Indian Journal of Experimental Biology

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VOLUME47

NUMBER6

JUNE2009

CODEN: IJEB (A6) 47(6) 385-522(2009)

ISSN: 0019-5189

 

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CONTENTS

 

Special issue on Emerging Trends in TB, HIV and Leishmaniasis

 

Foreword

387

     Patrick J Brennan

 

 

 

Preface

389

      Pawan Sharma

 

 

 

Review Articles

 

cAMP signaling in Mycobacterium tuberculosis

393

      Nisheeth Agarwal & William R Bishai

 

 

 

Interference of Mycobacterium tuberculosis with macrophage responses

401

      Nicole Scherr, Rajesh Jayachandran, Philipp Mueller & Jean Pieters

 

 

 

Role of cholesterol in Mycobacterium tuberculosis infection

407

      Maurine D Miner, Jennifer C Chang, Amit K Pandey, Christopher M Sassetti & David R Sherman

 

 

 

Immunobiology of leishmaniasis

412

      Umakant Sharma & Sarman Singh

 

 

 

Global HIV-1 molecular epidemiology with special reference to genetic analysis of HIV-1 subtypes circulating in North India: Functional and pathogenic implications of genetic variation

424

      Ujjwal Neogi, Vikas Sood, Snigdha Banerjee, Nilanjana Ghosh, Sachin Verma, Subodh Samrat, Yogeshwar Sharma, Abhishek Saxena, Sajid Husain, V G Ramachandran, S Das, Vijesh Sreedhar K, Nidhi Goel, Ajay Wanchu & Akhil C Banerjea

 

 

 

The Yin-Yang of TNFa in the guinea pig model of tuberculosis

432

      Lan H Ly & David N McMurray

 

 

 

Mini Review

 

Potential complications to TB vaccine testing in animal models

440

      Ian M Orme

 

Perspective

 

Need for more TB vaccine field sites

445

      Helen McShane

 

 

 

Papers

 

Peripheral blood based cPCR assay for diagnosing extra-pulmonary tuberculosis

447

      Rajiv Khosla, Alka Dwivedi, B C Sarin & P K Sehajpal

 

 

 

Comparison of performance of two DNA line probe assays for rapid detection of multidrug-resistant isolates of Mycobacterium tuberculosis

454

      Suhail Ahmad, Noura M Al-Mutairi & Eiman Mokaddas

 

 

 

Evaluation of whole blood IFNγ test using PPD and recombinant antigen challenge for diagnosis of pulmonary and extra-pulmonary tuberculosis

463

      Yatiraj Kalantri, Nanda Hemvani & D S Chitnis

 

 

 

Inhalable microparticles containing isoniazid and rifabutin target macrophages and ‘stimulate the phagocyte’ to achieve high efficacy

469

      Awadh Bihari Yadav, Rolee Sharmaa, Pavan Muttil, Amit Kumar Singh, Rahul        Kumar Verma, Mradul Mohan, Sanjay Kumar Patel & Amit Misra

 

 

 

High-throughput screening of amastigotes of Leishmania donovani clinical isolates against drugs using a colorimetric β-lactamase assay

475

      Swati Mandal, Mahendra Maharjan, Sudipto Ganguly, Mitali Chatterjee,

       Sarman Singh, Frederick S Buckner & Rentala Madhubala

 

 

 

A novel calcium binding protein in Mycobacterium tuberculosis—Potential target for trifluoperazin

480

        Sunaina Koul, Aruna Somayajulu, Meeta J Advani & Hemalatha Reddy

 

 

 

Anti-IL-10 mAb protection against experimental visceral leishmaniasis via induction of Th1 cytokines and nitric oxide

489

      Surajit Bhattacharjee, Gaurav Gupta, Parna Bhattacharya, Anupam Adhikari,         Suchandra Bhattacharya Majumdar & Subrata Majumdar

 

 

 

Identification of Mycobacterium tuberculosis-specific genomic regions encoding antigens inducing protective cellular immune responses

498

        Abu Salim Mustafa & Rajaa Al-Attiyah

 

 

 

Mycobacterium tuberculosis secreted antigen (MTSA-10) inhibits macrophage response to lipopolysaccharide by redox regulation of phosphatases

505

       Sandip Kumar Basu, Dhiraj Kumar, Niladri Ganguly, Kanury Venkata Subba

        Rao & Pawan Sharma

 

 

 

Note

 

Cross-resistance of Mycobacterium tuberculosis isolates among streptomycin, kanamycin and amikacin

520

       I Sugawara, J Zhang & C Li

 

 

 

Announcements

386

 

Announcements

 

National Workshop on Proteomic Studies

20-25 July 2009, Coimbatore , India

 

Organized by the Bharathiar University , Coimbatore and co-sponsored by the Department of Biotechnology, Govt. of India, the workshop is designed for the teachers and research scholars. The workshop will focus right from simple isolation and identification of proteins to detection by Western staining and over expression in bacterial system. All these protocols will be well designed to facilitate the high throughput production of purified proteins for the purpose of high-resolution three dimensional structure analyses. Those interested may send their application on a plain paper with forwarding letter from their Head of the Department to Dr . K. Sasikala , Convener or Dr. S. Kannan, Organizing Secretary , Department of Zoology & Animal Biotechnology, Bharathiar University, Coimbatore 641 046, India. Telephone: 0422 2428108; Mobile: 097872 32032; Fax: 0422 2425706; E-mail: skanmbt2008@yahoo.in 

 

—————————

 

Biotech Research Society, India (BRSI) Annual Awards-2008

 

Nominations are invited for the following BRSI Annual Awards- 2008 of the Biotech Research Society: 1. Young Scientist award, 2. Woman Scientist award, 3. BRSI Life Time award, 4. Industrial Medal award, 5. Fellow of BRSI, and 6. AU-CBT Excellence Awards for Research Scholars. The last date to receive the nominations is 31 July 2009.

 

The nomination form and other details can be obtained from the homepage ( www.brsi.in ) of the Society or can be obtained from Prof. Ashok Pandey, President-BRSI, Biotechnology Division, National Institute for Interdisciplinary Science and Technology (formerly, R R L), CSIR, Trivandrum 695 019, India ; Telephone: 0471-251 5279; Fax 0471-2491712; E-mail: ashokpandey56@yahoo.co.in.

 

Foreword

 

 

Text Box:  I am delighted to know that National Institute of Science Communications And Information Resources (NISCAIR), a Constituent Establishment of Council of Scientific & Industrial Research (CSIR), New Delhi, India is bringing out a special issue of its premier journal the Indian Journal of Experimental Biology (IJEB) on ‘Emerging trends in TB, HIV and Leishmaniasis’ to commemorate the third International Tuberculosis (TB) Symposium (ITBS-2008), held at the International Centre for Genetic Engineering and Biotechnology (ICGEB), New Delhi, in December 2008. The Symposium, organized by Dr. Pawan Sharma of ICGEB, was a successful event with a large number of presentations made by global leaders in TB research, deliberating current trends and future research in the areas of Diagnostics, Drug Development and Vaccines. I had the privilege of attending all three meetings and witnessed a dramatic growth in research presentations on the subject from India and abroad.

 

Some of these formal presentations are included in this special issue. They provide an excellent state-of-the-art on several cutting-edge topics in patho-biology of tuberculosis, cell biology of the host-pathogen interaction, and TB vaccine development. Several research articles bring out important findings on diagnostics of tuberculosis, an area so critical for the success of various control programs and for efficient monitoring of future clinical trials of new TB drugs and vaccines. Several of these presentations also address aspects of drug development, a topic being pursued in India by the scientists and scientific administrators on account of the presence of major pharmaceutical companies and  a rich source of natural products in India.

 

It is heartening to note that this issue also carries articles from leading Indian labs working on Leishmaniasis and HIV/AIDS.

 

Therefore, this volume provides an excellent cross-section of research trends emerging from some of the leading laboratories in the world on important infectious diseases—Tuberculosis, HIV/AIDS and Leishmaniasis. I trust that this volume will be quite interesting for the researchers/scientists working on these infectious diseases.

 

Dr Patrick J Brennan

University Distinguished Professor,

Colorado State University,

Fort Collins, CO, USA

Telephone: 970 491-6700

Fax: 970 491-1815

E-mail: Patrivk.Brennan@Colostate.edu

——————————

Preface

 


Text Box:  Infectious diseases continue to inflict a heavy toll on human health in terms of both morbidity and mortality. Tuberculosis (TB) and HIV/AIDS form a deadly duo; together they continue to be the leading cause of death due to infections across the world. This special issue of the Indian Journal of Experimental Biology (IJEB) is an attempt to provide a snapshot of the current status of some of the global research efforts to mitigate human misery due to TB, HIV/AIDS and Leishmaniasis, the latter being a group of infections caused by various Leishmania species (e.g., kala azar by L. donovani, oriental sore by L. major etc.). This special issue also celebrates the success of the third International TB Symposium (ITBS-2008) held at International Centre for Genetic Engineering and Biotechnology (ICGEB), New Delhi, in December 2008, on the “Emerging Trends in TB Research: Biomarkers/Diagnostics, Drugs and Vaccines”, with generous financial support from the Bill & Melinda Gates Foundation, Aeras Global TB Vaccine Foundation, Novartis Institute of Tropical Diseases, and Astra Zeneca India.

ITBS-2008, attended by nearly 275 delegates drawn from 12 different countries, provided an opportunity to ponder over as to how far we have come and how much still remains to be done to address the daunting issues in the diagnosis, treatment and prevention of tuberculosis since the discovery of Mycobacterium tuberculosis by the great Dr. Robert Koch some 125 years ago. The articles from some of the leading researchers working on TB have been presented in this special issue, covering wide spectrum of global research efforts aimed at developing more efficient TB diagnostics, drugs and vaccine candidates. In addition, acknowledged experts in the Leishmaniasis and HIV/AIDS research fields have also come forward to contribute to this special issue. I wish to thank all the authors for contributing their significant findings/ observations/ critical commentaries on a number of emerging topical issues in the infectious disease research.

I also wish to thank the entire editorial team of IJEB for their spirited initiative and hard work to bring out this excellent number. I congratulate them on the flawless outcome of their effort, and look forward to many more special issues of IJEB benefit the biology research community.

 

 

Dr Pawan Sharma

(Guest Editor)

Scientist

Immunology Group

ICGEB, New Delhi 110 067

Telephone: +91-011-26741680

Fax: +91-011-26162316

E-mail: pawan37@gmail.com

 

 

 

Author Index

Adhikari Anupam

489

Advani Meeta J

480

Agarwal Nisheeth

393

Ahmad Suhail

454

Al-Attiyah Rajaa

498

Al-Mutairi Noura M

454

 

 

Banerjea Akhil C

424

Banerjee Snigdha

424

Basu Sandip Kumar

505

Bhattacharjee Surajit

489

Bhattacharya  Parna

489

Bishai William R

393

Brennan Patrick J

387

Buckner Frederick S

475

 

 

Chang Jennifer C

407

Chatterjee Mitali

475

Chitnis D S

463

 

 

Das S

424

Dwivedi Alka

447

 

 

Ganguly Niladri

505

Ganguly Sudipto

475

Ghosh Nilanjana

424

Goel Nidhi

424

Gupta Gaurav

489

 

 

Hemvani Nanda

463

Husain Sajid

424

 

 

Jayachandran Rajesh

401

 

 

Kalantri Yatiraj

463

Khosla Rajiv

447

Koul Sunaina

480

Kumar Dhiraj

505

 

 

Li C

520

Ly Lan H

432

 

 

Madhubala Rentala

475

Maharjan Mahendra

475

Majumdar Subrata

489

Majumdar Suchandra
Bhattacharya

489

Mandal Swati

475

McMurray David N

432

McShane Helen

445

Miner Maurine D

407

Misra Amit

469

Mohan Mradul

469

Mokaddas Eiman

454

Mueller Philipp

401

Mustafa Abu Salim

498

Muttil Pavan

469

 

 

Neogi Ujjwal

424

 

 

Orme Ian M

440

 

 

Pandey Amit K

407

Patel Sanjay Kumar

469

Pieters Jean

401

 

 

Ramachandran V G

424

Rao Kanury Venkata Subba

505

Reddy Hemalatha

480

 

 

Samrat Subodh

424

Sarin B C

447

Sassetti Christopher M

407

Saxena Abhishek

424

Scherr Nicole

401

Sehajpal P K

447

Sharma Pawan

389, 505

Sharma Umakant

412

Sharma Yogeshwar

424

Sharmaa Rolee

469

Sherman David R

407

Singh Amit Kumar

469

Singh Sarman

412, 475

Somayajulu Aruna

480

Sood Vikas

424

Sreedhar K Vijesh

424

Sugawara I

520

 

 

Verma Rahul Kumar

469

Verma Sachin

424

 

 

Wanchu Ajay

424

 

 

Yadav Awadh Bihari

469

 

 

Zhang J

520

 

 

 

 

Keyword Index

Adenylate cyclase

393

Amastigotes

475

Amikacin

520

Animal model

440

Antigenic (MTSA-10)

505

Anti-IL-10 monoclonal
antibody

489

 

 

Calcium binding protein

480

Calmodulin

480

CFP-10

463

Cholesterol

407

Competitive PCR

447

CREB

393

Cross-resistance

520

Cyclic AMP

393

 

 

Drug screening

475

 

 

ESAT-6

463

Extra- pulmonary

 

tuberculosis

447, 463

 

 

Foxp3+ cells

440

 

 

GenoType MTBDR

454

Guinea pig

432

 

 

HIV/AIDS epidemic

424

IFN-g

463, 498

Δigr

407

IL-10

498

Immune defence

401

Immunobiology

412

Immunomodulation

412

Indian clinical isolates

475

Inhalable microparticles

469

INNO-LiPA Rif . TB

454

Isoniazid

469

 

 

Kanamycin

520

 

 

β-lactamase assay

475

Leishmania donovani

475

Leishmaniasis

412, 489

Line probe assays

454

Lipid metabolism

407

Lipopolysaccharide

505

Lung necrosis

440

 

 

Macrophage response

401

Macrophage

412, 469, 505

mce4

407

MDR-TB

520

Molecular epidemiology

424

Multidrug resistance

454

Mycobacterium

 

Tuberculosis

393, 401, 407, 432, 445, 447, 454, 469, 480, 498, 505

 

 

NAD-kinase

480

Nickel affinity column

480

Nitric oxide

489

Non-tubercular mycobacteria

463

 

 

PBMCs

498

PCR

447

Phagocyte

469

Phosphatase

505

Phosphodiesterase

393

PPD

463

 

 

RD antigens

498

Redox regulation

505

Rifabutin

469

 

 

Signal transduction

393

Streptomycin

520

 

 

T cell

440

TB

463, 469

TB vaccine

440

T-cell response

412

Th1/Th2 cells

489

TNFa

432

Trifluoperazine

480

 

 

Vaccines

445

Virulence

393

 

 

 

 

 

Correspondent author has been indicated by * sign

 

 

Review Articles

 

Indian Journal of Experimental Biology

Vol. 47, June 2009, pp 393-400

 

 

cAMP signaling in Mycobacterium tuberculosis

Nisheeth Agarwal & William R Bishai*

Departtment of Medicine, Johns Hopkins School of Medicine, CRB2, Rm 1.08,
1550 Orleans Street, Baltimore, Maryland 21231-1044, USA

 

cAMP is an important second messenger in both eukaryotic and prokaryotic organisms. Several bacterial pathogens have developed mechanisms to subvert eukaryotic cAMP signaling by injecting protein toxins that are themselves adenylate cyclases or by introducing toxins that modify host adenylate cyclases to an overexpression state. Curiously, Mycobacterium tuberculosis CDC1551 genome contains seventeen adenylate cyclase homologues suggesting that cAMP signaling is both relevant and complex in biology of M. tuberculosis. The present article provides an overview of the role of cAMP as a second messenger, discusses bacterial cAMP subversion mechanisms, and reviews the evidence currently available on cAMP-based signaling in M. tuberculosis.

 

Indian Journal of Experimental Biology

Vol. 47, June 2009, pp 401-406

 

 

 

Interference of Mycobacterium tuberculosis with macrophage responses

Nicole Scherr, Rajesh Jayachandran, Philipp Mueller & Jean Pieters*

Biozentrum, University of Basel, Klingelbergstrasse 50, 4056 Basel, Switzerland

 

Tuberculosis, caused by Mycobacterium tuberculosis, has become an important health and economic burden, with more than four thousand people succumbing to the disease every day. Thus, there is an urgent need to understand the molecular basis of this pathogen’s success in causing disease in humans, in order to develop new drugs superior to conventional drugs available at present. One reason why M. tuberculosis is such a dangerous microbe lies within its ability to survive within infected hosts, thereby efficiently circumventing host immune responses. Over the past few years, a number of mechanisms have been unravelled that are utilized by M. tuberculosis to survive within hosts and to avoid immune defence mechanisms. Several of these mechanisms have been described in this communication that may be useful for the development of novel compounds to treat tuberculosis.

 

 

Indian Journal of Experimental Biology

Vol. 47, June 2009, pp 407-411

 

 

 

 

Role of cholesterol in Mycobacterium tuberculosis infection

Maurine D Miner1, Jennifer C Chang1,2, Amit K Pandey3, Christopher M Sassetti3 & David R Sherman*1,4

1Seattle Biomedical Research Institute, Seattle, WA 98109, USA

2Center for Pharmaceutical Biotechnology, University of Illinois at Chicago, Chicago, IL 60607, USA

3Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, MA 01655, USA

4Interdisciplinary Program in Pathobiology, Department of Global Health, University of Washington, Seattle, WA 98195, USA

Mycobacterium tuberculosis (MTB) acquisition and utilization of nutrients within the host cell is poorly understood, although it has been hypothesized that host lipids probably play an important role in MTB survival. Cholesterol has recently been identified as an important lipid for mycobacterial infection. The mce4 transport system is required for cholesterol import into bacterial cells, and deletion of mce4 locus resulted in severe attenuation in a chronic mouse model of infection. However, it has remained unclear what additional bacterial functions were required for utilization of this sterol. We have found that the igr locus, which was previously found essential for intracellular growth and virulence of MTB, is required for cholesterol metabolism: igr-deficient bacteria cannot grow using cholesterol as a primary carbon source. The growth-inhibitory effect of cholesterol in vitro depends on cholesterol import, as the Δigr mutant growth defect during the early phase of disease is completely suppressed by mutating mce4, implicating cholesterol intoxication as the primary mechanism of attenuation. We conclude that M. tuberculosis metabolizes cholesterol throughout the course of infection, and that degradation of this sterol is crucial for bacterial persistence.

 

Indian Journal of Experimental Biology

Vol. 47, June 2009, pp 412-423

 

 

 

Immunobiology of leishmaniasis

Umakant Sharma & Sarman Singh*

Division of Clinical Microbiology, Department of Laboratory Medicine,
All India Institute of Medical Sciences, New Delhi 110 029, India

Leishmaniasis is a parasitic disease caused by various species of Leishmania, a unicellular kinetoplastid protozoan flagellate. It manifests mainly in 3 clinical forms; visceral leishmaniasis (VL), cutaneous leishmaniasis (CL) and mucocutaneous leishmaniasis (MCL), of which VL is the most severe form of the disease. VL is lethal if untreated and spontaneous cure is extremely rare. Cutaneous leishmaniasis usually has milder course and often results into a self-healing of ulcers. Resolution of leishmanial infection is dependent on the coordinated interactions between components of cell mediated immune response, specifically the activation of targeted T-cell populations for appropriate cytokine production and activation of macrophages. In murine model, the development of Th1 response is associated with control of infection, and Th2 response is associated with disease progression. However, Thl and Th2 dichotomy in the human system is not as distinct as in mice and the murine model does not strictly apply to human leishmaniasis. This review focuses the dichotomy of immune response against various clinical forms of the disease. An in-depth knowledge of sequences involved in the immune response to the parasite would help in designing prophylactic and therapeutic strategies against leishmaniasis.

 

Indian Journal of Experimental Biology

Vol. 47, June 2009, pp 424-431

 

 

 

Global HIV-1 molecular epidemiology with special reference to genetic analysis of HIV-1 subtypes circulating in North India: Functional and pathogenic implications of genetic variation

aUjjwal Neogi aVikas Sood, aSnigdha Banerjee, aNilanjana Ghosh, aSachin Verma, aSubodh Samrat,
aYogeshwar Sharma, aAbhishek Saxena, bSajid Husain, cV G Ramachandran, cS Das, dVijesh Sreedhar K,
dNidhi Goel, dAjay Wanchu & aAkhil C Banerjea*

aLaboratory of Virology II, National Institute of Immunology, New Delhi 110 067, India

bGuru Nanak Dev University, Amritsar 143 005, India

cUniversity College of Medical Sciences & GTB Hospital, Delhi 110 097, India

dPost Graduate Institute of Medical Education and Research (PGIMER), Chandigarh 160 012, India

HIV-1 displays extensive genetic diversity globally which poses challenge in designing a suitable antigen/immunogen to provoke desired protective immune response in host. HIV-1 mediated pathogenesis is complex and involves host genes, virus genes and other factors. A number of genetic subtypes have been identified based on sequence variations, largely in envelope region. Different genetic subtypes display variation in amino acid sequences with increasing incidence of subtype B, C, D and mosaic recombinants in India. They can potentially alter the functions of several proteins like Rev, Tat ,Vpr, Vif etc and thereby, influence HIV-1 mediated pathogenesis. Recent study has shown that LTR promoter region exhibits novel mosaic structures with segments from B/C Myanmar and India. This indicates rapid evolving nature of HIV-1 and causing epidemics due to existence of multiple subtypes in Indian region. These multiple subtypes show significant differences in various functions (gene activation, cell cycle arrest, RNA binding activities) compared to prototype subtype B genes. These differences may help in better understanding of unique features of HIV-1 epidemic in India.

 

Indian Journal of Experimental Biology

Vol. 47, June 2009, pp 432-439

 

 

  

The Yin-Yang of TNFa in the guinea pig model of tuberculosis

Lan H Ly & David N McMurray

Department of Microbial and Molecular Pathogenesis, Texas A & M System Health Science Center, College Station,
Texas, 77843 1114, USA

 

Tumour necrosis factor alpha (TNF-a) is a prototypic pro-inflammatory cytokine that has a central role in the initial host response to Mycobacterium tuberculosis infection. It is a key player in granuloma formation, macrophage activation, bacterial killing, and pathology in vivo. However, the exact mechanism has not been completely understood. This review summarizes the TNFa data acquired from the ‘gold standard’ guinea pig animal model of tuberculosis. While production of TNFa is widely accepted as beneficial to the host response, we have found that this hypothesis is just one side of the story. TNFa can up-regulate and down-regulate some key pro-inflammatory cytokines (IFNg, IL-12p40) and differentially modulate macrophage activation and intracellular bacterial growth. Neutralization of TNFa in vivo allows an anti-inflammatory TGFb-mediated response to develop. Furthermore, BCG vaccination modulates TNFa responses directly in the pulmonary granulomas to reduce tissue damage. The bipolar nature of TNFa should be considered as knowledge of this critical molecule continues to grow.

 

 

 

 

Mini Review

 

 

Indian Journal of Experimental Biology

Vol. 47, June 2009, pp 440-444

 

 

 

Potential complications to TB vaccine testing in animal models

Ian M Orme

Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins CO 80523, USA

 

Testing of new vaccines in animal models has certain advantages and disadvantages. As we better understand the complexity of the immune response to vaccines, new information may be complicating the assessment of the efficacy of new candidate vaccines. Four possible complications are discussed here, (i) induction of Foxp3+ T cells; (ii) induction of memory T cell subsets; (iii) location of extracellular organisms in lung necrosis; and (iv) protection against isolates of high/extreme immunopathology.

 

 

Perspective

 

Indian Journal of Experimental Biology

Vol. 47, June 2009, pp 445-446

  

 

Need for more TB vaccine field sites

 Helen McShane

Reader in Vaccinology and Wellcome Senior Clinical Fellow, The Jenner Institute, Old Road Campus Research Building,
Roosevelt Drive, Oxford OX3 7DQ

Efforts to control the tuberculosis (TB) epidemic have been challenged by both the geographical overlap with the HIV pandemic, and the emergence of multi - and extensively - drug-resistant strains of Mycobacterium tuberculosis. There is, therefore, an urgent global need for an improved vaccine. However, the development of an improved vaccine is scientifically and logistically challenging. Immunological correlates or biomarkers of protection are not known and there is no perfect preclinical animal model with which to predict success in humans. Indeed, vaccine development in general is time-consuming and costly. One of the many road-blocks to the development of new TB vaccines is the availability of field sites that are suitable for large scale Phase IIb/III efficacy testing. Because disease incidence is low, even though prevalence is high, Phase IIb efficacy trials involve several thousand subjects, and require lengthy follow-up. Phase III licensure trials will need to be even larger, and are likely to require the involvement of multiple field sites. There is currently inadequate capacity within high-burden TB countries to conduct these essential trials. We need to invest now to expand current capacity if we are to reduce the time taken to develop new vaccines.

 

 

 

Papers

 

 

Indian Journal of Experimental Biology

Vol. 47, June 2009, pp 447-453

 

 

 

Peripheral blood based C-PCR assay for diagnosing extra-pulmonary tuberculosis

Rajiv Khosla1a, Alka Dwivedi1b, B C Sarin2 & P K Sehajpal1*

1 Department of Molecular Biology and Biochemistry, Guru Nanak Dev University, Amritsar 143 005, India
2Department of Tuberculosis and Chest Diseases,
Sri Guru Ram Das Institute of Medical Sciences and Research, Amritsar 143 005, India

Received 8 February 2009

Extra pulmonary tuberculosis (EPTB) constitutes around 20% of all tuberculosis cases in India. Conventional methods are of limited use in diagnosing this form of the disease. Polymerase chain reaction (PCR) has emerged as a sensitive and specific tool for documenting the presence of Mycobacterium tuberculosis in clinical samples but lacks quantitative ability. The present study evaluates peripheral blood as an alternative clinical specimen for diagnosing EPTB. Peripheral blood samples from 38 EPTB and 89 non tuberculous subjects were analyzed for the presence of tubercle bacilli by MPB 64 gene based PCR method. The assay gave an overall sensitivity of 60.53% with negative predictive value of 76.92% which is superior to present gold standard of mycobacterial culture (10.53 and 72.36%). Additionally, 43.82% of non tuberculous subjects gave positive results with the PCR, thus mitigating the clinical utility of this test. An in-house Competitive PCR (C-PCR) assay was used to determine the mycobacterial load in peripheral blood from culture positive, culture negative EPTB patients and non tuberculous controls which ranged from 7498 – 12498, 602 – 4797 and 101 – 800 genome equivalent (ge)/mL, respectively. The data clearly demonstrated that C-PCR assay can furnish insightful information in diagnosing extra pulmonary disease.

 

Indian Journal of Experimental Biology

Vol. 47, June 2009, pp 454-462

 

 

  

Comparison of performance of two DNA line probe assays for rapid detection of multidrug-resistant isolates of Mycobacterium tuberculosis

Suhail Ahmad*, Noura M Al-Mutairi & Eiman Mokaddas

Department of Microbiology, Faculty of Medicine, Kuwait University, P.O. Box 24923, Safat, 13110, Kuwait

Received 18 December 2008

Infections with multidrug-resistant (resistant at least to rifampicin, RIF and isoniazid, INH) strains of Mycobacterium tuberculosis (MDR-TB) are associated with high case fatality rates. Rapid identification of MDR-TB strains is important for early institution of appropriate therapy. Two DNA line probe assays, GenoType MTBDR (GT-MTBDR) and INNO-LiPA Rif. TB (INNO-LiPA) were compared for their abilities to detect resistance to INH and RIF in 80 M. tuberculosis isolates. The test results were compared to those obtained by conventional drug susceptibility testing (DST), DNA sequencing and/or PCR-restriction fragment length polymorphism (RFLP) analysis of regions of interest of M. tuberculosis genome. Compared to the DST and katG codon 315 PCR-RFLP results, GT-MTBDR test results were concordant for INH resistance for 63 of 80 (78.7%) isolates. For RIF resistance, GT-MTBDR and INNO-LiPA test results were concordant with DST for 74 of 80 (92.5%) and 76 of 80 (95%) strains, respectively. The GT-MTBDR test results correlated with sequencing results for 77 of 80 (96.2%) while INNO-LiPA results for 79 of 80 (98.7%) isolates. Both the tests are useful for rapid detection of MDR-TB strains, however, GT-MTBDR assay offers the advantage of detecting the resistance to both INH and RIF simultaneously when MDR-TB is suspected.

 

Indian Journal of Experimental Biology

Vol. 47, June 2009, pp 463-468

 

  

Evaluation of whole blood IFNγ test using PPD and recombinant antigen challenge for diagnosis of pulmonary and extra-pulmonary tuberculosis

Yatiraj Kalantri, Nanda Hemvani & D S Chitnis*

Department of Microbiology and Immunology, Choithram Hospital and Research Centre, Indore 452 014, India

Received 23 January 2009

Quantiferon TB gold (QFT-G) with recombinant antigen cocktail is well evaluated for diagnosis of pulmonary tuberculosis (PTB). However, diagnosis of extra-pulmonary tuberculosis (EPTB) is more difficult due to limitations of conventional techniques. This study compares recombinant antigens based QFT-G and low cost PPD based interferon test for the diagnosis of PTB and EPTB. IFNγ release, with recombinant antigens and PPD, was assayed by ELISA from 140 cases of EPTB, 100 cases of PTB along with acid fast bacillus (AFB) detection, AFB culture on LJ and MGIT BACTEC. Sensitivity and specificity for QFT-G recombinant antigens was 84.29% and 96%, while for PPD based interferon was 70% and 84% for EPTB group. The sensitivity was far superior to AFB smear and culture for both the antigens. Nine samples were identified as non-tubercular mycobacteria (NTM) in the EPTB group and all were negative for QFT-G, but six of them were positive for PPD based test. Results of the study show that QFT-G using recombinant antigen is sensitive and specific for both PTB and EPTB diagnosis. The PPD based test is economic and offers comparable performance for PTB and EPTB diagnosis and also useful for diagnosis of NTM.

Indian Journal of Experimental Biology

Vol. 47, June 2009, pp 469-474

 

  

Inhalable microparticles containing isoniazid and rifabutin target macrophages and ‘stimulate the phagocyte’ to achieve high efficacy

Awadh Bihari Yadav, Rolee Sharmaa, Pavan Muttilb, Amit Kumar Singh, Rahul Kumar Verma, Mradul Mohan, Sanjay Kumar Patel & Amit Misra*

Pharmaceutics Division, Central Drug Research Institute, Lucknow 226 001, India

Received 3 February 2009

Macrophage responses to infection with Mycobacterium tuberculosis (MTB) and treatment with soluble isoniazid (INH) plus rifabutin (RFB) versus microparticles containing equivalent amounts of drugs were compared. It was investigated whether macrophages driven to alternative activation upon infection with MTB could be rescued to display the classical activation phenotype. It was established that microparticles sustain high levels of drugs in cytosol of macrophages for longer period as compared to soluble drugs. Microparticles co-localized with intracellular bacteria, and induced a variety of innate bactericidal responses, including induction of free radicals, alteration of mitochondrial membrane potential and apoptosis. The data strongly suggest that additional benefit may be derived from the nature of the drug delivery system, which fulfils Koch’s dictum ‘stimulate the phagocyte’ for curing tuberculosis.

 

Indian Journal of Experimental Biology

Vol. 47, June 2009, pp 475-479

 

 

 

High-throughput screening of amastigotes of Leishmania donovani clinical isolates against drugs using a colorimetric β-lactamase assay

Swati Mandal1, Mahendra Maharjan1, Sudipto Ganguly2, Mitali Chatterjee2, Sarman Singh3,
Frederick S Buckner4 & Rentala Madhubala1*

1School of Life Sciences, Jawaharlal Nehru University, New Delhi 110 067, India

2Institute of Post Graduate Medical Education and Research, Kolkata 700 020, India

3Department of Laboratory Medicine, All India Institute of Medical Sciences, New Delhi 110 029, India

4Department of Medicine, University of Washington, Seattle, Washington

Received 22 January 2009

A simple colorimetric β-lactamase assay for quantifying Leishmania amastigotes in macrophages grown in microtiter plates has been reported. The β-lactamase gene was integrated into the rRNA region of the genome, thereby allowing for high-level stable expression of the enzyme. Both visceral leishmaniasis (VL) and post-kala azar dermal leishmaniasis (PKDL) isolates were transfected with β-Lactamase gene. These β-lactamase-expressing promastigotes were used for infecting intracellular J774A.1 macrophages in vitro. Quantification was done by a colorimetric readout with CENTATM β-lactamase as substrate and with an optical density plate reader. The assay was carried out in 96-well plates. Results obtained demonstrate that this methodology could be a valuable high-throughput screening assay for checking efficacy of anti-leishmanial drugs in the clinical isolates.

 

Indian Journal of Experimental Biology

Vol. 47, June 2009, pp 480-488

 

  

A novel calcium binding protein in Mycobacterium tuberculosis—Potential target for trifluoperazine

Sunaina Koul, Aruna Somayajulu, Meeta J Advani & Hemalatha Reddy*

Department of Biochemistry, Sri Venkateswara College, Dhaula Kuan, New Delhi 110 021, India

Received 22 January 2009

Phenothiazines have been reported for anti-mycobacterial activity by inhibiting calcium binding proteins, potassium transport processes of phagolysosomes, NADH dependent oxygen consumption by M. tuberculosis membranes and DNA, and lipid synthesis of the bacterium. Thioridazine (TZ), chloropromazine (CPZ) and trifluoperazine (TFP) belong to the class of phenothiazines widely used as neuroleptic drugs. Trifluoperazine, a calmodulin antagonist in eukaryotes, binds to a similar protein containing prototypical EF hand to bind to calcium in M. tuberculosis. Calmodulin, a calcium binding protein, plays a critical role in regulating the activities of several enzymes in response to intracellular calcium levels. Since calmodulins are best characterized in eukaryotes as opposed to prokaryotes, the presence of calmodulin-like activity in M. tuberculosis, the causative agent of tuberculosis, is unknown. We have provided biochemical evidence that M. tuberculosis recombinant (r) Rv1211 gene product stimulates the activities of heterologous calcium-deficient NAD-kinase and bovine brain phosphodiesterase (PDE), much like the eukaryotic calmodulins. Further we have shown that EGTA, a calcium chelator, inhibits rRv1211-stimulated NAD-kinase and PDE activities. We have also shown that trifluoperazine interferes with the activation of NAD-kinase and PDE activities by Rv1211. Using a bioinformatics approach, we have shown that Rv1211 contains one prototypical calcium-binding EF-hand motif, a characteristic feature of calmodulins. Based on these data, we conclude that Rv1211 encodes a protein with calmodulin-like activity (CAMLP) in the human pathogen M. tuberculosis and acts as a potential target for trifluoperazine.

 

Indian Journal of Experimental Biology

Vol. 47, June 2009, pp 489-497

 

  

Anti-IL-10 mAb protection against experimental visceral leishmaniasis via induction of Th1 cytokines and nitric oxide

Surajit Bhattacharjee, Gaurav Gupta, Parna Bhattacharya, Anupam Adhikari,
Suchandra Bhattacharya Majumdar & Subrata Majumdar*

Division of Molecular Medicine, Bose Institute, Kolkata, 700 032, India

Received 6 February 2009

Visceral leishmaniasis is characterized by severe immune suppression of the host. This suppression of the host immune system is primarily mediated by the immunosuppressive cytokine Interleukin-10 (IL-10), whose levels are significantly up-regulated during leishmaniasis. This immune suppression is reflected at the level of T-cell dysfunction and abrogation of leishmaniacidal molecules along with a dampened Th1 cytokine response. In the present study, we showed in vivo neutralization of IL-10 by administration of anti IL-10 monoclonal antibodies (mAb) could confer protection against leishmanial pathogenesis. This protective response was primarily mediated by a strong induction of T cell proliferation along with a Th1 biased cytokine response which was further aided by the generation of, leishmanicidal molecules, nitric oxide.

 

Indian Journal of Experimental Biology

Vol. 47, June 2009, pp 498-504 

 

 

Identification of Mycobacterium tuberculosis-specific genomic regions encoding antigens inducing protective cellular immune responses

Abu Salim Mustafa* & Rajaa Al-Attiyah

Department of Microbiology, Faculty of Medicine, Kuwait University, Kuwait

Received 4 January 2009

Comparative genomic studies have identified 11 regions of difference (RD1, RD4, RD5, RD6, RD7, RD9, RD10, RD11, RD12, RD13 and RD15) in Mycobacterium tuberculosis genome which are absent in all vaccine strains of M. bovis BCG. The proteins encoded by genes predicted in these RDs could be useful as protective vaccines and/or exacerbate the disease process by inducing cellular immune responses involved in protection and pathogenesis of tuberculosis. In our studies, by using pools of overlapping synthetic peptides covering the sequence of putative proteins encoded by genes predicted in each RD, we have determined the cellular immune responses in relation to antigen-induced proliferation and secretion of the protective Th1 cytokine IFN-g and the pathologic Th2 cytokine IL-10 by peripheral blood mononuclear cells of tuberculosis patients and healthy humans. It has been observed that peptides of RD1pool induced the highest antigen-induced proliferation and IFN-g responses, whereas the peptides of RD12pool and RD13pool induced the highest IL-10 responses. Furthermore, addition of RD12pool and RD13pool to peripheral blood mononuclear cells (PBMCs) cultures inhibited the RD1pool-induced secretion of IFN-g by PBMCs of healthy humans. These results suggest the relevance of RD1-encoded proteins in protection and RD12- and RD13-encoded proteins in pathogenesis of tuberculosis.

 

Indian Journal of Experimental Biology

Vol. 47, May 2009, pp 505-519

 

 

 Mycobacterium tuberculosis secreted antigen (MTSA-10) inhibits macrophage response to lipopolysaccharide by redox regulation of phosphatases

 

Sandip Kumar Basu1, Dhiraj Kumar, Niladri Ganguly2, Kanury Venkata Subba Rao & Pawan Sharma*

Immunology Group, International Centre for Genetic Engineering and Biotechnology,
ICGEB Campus, Aruna Asaf Ali Marg, New Delhi 110 067, India.

Received 14 January 2009

The present study was undertaken to investigate the possible role of a 10-kDa, secretory antigenic protein of Mtb (MTSA-10) in regulating macrophase response to lipopolysacchride (LPS). MTSA-10 inhibited the lipopolysaccharide (LPS)-induced oxidant species generation in the macrophage. Treatment of macrophages with MTSA-10 activated their protein tyrosine phosphatases (PTPs) in a redox-regulated fashion. These activated phosphatases then interfered with the early events of LPS signaling and lower the strength and magnitude of the signal generated, thereby preventing macrophages from making an effective immune response. Mycobacterium tuberculosis Region of Deletion-1 (RD-1)-specific secretory antigen MTSA-10 (encoded by ORF Rv3874 of Mtb genome) modulated the macrophage signaling machinery and prevented it from responding to further activation by LPS.

 

 

 

Notes

 

  

Indian Journal of Experimental Biology

Vol. 47, June 2009, pp 520-522

 

  

Cross-resistance of Mycobacterium tuberculosis isolates among streptomycin, kanamycin and amikacin

I Sugawara1*, J Zhang2 & C Li2

1Mycobacterial Reference Center, The Research Institute of Tuberculosis, 3-1-24 Matsuyama, Kiyose, Tokyo 204-0022, Japan

2Beijing Tuberculosis and Lung Tumor Research Institute, Beijing, China

Received 8 December 2008

Seventy-four streptomycin (SM)-resistant M. tuberculosis clinical isolates were subjected to cross-resistance drug testing against two major aminoglycosides, kanamycin (KM) and amikacin (AMK). Among them, 15 clinical isolates (20.3%) were resistant to both KM and AMK. Fifteen (80%) of 19 KM-resistant isolates were AMK-resistant. Fifteen SM, KM, and AMK resistant isolates harbored rrs mutation, but only two had rrs and rpsL double mutations. Low-level SM resistance was associated with rpsL mutation, whereas high-level SM resistance was linked to rrs mutation.