasmid Encoding NDM-1 from Escherichia coli ST131

Background: The current spread of the gene encoding the metallo-ß-lactamase NDM-1 in Enterobacteriaceae is linked to a variety of surrounding genetic structures and plasmid scaffolds. Methodology: The whole sequence of plasmid pGUE-NDM carrying the bla NDM-1 gene was determined by high-density pyrosequencing and a genomic comparative analysis with other blaNDM-1-negative IncFII was performed. Principal Findings: Plasmid pGUE-NDM replicating in Escherichia coli confers resistance to many antibiotic molecules including b-lactams, aminoglycosides, trimethoprim, and sulfonamides. It is 87,022 bp in-size and carries the two b-lactamase genes bla NDM-1 and bla OXA-1, together with three aminoglycoside resistance genes aacA4, aadA2, and aacC2. Comparative analysis of the multidrug resistance locus contained a module encompassing the blaNDM-1 gene that is actually conserved among different structures identified in other enterobacterial isolates. This module was constituted by the blaNDM-1 gene, a fragment of insertion sequence ISAba125 and a bleomycin resistance encoding gene. Significance: This is the first characterized bla NDM-1-carrying IncFII-type plasmid. Such association between the bla NDM-1 gene and an IncFII-type plasmid backbone is extremely worrisome considering that this plasmid type is known to sprea

Multiple Aggregates and Aggresomes of C-Terminal Truncated Human αA-Crystallins in Mammalian Cells and Protection by αB-Crystallin

Cleavage of 11 (αA162), 5 (αA168) and 1 (αA172) residues from the C-terminus of αA-crystallin creates structurally and functionally different proteins. The formation of these post-translationally modified αA-crystallins is enhanced in diabetes. In the present study, the fate of the truncated αA-crystallins expressed in living mammalian cells in the presence and absence of native αA- or αB-crystallin has been studied by laser scanning confocal microscopy (LSM).YFP tagged αAwt, αA162, αA168 and αA172, were individually transfected or co-transfected with CFP tagged αAwt or αBwt, expressed in HeLa cells and studied by LSM. Difference in protein aggregation was not caused by different level of α-crystallin expression because Western blotting results showed nearly same level of expression of the various α-crystallins. The FRET-acceptor photo-bleaching protocol was followed to study in situ protein-protein interaction. αA172 interacted with αAwt and αBwt better than αA168 and αA162, interaction of αBwt being two-fold stronger than that of αAwt. Furthermore, aggresomes were detected in cells individually expressing αA162 and αA168 constructs and co-expression with αBwt significantly sequestered the aggresomes. There was no sequestration of aggresomes with αAwt co-expression with the truncated constructs, αA162 and αA168. Double immunocytochemistry technique was used for co-localization of γ-tubulin with αA-crystallin to demonstrate the perinuclear aggregates were aggresomes.αA172 showed the strongest interaction with both αAwt and αBwt. Native αB-crystallin provided protection to partially unfolded truncated αA-crystallins whereas native αA-crystallin did not. Aggresomes were detected in cells expressing αA162 and αA168 and αBwt co-expression with these constructs diminished the aggresome formation. Co-localization of γ-tubulin in perinuclear aggregates validates for aggresomes

Incidence and Risk Factors of Serious Adverse Events during Antituberculous Treatment in Rwanda: A Prospective Cohort Study

BACKGROUND: Tuberculosis (TB) and TB-human immunodeficiency virus infection (HIV) coinfection is a major public health concern in resource-limited settings. Although TB treatment is challenging in HIV-infected patients because of treatment interactions, immunopathological reactions, and concurrent infections, few prospective studies have addressed this in sub-Saharan Africa. In this study we aimed to determine incidence, causes of, and risk factors for serious adverse events among patients on first-line antituberculous treatment, as well as its impact on antituberculous treatment outcome. METHODS AND FINDINGS: Prospective observational cohort study of adults treated for TB at the Internal Medicine department of the Kigali University Hospital from May 2008 through August 2009. Of 263 patients enrolled, 253 were retained for analysis: median age 35 (Interquartile range, IQR 28-40), 55% male, 66% HIV-positive with a median CD4 count 104 cells/mm(3) (IQR 44-248 cells/mm(3)). Forty percent had pulmonary TB, 43% extrapulmonary TB and 17% a mixed form. Sixty-four (26%) developed a serious adverse event; 58/167 (35%) HIV-infected vs. 6/86 (7%) HIV-uninfected individuals. Commonest events were concurrent infection (n = 32), drug-induced hepatitis (n = 24) and paradoxical reactions/TB-IRIS (n = 23). HIV-infection (adjusted Hazard Ratio, aHR 3.4, 95% Confidence Interval, CI 1.4-8.7) and extrapulmonary TB (aHR 2, 95%CI 1.1-3.7) were associated with an increased risk of serious adverse events. For TB/HIV co-infected patients, extrapulmonary TB (aHR 2.0, 95%CI 1.1-3.9) and CD4 count <100 cells/mm3 at TB diagnosis (aHR 1.7, 95%CI 1.0-2.9) were independent predictors. Adverse events were associated with an almost two-fold higher risk of unsuccessful treatment outcome at 6 months (HR 1.89, 95%CI 1.3-3.0). CONCLUSION: Adverse events frequently complicate the course of antituberculous treatment and worsen treatment outcome, particularly in patients with extrapulmonary TB and advanced immunodeficiency. Concurrent infection accounts for most events. Our data suggest that deterioration in a patient already receiving antituberculous treatment should prompt an aggressive search for additional infections

Third generation cephalosporin use in a tertiary hospital in Port of Spain, Trinidad: need for an antibiotic policy

BACKGROUND: Tertiary care hospitals are a potential source for development and spread of bacterial resistance being in the loop to receive outpatients and referrals from community nursing homes and hospitals. The liberal use of third-generation cephalosporins (3GCs) in these hospitals has been associated with the emergence of extended-spectrum beta- lactamases (ESBLs) presenting concerns for bacterial resistance in therapeutics. We studied the 3GC utilization in a tertiary care teaching hospital, in warded patients (medical, surgical, gynaecology, orthopedic) prescribed these drugs. METHODS: Clinical data of patients (≥ 13 years) admitted to the General Hospital, Port of Spain (POSGH) from January to June 2000, and who had received 3GCs based on the Pharmacy records were studied. The Sanford Antibiotic Guide 2000, was used to determine appropriateness of therapy. The agency which procures drugs for the Ministry of Health supplied the cost of drugs. RESULTS: The prevalence rate of use of 3GCs was 9.5 per 1000 admissions and was higher in surgical and gynecological admissions (21/1000) compared with medical and orthopedic (8 /1000) services (p < 0.05). Ceftriaxone was the most frequently used 3GC. Sixty-nine (36%) patients without clinical evidence of infection received 3Gcs and prescribing was based on therapeutic recommendations in 4% of patients. At least 62% of all prescriptions were inappropriate with significant associations for patients from gynaecology (p < 0.003), empirical prescribing (p < 0.48), patients with undetermined infection sites (p < 0.007), and for single drug use compared with multiple antibiotics (p < 0.001). Treatment was twice as costly when prescribing was inappropriate CONCLUSIONS: There is extensive inappropriate 3GC utilization in tertiary care in Trinidad. We recommend hospital laboratories undertake continuous surveillance of antibiotic resistance patterns so that appropriate changes in prescribing guidelines can be developed and implemented. Though guidelines for rational antibiotic use were developed they have not been re-visited or encouraged, suggesting urgent antibiotic review of the hospital formulary and instituting an infection control team. Monitoring antibiotic use with microbiology laboratory support can promote rational drug utilization, cut costs, halt inappropriate 3GC prescribing, and delay the emergence of resistant organisms. An ongoing antibiotic peer audit is suggested

The Cost-Effectiveness of Tuberculosis Preventive Therapy for HIV-Infected Individuals in Southern India: A Trial-Based Analysis

Regimens for isoniazid-based preventive therapy (IPT) for tuberculosis (TB) in HIV-infected individuals have not been widely adopted given concerns regarding efficacy, adherence and drug resistance. Further, the cost-effectiveness of IPT has not been studied in India.We used an HIV/TB model to project TB incidence, life expectancy, cost and incremental cost-effectiveness of six months of isoniazid plus ethambutol (6EH), thirty-six months of isoniazid (36H) and no IPT for HIV-infected patients in India. Model input parameters included a median CD4 count of 324 cells/mm(3), and a rate ratio of developing TB of 0.35 for 6EH and 0.22 for 36H at three years as compared to no IPT. Results of 6EH and 36H were also compared to six months of isoniazid (6H), three months of isoniazid plus rifampin (3RH) and three months of isoniazid plus rifapentine (3RPTH).Projected TB incidence decreased in the 6EH and 36H regimens by 51% and 62% respectively at three-year follow-up compared to no IPT. Without IPT, projected life expectancy was 136.1 months at a lifetime per person cost of $5,630. 6EH increased life expectancy by 0.8 months at an additional per person cost of$100 (incremental cost-effectiveness ratio (ICER) of $1,490/year of life saved (YLS)). 36H further increased life expectancy by 0.2 months with an additional per person cost of$55 (ICER of $3,120/YLS). The projected clinical impact of 6EH was comparable to 6H and 3RH; however when compared to these other options, 6EH was no longer cost-effective given the high cost of ethambutol. Results were sensitive to baseline CD4 count and adherence.Three, six and thirty-six-month regimens of isoniazid-based therapy are effective in preventing TB. Three months of isoniazid plus rifampin and six-months of isoniazid are similarly cost-effective in India, and should be considered part of HIV care