In vivo induction of IL-6 by administration of exogenous cytokines and detection of de novo serum levels of IL-6 in tumor-bearing mice

We investigated the capacity of several recombinant cytokines to induce IL-6 in vivo in both normal and tumor-bearing (TB) mice. Intravenous administration of human rhTNF-alpha, rhIL-1, rhIL-2, rhIFN-alpha A/D, and rmIFN-gamma were all capable of inducing circulating IL-6. rhTNF-alpha administration caused the greatest induction of IL-6. TB animals consistently produced more IL-6 in response to rhTNF-alpha than did normal mice (2 h after 4 micrograms rhTNF-alpha, TB = 24,100 HGF U/ml, non-TB = 3600 HGF U/ml of IL-6). A single daily i.v. dose of rhTNF-alpha (4 micrograms/mouse/day) for 5 days led to decreased IL-6 induction in TB animals by day 3 of treatment (peak levels of IL-6, day 1 = 72,800 HGF U/ml, day 3 = 23,400 HGF U/ml, day 5 = 26,400 HGF U/ml). rhIL-1 administration also resulted in considerable IL-6 production, although peak values were less than those resulting from administration of rhTNF-alpha. Administration of rhIL-1 induced similar IL-6 levels (TB = 10,025 and non-TB = 10,600 HGF U/ml) in TB and normal mice. Single high doses of rhIL-2, rhIFN-alpha A/D, and rmIFN-gamma induced lower but consistent levels of circulating IL-6 in mice with and without tumor. In addition, the sera of untreated TB mice contained levels of IL-6 which paralleled the extent of tumor burden (serum IL-6 in day 30 MCA 106 TB mice = 420 HGF U/ml). The detection of de novo IL-6 was also confirmed in animals bearing tumors of different histologies (the MCA 102 sarcoma, MCA 38 adenocarcinoma, and B16 melanoma). At no time was IL-6 measurable in the sera of untreated normal mice. The identification of IL-6 was verified by neutralization studies using specific antimurine IL-6 antibody. Although the exact role of IL-6 in TB animals remains to be elucidated, its known pleotrophic immune and metabolic effects may be important in the host response to malignancy.     

Direct rapid diagnosis of rifampicin-resistant M. tuberculosis infection in clinical samples by line probe assay (INNO LiPA Rif-TB)

M. tuberculosis is one of the leading causes of death worldwide and Multi Drug Resistant Tuberculosis (MDR-TB) is associated with a high case-fatality rate. Rapid identification of resistant strains is crucial to institute prompt appropriate therapy, and prevent the development of further resistance and spreading of MDR strains. The INNO-LiPA Rif. TB is a commercial reverse hybridisation line probe assay designed for rapid detection of rpoB gene mutations in clinical isolates. We applied this test directly to 44 smear-positive and 45 smear-negative clinical specimens collected from patients suspected of active TB. The capability of this technique to correctly identify local MDR-TB strains was tested on 50 MDR strains isolated in Italy. Results of the test were compared to conventional antibiogram performed on isolated strains. The concordance rate of the LiPA test results on clinical specimens with those obtained with "in vitro" sensitivity was 100%. These results show that the LiPA test can be useful in rapid detection and prompt management of tuberculosis when MDR disease is suspected.     

Elucidating Emergence and Transmission of Multidrug-Resistant Tuberculosis in Treatment Experienced Patients by Whole Genome Sequencing

Background

Understanding the emergence and spread of multidrug-resistant tuberculosis (MDR-TB) is crucial for its control. MDR-TB in previously treated patients is generally attributed to the selection of drug resistant mutants during inadequate therapy rather than transmission of a resistant strain. Traditional genotyping methods are not sufficient to distinguish strains in populations with a high burden of tuberculosis and it has previously been difficult to assess the degree of transmission in these settings. We have used whole genome analysis to investigate M. tuberculosis strains isolated from treatment experienced patients with MDR-TB in Uganda over a period of four years.

Methods and Findings

We used high throughput genome sequencing technology to investigate small polymorphisms and large deletions in 51 Mycobacterium tuberculosis samples from 41 treatment-experienced TB patients attending a TB referral and treatment clinic in Kampala. This was a convenience sample representing 69% of MDR-TB cases identified over the four year period. Low polymorphism was observed in longitudinal samples from individual patients (2-15 SNPs). Clusters of samples with less than 50 SNPs variation were examined. Three clusters comprising a total of 8 patients were found with almost identical genetic profiles, including mutations predictive for resistance to rifampicin and isoniazid, suggesting transmission of MDR-TB. Two patients with previous drug susceptible disease were found to have acquired MDR strains, one of which shared its genotype with an isolate from another patient in the cohort.

Conclusions

Whole genome sequence analysis identified MDR-TB strains that were shared by more than one patient. The transmission of multidrug-resistant disease in this cohort of retreatment patients emphasises the importance of early detection and need for infection control. Consideration should be given to rapid testing for drug resistance in patients undergoing treatment to monitor the emergence of resistance and permit early intervention to avoid onward transmission.     

Treatment of tuberculosis in a region with high drug resistance: outcomes, drug resistance amplification and re-infection

Introduction

Emerging antituberculosis drug resistance is a serious threat for tuberculosis (TB) control, especially in Eastern European countries.

Methods

We combined drug susceptibility results and molecular strain typing data with treatment outcome reports to assess the influence of drug resistance on TB treatment outcomes in a prospective cohort of patients from Abkhazia (Georgia). Patients received individualized treatment regimens based on drug susceptibility testing (DST) results. Definitions for antituberculosis drug resistance and treatment outcomes were in line with current WHO recommendations. First and second line DST, and molecular typing were performed in a supranational laboratory for Mycobacterium tuberculosis (MTB) strains from consecutive sputum smear-positive TB patients at baseline and during treatment.

Results

At baseline, MTB strains were fully drug-susceptible in 189/326 (58.0%) of patients. Resistance to at least H or R (PDR-TB) and multidrug-resistance (MDR-TB) were found in 69/326 (21.2%) and 68/326 (20.9%) of strains, respectively. Three MDR-TB strains were also extensively resistant (XDR-TB). During treatment, 3/189 (1.6%) fully susceptible patients at baseline were re-infected with a MDR-TB strain and 2/58 (3.4%) PDR-TB patients became MDR-TB due to resistance amplification. 5/47 (10.6%) MDR- patients became XDR-TB during treatment. Treatment success was observed in 161/189 (85.2%), 54/69 (78.3%) and 22/68 (32.3%) of patients with fully drug susceptible, PDR- and MDR-TB, respectively. Development of ofloxacin resistance was significantly associated with a negative treatment outcome.

Conclusion

In Abkhazia, a region with high prevalence of drug resistant TB, the use of individualized MDR-TB treatment regimens resulted in poor treatment outcomes and XDR-TB amplification. Nosocomial transmission of MDR-TB emphasizes the importance of infection control in hospitals.     

Low dose aerosol fitness at the innate phase of murine infection better predicts virulence amongst clinical strains of Mycobacterium tuberculosis

Background

Evaluation of a quick and easy model to determine the intrinsic ability of clinical strains to generate active TB has been set by assuming that this is linked to the fitness of Mycobacterium tuberculosis strain at the innate phase of the infection. Thus, the higher the bacillary load, the greater the possibility of inducting liquefaction, and thus active TB, once the adaptive response is set.

Methodology/Principal Findings

The virulence of seven clinical Mycobacterium tuberculosis strains isolated in Spain was tested by determining the bacillary concentration in the spleen and lung of mice at weeks 0, 1 and 2 after intravenous (IV) inoculation of 10⁴ CFU, and by determining the growth in vitro until the stationary phase had been reached. Cord distribution automated analysis showed two clear patterns related to the high and low fitness in the lung after IV infection. This pattern was not seen in the in vitro fitness tests, which clearly favored the reference strain (H37Rv). Subsequent determination using a more physiological low-dose aerosol (AER) inoculation with 10² CFU showed a third pattern in which the three best values coincided with the highest dissemination capacity according to epidemiological data.

Conclusions/Significance

The fitness obtained after low dose aerosol administration in the presence of the innate immune response is the most predictive factor for determining the virulence of clinical strains. This gives support to a mechanism of the induction of active TB derived from the dynamic hypothesis of latent tuberculosis infection.     

Heightened Vulnerability to MDR-TB Epidemics after Controlling Drug-Susceptible TB

Background

Prior infection with one strain TB has been linked with diminished likelihood of re-infection by a new strain. This paper attempts to determine the role of declining prevalence of drug-susceptible TB in enabling future epidemics of MDR-TB.

Methods

A computer simulation of MDR-TB epidemics was developed using an agent-based model platform programmed in NetLogo (See http://mdr.tbtools.org/). Eighty-one scenarios were created, varying levels of treatment quality, diagnostic accuracy, microbial fitness cost, and the degree of immunogenicity elicited by drug-susceptible TB. Outcome measures were the number of independent MDR-TB cases per trial and the proportion of trials resulting in MDR-TB epidemics for a 500 year period after drug therapy for TB is introduced.

Results

MDR-TB epidemics propagated more extensively after TB prevalence had fallen. At a case detection rate of 75%, improving therapeutic compliance from 50% to 75% can reduce the probability of an epidemic from 45% to 15%. Paradoxically, improving the case-detection rate from 50% to 75% when compliance with DOT is constant at 75% increases the probability of MDR-TB epidemics from 3% to 45%.

Conclusions

The ability of MDR-TB to spread depends on the prevalence of drug-susceptible TB. Immunologic protection conferred by exposure to drug-susceptible TB can be a crucial factor that prevents MDR-TB epidemics when TB treatment is poor. Any single population that successfully reduces its burden of drug-susceptible TB will have reduced herd immunity to externally or internally introduced strains of MDR-TB and can experience heightened vulnerability to an epidemic. Since countries with good TB control may be more vulnerable, their self interest dictates greater promotion of case detection and DOTS implementation in countries with poor control to control their risk of MDR-TB.     

Self-reported risks for multiple-drug resistance among new tuberculosis cases: implications for drug susceptibility screening and treatment

Background

Multiple drug-resistance in new tuberculosis (TB) cases accounts for the majority of all multiple drug-resistant TB (MDR-TB) worldwide. Effective control requires determining which new TB patients should be tested for MDR disease, yet the effectiveness of global screening recommendations of high-risk groups is unknown.

Methods

Sixty MDR-TB cases with no history of previous TB treatment, 80 drug-sensitive TB and 80 community-based controls were recruited in Lima, Peru between August and December, 2008 to investigate whether recommended screening practices identify individuals presenting with MDR-TB. Odd ratios (OR) and 95% confidence intervals (CI) were calculated using logistic regression to study the association of potential risk factors with case/control variables.

Results

MDR-TB cases did not differ from drug-sensitive TB and community controls in rates of human immunodeficiency virus infection, reported hospital or prison visits in the 3 years prior to diagnosis. MDR-TB cases were more likely than drug-sensitive TB controls to have had a recent MDR-TB household contact (OR 4.66, (95% CI 1.56–13.87)); however, only 15 cases (28.3%) reported this exposure. In multivariate modeling, recent TB household contact, but not contact with an MDR-TB case, remained predictive of MDR-TB, OR 7.47, (95% CI 1.91–29.3). Living with a partner rather than parents was associated with a lower risk of MDR-TB, OR 0.15, (95% CI 0.04–0.51).

Conclusion

Targeted drug susceptibility testing (DST) linked to reported MDR-TB contact or other high-risk exposures does not identify the majority of new TB cases with MDR disease in Lima where it is endemic. All new TB cases should be screened with DST to identify MDR patients. These findings are likely applicable to other regions with endemic MDR-TB.     

High prevalence of primary multidrug resistant tuberculosis in persons with no known risk factors

Introduction

In high multidrug resistant (MDR) tuberculosis (TB) prevalence areas, drug susceptibility testing (DST) at diagnosis is recommended for patients with risk factors for MDR. However, this approach might miss a substantial proportion of MDR-TB in the general population. We studied primary MDR in patients considered to be at low risk of MDR-TB in Lima, Peru.

Methods

We enrolled new sputum smear-positive TB patients who did not report any MDR-TB risk factor: known exposure to a TB patient whose treatment failed or who died or who was known to have MDR-TB; immunosuppressive co-morbidities, ex prison inmates; prison and health care workers; and alcohol or drug abuse. A structured questionnaire was applied to all enrolled participants to confirm the absence of these factors and thus minimize underreporting. Sputum from all participants was cultured on Löwenstein-Jensen media and DST for first line drugs was performed using the 7H10 agar method.

Results

Of 875 participants with complete data, 23.2% (203) had risk factors for MDR-TB elicited after enrolment. Among the group with no reported risk factors who had a positive culture, we found a 6.3% (95%CI 4.4–8.3) (37/584) rate of MDR-TB. In this group no epidemiological characteristics were associated with MDR-TB. Thus, in this group, multidrug resistance occurred in patients with no identifiable risk factors.

Conclusions

We found a high rate of primary MDR-TB in a general population with no identifiable risk factors for MDR-TB. This suggests that in a high endemic area targeting patients for MDR-TB based on the presence of risk factors is an insufficient intervention.     

Epidemiology and Clinical Characteristics of Pediatric Drug-Resistant Tuberculosis in Chongqing,China

To gain insight into the epidemiology of childhood drug resistant tuberculosis (DR-TB) in China that has the second largest burden of TB and the largest number of multidrug resistant (MDR) TB cases in the world, we performed the cross-sectional study to investigate drug resistance of four first-line anti-TB drugs (isoniazid, rifampicin, streptomycin and ethambutol) using Mycobacterium tuberculosis isolates from 196 culture-confirmed pediatric TB cases diagnosed in the Children’s Hospital of Chongqing Medical University, China during 2008–2013. Univariate and multivariate logistic regression analyses were performed to assess the associations between patient demographic and clinical characteristics and DR-and MDR-TB, respectively. Twenty-eight percent (56/196) of the study patients exhibited resistance to at least one of the four first-line anti-TB drugs tested. MDR was found in 4.6% (9/196) of the study patients. More than half (5/9, 55.6%) of the MDR cases were from a single county of Chongqing. A significant association was found between being acid-fast bacilli-smear negative and DR-TB (adjusted OR, 2.33; 95% CI, 1.13–4.80) and between having concurrent thoracic-extrathoracic involvement and MDR-TB (adjusted OR, 9.49; 95% CI, 1.05–85.92), respectively. The findings of this study indicate that the rate of DR is high among pediatric TB patients in Chongqing and suggest an urgent need for studies to identify MDR transmission hotspots in Chongqing, thereby contributing to the control DR- and MDR-TB epidemics in China. The study also generates new insight into the pathogenesis of DR and MDR M. tuberculosis strains and highlights the importance of studying childhood TB to the goal of global TB control.     

Experimental study of the prophylactic anti-influenza and interferon-inducing activity of epsilon-aminocaproic acid

Epsilon-aminocaproic acid, an original antifibrinolytic had a rapid and prolonged protective effect, thus lowering the death rate of experimental mice contaminated with virulent strains of the influenza virus. The protective effect of the acid was observed after its intraperitoneal administration in a dose of 20 mg/mouse 24 hours before contamination by the strain with the H3N2 antigenic formula and after intranasal application of 15 mg of epsilon-aminocaproic acid divided into 2 doses 2 days before contamination by the strain with the H1N1 antigenic formula. In the animals contaminated by the H1N1 influenza virus 5 days after subcutaneous administration of the drug in a dose of 30 mg there was detected in the lungs a much lower number of the infectious viruses 3 days after the contamination. It was shown that 3 and 6 weeks after initiation of the 5-day treatment course with subcutaneous administration of epsilon-aminocaproic acid in a daily dose of 90 mg/mouse the animal resistance to the H1N1 influenza virus increased. No interferonogenic activity after administration of epsilon-aminocaproic acid was observed in the mice.     

Reduced Virulence of an Extensively Drug-Resistant Outbreak Strain of Mycobacterium tuberculosis in a Murine Model

Bacterial drug resistance is often associated with a fitness cost. Large outbreaks of multidrug-resistant (MDR) and extensively drug-resistant (XDR) TB have been described that predominately affect persons with HIV infection. We obtained four closely-related Mycobacterium tuberculosis strains (genotype F15/LAM4/KZN) from an outbreak in KwaZulu-Natal (KZN), South Africa, including drug-sensitive, MDR, and XDR clinical isolates. We compared the virulence of these strains in a murine model of aerosol M. tuberculosis infection for four phenotypes: (1) competitive in vivo growth in lung and spleen, (2) non-competitive in vivo growth in lung and spleen, (3) murine survival time, and (4) lung pathology. When mixtures of sensitive, MDR, and XDR KZN strains were aerosolized (competitive model), lung CFUs were similar at 60 days after infection, and spleen CFUs were ordered as follows: sensitive > MDR > XDR. When individual strains were aerosolized (non-competitive model), modest differences in lung and spleen CFUs were observed with the same ordering. C57BL/6, C3H/FeJ, and SCID mice all survived longer after infection with MDR as compared to sensitive strains. SCID mice infected with an XDR strain survived longer than those infected with MDR or sensitive strains. Lung pathology was reduced after XDR TB infection compared to sensitive or MDR TB infection. In summary, increasing degrees of drug resistance were associated with decreasing murine virulence in this collection of KZN strains as measured by all four virulence phenotypes. The predominance of HIV-infected patients in MDR and XDR TB outbreaks may be explained by decreased virulence of these strains in humans.     

Comparative genomic analyses of multi-drug resistant Mycobacterium tuberculosis from Nepal and other geographical locations

Nepal exhibits a tuberculosis (TB) incidence rate that is comparable to neighbouring high TB incidence countries. In addition, it records >500 cases of multi-drug resistant (MDR) TB each year. The objective of this study was to perform whole-genome bioinformatic analysis on MDR-TB isolates from Nepal (n = 19) to identify the specific mutations underlying their phenotypic resistance. In addition, we examined the dominant genotype among the Nepal MDR-TB isolates, the East-Asian Beijing sub-lineage, to determine its relatedness to a panel of 1274 genomes of international strains available from public databases. These analyses provided evidence that the XDR-TB isolates in our collection were not derived from importation of primary XDR-TB to Nepal but were more likely the result of acquisition of second-line drug resistance in Nepal. Resistance to fluoroquinolones was detected among a high proportion of the Nepal isolates. This has implications for the management of TB, including appropriate antimicrobial stewardship and susceptibility testing for fluoroquinolones and other second-line TB drugs, to minimise the development of XDR-TB among Nepal TB cases.     

Transmission of MDR and XDR Tuberculosis in Shanghai,China

Background

Multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB) are global health problems. We sought to determine the characteristics, prevalence, and relative frequency of transmission of MDR and XDR TB in Shanghai, one of the largest cities in Asia.

Methods

TB is diagnosed in district TB hospitals in Shanghai, China. Drug susceptibility testing for first-line drugs was performed for all culture positive TB cases, and tests for second-line drugs were performed for MDR cases. VNTR-7 and VNTR-16 were used to genotype the strains, and prior treatment history and treatment outcomes were determined for each patient.

Results

There were 4,379 culture positive TB cases diagnosed with drug susceptibility test results available during March 2004 through November 2007. 247 (5.6%) were infected with a MDR strain of M. tuberculosis and 11 (6.3%) of the 175 MDR patients whose isolate was tested for susceptibility to second-line drugs, were XDR. More than half of the patients with MDR and XDR were newly diagnosed and had no prior history of TB treatment. Nearly 57% of the patients with MDR were successfully treated.

Discussion

Transmission of MDR and XDR strains is a serious problem in Shanghai. While a history of prior anti-TB treatment indicates which individuals may have acquired MDR or XDR TB, it does not accurately predict which TB patients have disease caused by transmission of MDR and XDR strains. Therefore, universal drug susceptibility testing is recommended for new and retreatment TB cases.     

Predictors of multidrug- and extensively drug-resistant tuberculosis in a high HIV prevalence community

Background

Multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB) have emerged in high-HIV-prevalence settings, which generally lack laboratory infrastructure for diagnosing TB drug resistance. Even where available, inherent delays with current drug-susceptibility testing (DST) methods result in clinical deterioration and ongoing transmission of MDR and XDR-TB. Identifying clinical predictors of drug resistance may aid in risk stratification for earlier treatment and infection control.

Methods

We performed a retrospective case-control study of patients with MDR (cases), XDR (cases) and drug-susceptible (controls) TB in a high-HIV-prevalence setting in South Africa to identify clinical and demographic risk factors for drug-resistant TB. Controls were selected in a 1∶1∶1 ratio and were not matched. We calculated odds ratios (OR) and performed multivariate logistic regression to identify independent predictors.

Results

We enrolled 116, 123 and 139 patients with drug-susceptible, MDR, and XDR-TB. More than 85% in all three patient groups were HIV-infected. In multivariate analysis, MDR and XDR-TB were each strongly associated with history of TB treatment failure (adjusted OR 51.7 [CI 6.6-403.7] and 51.5 [CI 6.4–414.0], respectively) and hospitalization more than 14 days (aOR 3.8 [CI 1.1–13.3] and 6.1 [CI 1.8–21.0], respectively). Prior default from TB treatment was not a risk factor for MDR or XDR-TB. HIV was a risk factor for XDR (aOR 8.2, CI 1.3–52.6), but not MDR-TB. Comparing XDR with MDR-TB patients, the only significant risk factor for XDR-TB was HIV infection (aOR 5.3, CI 1.0–27.6).

Discussion

In this high-HIV-prevalence and drug-resistant TB setting, a history of prolonged hospitalization and previous TB treatment failure were strong risk factors for both MDR and XDR-TB. Given high mortality observed among patients with HIV and drug-resistant TB co-infection, previously treated and hospitalized patients should be considered for empiric second-line TB therapy while awaiting confirmatory DST results in settings with a high-burden of MDR/XDR-TB.     

Efficacy of therapeutic and prophylactic actions of ultralow doses of antibodies to gamma-interferon in experimental murine model of herpes virus]

The process of the disease due to herpes simplex virus types 1 and 2 (HSV-1 and 2) was studied on white uninbred mice weighing 10 to 12 g. The animals were infected intracerebrally or intraperitoneally. Intraperitoneal contamination of the animals with MS strain of HSV-2 was used for the experimental model of the herpes simplex infection. The prophylactic antiherpes action of ultralow doses of the human gamma-interferon antibodies (ULD of anti-IFN-gamma) at a course of its intragastral administration was evaluated. The preparation was shown to have a significant (p < 0.05) protective effect in a dose of 10 LD50, evident from a 10-fold decrease of the HSV-2 accumulation in the brain, a lower percentage of the animal deaths and an increase of the average lifespan of the animals by 3.3 days. The study of the therapeutic action of ULD of anti-IFN-gamma at a course of its intragastral administration showed that the preparation had no significant positive effect on the disease process in the animals infected with HSV-2 in a dose of 10 LD50. However, a positive effect associated with delayed virus replication in the brain was observed in the study on the therapeutic effect of ULD of anti-IFN-gamma after its intragastral administration to the mice infected with a sublethal dose of the virus.     

Therapeutic effect of some antibiotics on experimental staphylococcal infection and its correlation with in vitro activity of antibiotics in sub-inhibitory concentration against Staphylococcus aureus strains

The aim of the study was to demonstrate of whether the therapeutic effects of antibiotics depend on their in vitro activity in sub-inhibitory concentrations against staphylococci. Cloxacillin, gentamicin and lincomycin were used in the study. Groups of S. aureus strains, containing 6 strains with similar MIC values each but different sensitivity to sub-inhibitory antibiotic concentrations (sub-MIC) were selected (a total of 36 trains): i. strains increasing their sensitivity to phagocytosis and bactericidal activity of rabbit leukocytes after incubation with an antibiotic in 0.1 MIC concentration, ii. strains with sensitivity to the above factors unaffected by incubation with an antibiotic in 0.5 MIC concentration. The doses of staphylococci causing death of 90-100% of Swiss albino mice 10 days after i.p. infection were determined. The injected doses (LD 90-100) and various doses of antibiotics were used to determine ED50 values as well as the survival rate of the mice with experimental staphylococcal infections after treatment with these antibiotics. It was demonstrated that effective doses (ED 50) of the antiboitics were significantly lower when the antibiotics were administered once to mice infected with strains S. aureus sensitive to sub-MIC concentrations of the investigated antibiotics than for mice infected with strains resistant to their sub-MIC concentrations. Similar correlations were observed in mice which were given the antibiotics several times (for 7 days): the percentage of the surviving mice was higher in the group infected with sub-MIC sensitive strains. The therapeutic effect of cloxacillin, gentamicin and lincomycin demonstrated a significant correlation with the S. aureus strains used to induce the infections and their sensitivity, or lack of sensitivity in vitro, to phagocytosis and bactericdal activity of leukocytes in the presence of antibiotics in sub-MIC concentrations.     

A single administration of adenoviral-mediated HGF cDNA permits survival of mice from acute hepatic failure

Heptatocyte growth factor (HGF) having a variety of biological activity was suggested as a protective agent against acute toxic hepatic injury or a potentially therapeutic agent. For the efficient in vivo application of this factor, we employed adenoviral-mediated HGF gene delivery system. In this study, we constructed E1-deleted recombinant adenovirus carrying cDNA of human HGF (Ad.hHGF) and elucidated that HGF was efficiently expressed in the liver of C57/BL mice. A mouse model of acute hepatic failure was induced by high dose (1000mg/kg) of thioacetamide (TA) administration. Mice infected with Ad.hHGF showed a dramatic resistance to TA-induced acute hepatic injury. Serum ALT was increased transiently and then the level was normalized in Ad.hHGF-infected mice with TA administration. Furthermore, the survival rate was remarkably enhanced in the mice infected with Ad.hHGF. In the histological examination, massive hepatic necrosis induced by TA was almost completely protected by HGF produced by Ad.hHGF. Our results indicate that a single dose of HGF-encoding adenoviral vector maintained liver function and prevented the progression of liver necrosis in a mouse model of acute hepatic failure.     

Comparison of different delivery systems of DNA vaccination for the induction of protection against tuberculosis in mice and guinea pigs

The great challenges for researchers working in the field of vaccinology are optimizing DNA vaccines for use in humans or large animals and creating effective single-dose vaccines using appropriated controlled delivery systems. Plasmid DNA encoding the heat-shock protein 65 (hsp65) (DNAhsp65) has been shown to induce protective and therapeutic immune responses in a murine model of tuberculosis (TB). Despite the success of naked DNAhsp65-based vaccine to protect mice against TB, it requires multiple doses of high amounts of DNA for effective immunization. In order to optimize this DNA vaccine and simplify the vaccination schedule, we coencapsulated DNAhsp65 and the adjuvant trehalose dimycolate (TDM) into biodegradable poly (DL-lactide-co-glycolide) (PLGA) microspheres for a single dose administration. Moreover, a single-shot prime-boost vaccine formulation based on a mixture of two different PLGA microspheres, presenting faster and slower release of, respectively, DNAhsp65 and the recombinant hsp65 protein was also developed. These formulations were tested in mice as well as in guinea pigs by comparison with the efficacy and toxicity induced by the naked DNA preparation or BCG. The single-shot prime-boost formulation clearly presented good efficacy and diminished lung pathology in both mice and guinea pigs.     

A population-based study of first and second-line drug-resistant tuberculosis in a high-burden area of the Mexico/United States border

The resistance of 139 Mycobacterium tuberculosis (MTB) isolates from the city of Monterrey, Northeast Mexico, to first and second-line anti-TB drugs was analysed. A total of 73 isolates were susceptible and 66 were resistant to anti-TB drugs. Monoresistance to streptomycin, isoniazid (INH) and ethambutol was observed in 29 cases. Resistance to INH was found in 52 cases and in 29 cases INH resistance was combined with resistance to two or three drugs. A total of 24 isolates were multidrug-resistant (MDR) resistant to at least INH and rifampicin and 11 MDR cases were resistant to five drugs. The proportion of MDR-TB among new TB cases in our target population was 0.72% (1/139 cases). The proportion of MDR-TB among previously treated cases was 25.18% (35/139 cases). The 13 polyresistant and 24 MDR isolates were assayed against the following seven second-line drugs: amikacin (AMK), kanamycin (KAN), capreomycin (CAP), clofazimine (CLF), ethionamide (ETH), ofloxacin (OFL) and cycloserine (CLS). Resistance to CLF, OFL or CLS was not observed. Resistance was detected to ETH (10.80%) and to AMK (2.70%), KAN (2.70%) and CAP (2.70%). One isolate of MDR with primary resistance was also resistant to three second-line drugs. Monterrey has a high prevalence of MDR-TB among previously treated cases and extensively drug-resistant-MTB strains may soon appear.     

Ability of Innate Defence Regulator Peptides IDR-1002, IDR-HH2 and IDR-1018 to Protect against Mycobacterium tuberculosis Infections in Animal Models

Tuberculosis is an ongoing threat to global health, especially with the emergence of multi drug-resistant (MDR) and extremely drug-resistant strains that are motivating the search for new treatment strategies. One potential strategy is immunotherapy using Innate Defence Regulator (IDR) peptides that selectively modulate innate immunity, enhancing chemokine induction and cell recruitment while suppressing potentially harmful inflammatory responses. IDR peptides possess only modest antimicrobial activity but have profound immunomodulatory functions that appear to be influential in resolving animal model infections. The IDR peptides HH2, 1018 and 1002 were tested for their activity against two M. tuberculosis strains, one drug-sensitive and the other MDR in both in vitro and in vivo models. All peptides showed no cytotoxic activity and only modest direct antimicrobial activity versus M. tuberculosis (MIC of 15–30 µg/ml). Nevertheless peptides HH2 and 1018 reduced bacillary loads in animal models with both the virulent drug susceptible H37Rv strain and an MDR isolate and, especially 1018 led to a considerable reduction in lung inflammation as revealed by decreased pneumonia. These results indicate that IDR peptides have potential as a novel immunotherapy against TB.