Quantitative detection of mycobacterial mannophosphoinositides in tuberculosis patients by real-time immuno-PCR assay.

A real-time immuno-PCR assay was deliberated to detect mycobacterial mannophosphoinositides (PIMs). A dynamic range of PIMs (0.9 pg/mL-10 ng/mL) was detected in TB patients, wherein 88.2% and 81.1% sensitivities were obtained in pulmonary TB and extrapulmonary TB respectively, with 96-96.4% specificity. This assay may translate into a diagnostic kit.

Streptomycin sulphate loaded solid lipid nanoparticles show enhanced uptake in macrophage, lower MIC in Mycobacterium and improved oral bioavailability.

Present study addresses the challenge of incorporating hydrophilic streptomycin sulphate (STRS; log P -6.4) with high dose (1 g/day) into a lipid matrix of SLNs. Cold high-pressure homogenization technique used for SLN preparation achieved 30% drug loading and 51.17 ± 0.95% entrapment efficiency. Polyethylene glycol 600 as a supporting-surfactant assigned small size (218.1 ± 15.46 nm) and mucus-penetrating property. It was conceived to administer STRS-SLNs orally rather than intramuscularly. STRS-SLNs remained stable on incubation for varying times in SGF or SIF. STRS-SLNs were extensively characterised for microscopic (TEM and AFM), thermal (DSC), diffraction (XRD) and spectroscopic (NMR and FTIR) properties and showed zero-order controlled release. Enhanced (60 times) intracellular uptake was observed in THP-1 and Pgp expressing LoVo and DLD-1 cell lines, using fluorescein-SLNs. Presence of SLNs in LoVo cells was also revealed by TEM studies. STRS-SLNs showed 3 times reduction in MIC against Mycobacterium tuberculosis H37RV (256182) in comparison to free STRS. It also showed better activity against both M. bovis BCG and Mycobacterium tuberculosis H37RV (272994) in comparison to free STRS. Cytotoxicity and acute toxicity studies (OECD 425 guidelines) confirmed in vitro and in vivo safety of STRS-SLNs. Single-dose oral pharmacokinetic studies in rat plasma using validated LCMS/MS technique or the microbioassay showed significant oral absorption and bioavailability (160% - 710% increase than free drug).

Preclinical Explorative Assessment of Celecoxib-Based Biocompatible Lipidic Nanocarriers for the Management of CFA-Induced Rheumatoid Arthritis in Wistar Rats.

Celecoxib (CXB), a COX-2 inhibitor, is primarily indicated for long-term treatment of rheumatoid arthritis (RA). The effective therapeutic efficacy of CXB on RA via oral administration shows adverse systemic complications, and therefore, local application of CXB has been recommended. The aim of the present study was to develop and characterize solid lipid nanoparticles (SLNs) with enhanced skin permeation potential of CXB. The particle size, polydispersity index (PDI), and percentage drug entrapment (PDE) of the developed SLNs (CXB-SLNs) were found to be 240 nm, < 0.3, and ~ 86% respectively. The developed SLNs exhibited sustained release up to 70% at the end of 48 h. Drug permeation was found to be 45% for SLN gel and 31% for conventional gel. The dermatokinetic studies also confirmed enhanced permeation of CXB in the epidermis and dermis and revealed superiority of the developed SLN gel vis-à-vis the conventional gel. Further, in the CFA-induced arthritis rat model, % arthritis index (AI) of the CXB-SLN gel formulation was found to be very less (18.54%) as compared to untreated (187.34%) and conventional gel-treated (91.61%) animals. In conclusion, the current study can provide a suitable alternative for the development of an effective topical formulation of CXB in lipid nanocarriers.

Serodiagnostic potential of immuno-PCR using a cocktail of mycobacterial antigen 85B, ESAT-6 and cord factor in tuberculosis patients.

A novel indirect immuno-polymerase chain reaction (I-PCR) assay was developed for the detection of circulating anti-Ag85B (antigen 85B, Rv1886c), anti-ESAT-6 (early secretory antigenic target-6, Rv3875) and anti-cord factor (trehalose 6,6'-dimycolate) antibodies from the sera samples of pulmonary tuberculosis (PTB) and extrapulmonary tuberculosis (EPTB) patients and the results were compared with an analogous enzyme-linked immunosorbent assay (ELISA). We covalently attached the amino-modified reporter DNA to the dithiothreitol (DTT)-reduced anti-human IgG antibody through a chemical linker succinimidyl 4-[N-maleimidomethyl]-cyclohexane-1-carboxylate (SMCC). The detection of cocktail of anti-Ag85B, anti-ESAT-6 and anti-cord factor antibodies was found to be superior to the detection of individual antibodies. The sensitivities of 89.5% and 77.5% with I-PCR and 70.8% and 65% with ELISA were observed in smear-positive and smear-negative PTB cases, respectively with high specificity (90.9%). On the other hand, a sensitivity of 77.5% with I-PCR and 65% with ELISA was observed in EBTB cases. The detection of cocktail of antibodies by I-PCR is likely to improve the utility of existing algorithms for TB diagnosis.

Comparative Evaluation of Several Gene Targets for Designing a Multiplex-PCR for an Early Diagnosis of Extrapulmonary Tuberculosis.

PURPOSE: Diagnosis of extrapulmonary tuberculosis (EPTB) poses serious challenges. A careful selection of appropriate gene targets is essential for designing a multiplex-polymerase chain reaction (M-PCR) assay. MATERIALS AND METHODS: We compared several gene targets of Mycobacterium tuberculosis, including IS6110, devR, and genes encoding MPB-64 (mpb64), 38kDa (pstS1), 65kDa (hsp65), 30kDa (fbpB), ESAT-6 (esat6), and CFP-10 (cfp10) proteins, using PCR assays on 105 EPTB specimens. From these data, we chose the two best gene targets to design an M-PCR. RESULTS: Among all gene targets tested, mpb64 showed the highest sensitivity (84% in confirmed cases and 77.5% in clinically suspected cases), followed by IS6110, hsp65, 38kDa, 30kDa, esat6, cfp10, and devR. We used mpb64+IS6110 for designing an M-PCR assay. Our M-PCR assay demonstrated a high sensitivity of 96% in confirmed EPTB cases and 88.75% in clinically suspected EPTB cases with a high specificity of 100%, taking clinical diagnosis as the gold standard. CONCLUSION: These M-PCR results along with the clinical findings may facilitate an early diagnosis of EPTB patients and clinical management of disease.

Diagnosis of pulmonary and extrapulmonary tuberculosis based on detection of mycobacterial antigen 85B by immuno-PCR.

We developed a novel indirect sandwich immuno-polymerase chain reaction (I-PCR) assay for the detection of mycobacterial antigen 85B (Ag85B, 30kDa, Rv1886c) in pulmonary tuberculosis (PTB) and extrapulmonary tuberculosis (EPTB) patients. The amino-modified reporter DNA was covalently attached with the antidetection antibody through a heterobifunctional cross-linking agent succinimidyl 4-[N-maleimidomethyl]-cyclohexane-1-carboxylate. The detection limit of Ag85B by I-PCR was found to be 1 femtogram (fg)/mL, which was 10(6)-fold lower than an analogous enzyme-linked immunosorbent assay (ELISA). The sensitivities of 85% and 77% with I-PCR and 77.6% and 62.5% with ELISA were observed in smear-positive and smear-negative PTB patients, respectively, with high specificity. On the other hand, sensitivities of 84% and 63.7% with I-PCR and 68% and 47.5% with ELISA were observed in confirmed and clinically suspected EPTB cases, respectively, with high specificity.

Detection of potential microbial antigens by immuno-PCR (PCR-amplified immunoassay).

Immuno-PCR (PCR-amplified immunoassay; I-PCR) is a novel ultrasensitive method combining the versatility of ELISA with the sensitivity of nucleic acid amplification of PCR. The enormous exponential amplification power of PCR in an I-PCR assay leads to at least a 10(2)-10(4)-fold increase in sensitivity compared with an analogous ELISA. I-PCR has been used to detect many biological molecules such as proto-oncogenes, toxins, cytokines, hormones, and biomarkers for autoimmune and Alzheimer's diseases, as well as microbial antigens and antibodies, and it can be adapted as a novel diagnostic tool for various infectious and non-infectious diseases. Quantitative real-time I-PCR has the potential to become the most analytically sensitive method for the detection of proteins. The sensitivity and specificity of a real-time I-PCR assay can be enhanced further with the use of magnetic beads and nanoparticles. This review is primarily focused on the detection of potential viral, bacterial and parasitic antigens by I-PCR assay, thus enabling their application for immunological research and for early diagnosis of infectious diseases.

Poly(lactide-co-glycolide)-rifampicin nanoparticles efficiently clear Mycobacterium bovis BCG infection in macrophages and remain membrane-bound in phago-lysosomes.

Nanoparticles (NPs) are increasingly used as biodegradable vehicles to selectively deliver therapeutic agents such as drugs or antigens to cells. The most widely used vehicle for this purpose is based on copolymers of lactic acid and glycolic acid (PLGA) and has been extensively used in experiments aimed at delivering antibiotics against Mycobacterium tuberculosis in animal models of tuberculosis. Here, we describe fabrication of PLGA NPs containing either a high concentration of rifampicin or detectable levels of the green fluorescent dye, coumarin-6. Our goal here was twofold: first to resolve the controversial issue of whether, after phagocytic uptake, PLGA NPs remain membrane-bound or whether they escape into the cytoplasm, as has been widely claimed. Second, we sought to make NPs that enclosed sufficient rifampicin to efficiently clear macrophages of infection with Mycobacterium bovis BCG. Using fluorescence microscopy and immuno-electron microscopy, in combination with markers for lysosomes, we show that BCG bacteria, as expected, localized to early phagosomes, but that at least 90% of PLGA particles were targeted to, and remained in, low pH, hydrolase-rich phago-lysosomes. Our data collectively argue that PLGA NPs remain membrane-enclosed in macrophages for at least 13 days and degrade slowly. Importantly, provided that the NPs are fabricated with sufficient antibiotic, one dose given after infection is sufficient to efficiently clear the BCG infection after 9-12 days of treatment, as shown by estimates of the number of bacterial colonies in vitro.

Diagnosis of extrapulmonary tuberculosis by PCR.

During the last two decades, the resurgence of tuberculosis (TB) has been documented in both developed and developing nations, and much of this increase in TB burden coincided with human immunodeficiency virus (HIV) epidemics. Since then, the disease pattern has changed with a higher incidence of extrapulmonary tuberculosis (EPTB) as well as disseminated TB. EPTB cases include TB lymphadenitis, pleural TB, TB meningitis, osteoarticular TB, genitourinary TB, abdominal TB, cutaneous TB, ocular TB, TB pericarditis and breast TB, although any organ can be involved. Diagnosis of EPTB can be baffling, compelling a high index of suspicion owing to paucibacillary load in the biological specimens. A negative smear for acid-fast bacilli, lack of granulomas on histopathology and failure to culture Mycobacterium tuberculosis do not exclude the diagnosis of EPTB. Novel diagnostic modalities such as nucleic acid amplification (NAA) can be useful in varied forms of EPTB. This review is primarily focused on the diagnosis of several clinical forms of EPTB by polymerase chain reaction (PCR) using different gene targets.

Evaluation of Aro-Tal-AST Complex Protein as a Marker for Differential Diagnosis of Mycobacterium Avium Infection.

PURPOSE: Conventional diagnostic techniques for detecting Mycobacterium avium infection are far from satisfactory. As serodiagnostic tests for M. avium infection have been shown to be simple and rapid, the present study was carried out to identify and evaluate M. avium secretory protein(s) of diagnostic potential. MATERIALS AND METHODS: Initially, by differential immunoblotting, a specific protein band of 45-50 kDa was recognized. Anion exchange column chromatography was used for purification of proteins. After fractionation, blast search was carried out. Further immunoreactivity studies were done with M. avium and Mycobacterium tuberculosis infected mice sera. Clinical utilization was confirmed by conducting indirect enzyme-linked immunosorbent assay (ELISA) with serum samples from mycobacterial infected patients. RESULTS: A complex of three proteins (Aro-Tal-AST) of molecular weight ~48 kDa, shown to be Aro A homologue (Aro), transaldolase (Tal) and aspartate transaminase (AST) by blast search was separated. Immunoreactivity studies of purified complex protein with mice sera confirmed it to be specific for M. avium infection. Indirect ELISA with patient samples further confirmed it to be M. avium infection specific. CONCLUSION: Aro-Tal-AST protein is specifically recognized by patients infected with M. avium and can be used as a marker for simple and rapid ELISA based tests for differential diagnosis of M. avium infection in patients with M. avium complex (MAC).

Immunotherapeutic role of Ag85B as an adjunct to antituberculous chemotherapy.

BACKGROUND: Immunotherapy to enhance the efficiency of the immune response in tuberculosis patients and to eliminate the persisters could be an additional valuable strategy to complement anti-mycobacterial chemotherapy. This study was designed to assess the immunotherapeutic potential of Ag85B as an adjunct to chemotherapy and its effect against active and persister bacteria left after therapy in mouse model of tuberculosis. METHODS: 6-8 week old female Balb/c mice were infected with Mycobacterium tuberculosis and treated with chemotherapy or immunotherapy. Protective efficacy was measured in terms of bacterial counts in lungs and spleen. Immune correlates of protection in terms of Th1 and Th2 cytokines were measured by ELISA. RESULTS: Therapeutic effect of Ag85B was found to be comparable to that of short term dosage of antituberculous drugs (ATDs). The therapeutic effect of ATDs was augmented by the simultaneous treatment with rAg85B and moreover therapy with this protein allowed us to reduce ATD dosage. This therapy was found to be effective even in case of drug persisters. The levels of antigen specific IFNγ and IL-12 were significantly increased after immunotherapy as compared to the basal levels; moreover antigen specific IL-4 levels were depressed on immunotherapy with Ag85B. CONCLUSION: We demonstrated in this study that the new combination approach using immunotherapy and concurrent chemotherapy should offer several improvements over the existing regimens to treat tuberculosis. The therapeutic effect is associated not only with initiating a Th1 response but also with switching the insufficient Th2 immune status to the more protective Th1 response.

Nonspecificity of 35 kDa protein: a proposed marker for the differential diagnosis of M. avium infection in the Indian population.

OBJECTIVE: The subunit vaccine strategies and development of various diagnostic reagents for Mycobacterium avium infection relies on the presence of secreted, species-specific mycobacterial antigens. The M. avium 35 kDa protein has been suggested as a candidate for vaccine/diagnostic reagent, specifically for M. avium infection. The present study was conducted to evaluate the diagnostic specificity of the M. avium 35 kDa protein in the Indian population. MATERIALS AND METHODS: Culture filtrate proteins were isolated by growing the bacilli in modified Youman's medium. The 35 kDa protein was purified by high-resolution preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and a blast search was carried out. Western blotting was performed with either monoclonal antibody CS-38 or serum samples of tuberculosis (TB) patients. The 35 kDa-specific immunoglobulin G antibody titer was estimated in the sera of TB patients and healthy individuals by indirect enzyme-linked immunosorbent assay (ELISA). RESULTS: Despite the absence of gene for the 35 kDa protein, the sera of TB patients and TB patient's contacts nonspecifically recognize it. Of 109 TB patients tested, the sera of 84 patients in ELISA (percentage recognition = 87.5%) and 27 of 29 TB patients tested in western immunoblotting (percentage recognition = 93.10%) recognized the M. avium 35 kDa protein, while with sera of TB patient's contacts, the recognition was 50%. CONCLUSION: Contrary to Western studies, the M. avium 35 kDa protein does not seem to be a good candidate for the specific diagnosis of M. avium infection in the Indian population.

Nanobead-based interventions for the treatment and prevention of tuberculosis.

Tuberculosis (TB), caused by Mycobacterium tuberculosis, is one of the most devastating bacterial diseases to affect humans. M. tuberculosis is a robust pathogen that has evolved the capacity to survive and grow inside macrophage phagosomes. A cocktail of antibiotics has long been successfully used against M. tuberculosis but is becoming less effective owing to the emergence of multidrug resistance. The only available preventive vaccine, using Mycobacterium bovis bacille Calmette-Guérin, is considered to be ineffective against adult pulmonary TB, the most prevalent form of the disease. Here, we review the potential use of biodegradable nanoparticle-based anti-TB drug delivery systems that have been shown to be more effective against M. tuberculosis in animal models than conventional antibiotic treatment regimens. This technology also has substantial potential for vaccination and other therapeutic strategies against TB and other infectious diseases.

Peptide deformylase--a promising therapeutic target for tuberculosis and antibacterial drug discovery.

BACKGROUND: Tuberculosis (TB) remains the most important infectious disease causing morbidity and death, due to the human pathogen Mycobacterium tuberculosis. The emergence of multi-drug-resistant and extensively-drug-resistant forms of TB have resulted in an increase in the number of TB cases. Thus, there is an urgent need to identify new drugs with novel targets to ensure future therapeutic success. Studies have indicated that peptide deformylase is an interesting potential candidate for discovering antimicrobial agents. OBJECTIVE: To explore the role of peptide deformylase, a highly conserved metalloprotease and an essential enzyme in bacterial life cycle, as a target for antibacterial as well as antimycobacterial drug development. METHODS: This review is based on recent published literature and online resources related to peptide deformylase inhibitors and their antibacterial potential. RESULTS/CONCLUSION: Peptide deformylase is an emerging therapeutic target for the treatment of tuberculosis and peptide deformylase inhibitors can act as potential future antibacterial agents.

Adjunct immunotherapy with Ag85 complex proteins based subunit vaccine in a murine model of Mycobacterium tuberculosis infection.

This study was designed to evaluate the immunotherapeutic potential of Mycobacterium tuberculosis Ag85AB emulsified with unmethylated CpG motif-containing oligonucleotide (CpG-ODN) and dimethyldioctadecylammonium bromide (DDA) adjuvants (Ag85AB-CpG-DDA) in conjunction with antituberculous drugs. Ag85 complex proteins of M. tuberculosis purified from total culture filtrate and purified proteins were emulsified with CpG-ODN and DDA adjuvants. Mice were infected with M. tuberculosis H37 Rv and left for 30 days to establish infection. These mice were named 'tuberculous mice'. Tuberculous mice were treated with Ag85AB-CpG-DDA alone or in conjunction with antituberculous drugs. Treatment of tuberculous mice with Ag85AB-CpG-DDA in conjunction with antituberculous drugs reduced significant bacilli burden in lung and spleen. Moreover, treatment of tuberculous mice with Ag85AB-CpG-DDA induced higher production of type-I cytokines, generated more CD44-positive T cells and suppresses secretion of IL-4 as compared with untreated animals. In conclusion, this study shows that Ag85AB-CpG-DDA formulation may act as a potential future therapeutic regimen in conjunction with antituberculous drugs.

Is intranasal vaccination a feasible solution for tuberculosis?

Bacille Calmette-Guerin (BCG) vaccine has been the only licensed tuberculosis (TB) vaccine administered to humans and, until today, more than 3 billion people have received BCG. However, despite the use of BCG, TB remains a global epidemic with a third of the world population being infected. Regardless of the protection induced by BCG in childhood TB, BCG vaccination fails to protect against pulmonary TB in adults, which represents more than 85% of the total TB burden. Therefore, the development of safe and efficacious TB vaccines that can confer potent protection in the lung mucosa has remained a major challenge to TB vaccinologists. Intranasal vaccination by different antigen formulations has shown promising results in the augmentation of immunity and the combat of the pathogens at the site of infection. This article will focus on the potential of intranasal vaccination and mucosal adjuvants for the development of new-generation TB vaccines.

Identification and purification of CREB like protein in Candida albicans.

cAMP response element binding protein (CREB) belongs to ATF/CREB family of transcription factors, which are bonafide targets of cAMP-PKA signalling pathway in mammalian cells. CREB is known to regulate the genes involved in transcription, cell cycle, cell survival, neurotransmitter, growth factors and immune regulation. But there is no evidence of presence of ATF/CREB family members in Candida albicans. In the present study, CREB like transcription factor has been identified and purified in C. albicans. The putative CREB was observed to have different molecular mass (47 kDa) as compared to its mammalian counterpart (43 kDa). Both forms of CREB (CREB and phosphorylated CREB) were detected in C. albicans and phosphorylation of CREB was found to be a function of cAMP levels and protein kinase A activity within this organism. CREB protein was purified by sequence-specific CRE-DNA affinity chromatography. Purified CREB exhibited characteristic CRE binding activity as revealed by electrophoretic mobility shift assay and gave reactivity with CREB antibodies. CREB protein was phosphorylated by purified catalytic subunit of PKA under in vitro conditions. To the best of our knowledge, this study reports for the first time identification of CREB like protein as an important component of cAMP signalling pathway in C. albicans.

Nanoparticle-based oral drug delivery system for an injectable antibiotic - streptomycin. Evaluation in a murine tuberculosis model.

BACKGROUND: To develop an oral drug delivery system for an injectable antibiotic, streptomycin. METHODS: Poly-lactide-co-glycolide (PLG) nanoparticles encapsulating streptomycin were prepared by the multiple emulsion technique and administered orally to mice for biodistribution and chemotherapeutic studies. RESULTS: The mean particle size was 153.12 nm with 32.12 +/- 4.08% drug encapsulation and 14.28 +/- 2.83% drug loading. Streptomycin levels were maintained for 4 days in the plasma and for 7 days in the organs following a single oral administration of PLG nanoparticles. There was a 21-fold increase in the relative bioavailability of PLG-encapsulated streptomycin compared with intramuscular free drug. In Mycobacterium tuberculosis H(37)Rv infected mice, eight doses of the oral streptomycin formulation administered weekly were comparable to 24 intramuscular injections of free streptomycin. Further, the nanoparticle formulation did not result in nephrotoxicity as assessed on a biochemical basis. CONCLUSION: Our results suggest that nanoencapsulation might be useful for developing a suitable oral dosage form for streptomycin and perhaps for other antibiotics that are otherwise injectable.

Chemotherapeutic evaluation of alginate nanoparticle-encapsulated azole antifungal and antitubercular drugs against murine tuberculosis.

The present study was designed to evaluate the chemotherapeutic potential of alginate nanoparticle-encapsulated econazole and antitubercular drugs (ATDs) against murine tuberculosis. Alginate nanoparticles encapsulating econazole and ATDs were prepared by the cation-induced controlled gelification of alginate and were characterized. Drugs were analyzed by high-performance liquid chromatography. All the ATDs were detected above minimum inhibitory concentrations for as long as 15 days and econazole until the day 8 in organs (lungs, liver, and spleen) after administration of encapsulated drugs, whereas free drugs remained detectable for only 12 to 24 hours. Eight doses of alginate nanoparticle-encapsulated econazole or 112 doses of free econazole reduced bacterial burden by more than 90% in the lungs and spleen of mice infected with Mycobacterium tuberculosis. Econazole (free or encapsulated) could replace rifampicin and isoniazid during chemotherapy of murine tuberculosis. Alginate nanoparticles reduced the dosing frequency of azoles and ATDs by 15-fold. Alginate nanoparticles are the ideal carriers of azole and antitubercular drugs, which can reduce dosing frequency of azoles as well as ATDs for the better management of tuberculosis.

cAMP regulates vegetative growth and cell cycle in Candida albicans.

We demonstrate here the regulatory role of cAMP in cell cycle of Candida albicans. cAMP was found to be a positive signal for growth and morphogenesis. Phosphodiesterase inhibitor aminophylline exhibited significant effects, i.e., increased growth, as well as induced morphogenesis. Atropine and trifluoperazine negatively regulated (inhibited) growth and did not induce morphogenesis. These changes were attributed to increase in cAMP levels and protein kinase A (PKA) activity in presence of aminophylline, while reduction was observed in atropine and trifluoperazine (TFP) grown cells. Alteration in cAMP signaling pathway affected the cell cycle progression in Candida albicans. Increased cAMP levels in aminophylline grown cells reduced the duration of cell cycle by inciting the cell cycle-specific expression of G1 cyclins (CLN1 and CLN2). However atropine and trifluoperazine delayed the expression of G1 cyclins and hence prolonged the cell cycle. Implication of cAMP signaling pathway in both the cell cycle and morphogenesis further opened the channels to explore the potential of this pathway to serve as a target for development of new antifungal drugs.