Search

THIS STUDY AIMS TO COMPARE THE EFFICACY OF ANTISEPTIC DRESSINGS, HYPERBARIC OXYGEN THERAPY, AND RECOMBINANT HUMAN PLATELET DERIVED GROWTH FACTOR (RHPDGF) FOR TWO REASONS: i) to reduce the incidence of lower limb amputations in diabetic foot ulcer; ii) to limit the duration of stay in the hospital. A prospective randomized trial was conducted on 60 patients with stage III and IV diabetic foot ulcers (International Association of Enterostomal Therapy classification) and patients were divided randomly in three different therapy groups - antiseptics, hyperbaric oxygen therapy, recombinant platelet derived growth factor, with 20 patients in each group. Patients were managed initially on inpatient and then on outpatient basis till the ulcer healed completely. Results among three groups were compared using unpaired T test and the level of significance was set at P<0.05 using ANOVA. This study compares the efficacy of hyperbaric oxygen therapy, antiseptic dressings, and rhPDGF in grade III and IV diabetic foot ulcers. P value (0.0348) was significant for complete wound contraction while p value healing time (0.6534) and ulcer size (0.0593) in the groups was not significant. PDGF is safe, effective and easy to apply. Results are comparable with hyperbaric oxygen (HBO) therapy and cost of treatment is lower than other therapies. Diabetic foot ulcer management requires multidisciplinary and aggressive approach. PDGF should be recommended for all grade III and IV diabetic foot ulcer at least 8 weeks old. HBO is equally good an option but has limitations and side effects.

The overall goal of the present study was to determine the effects of different doses of (+)-methamphetamine (meth) on locomotor activity of Balb/C mice. Four experiments were designed to test a wide range of meth doses in BALB/c female mice. In Experiment 1, we examined locomotor activity induced by an acute administration of low doses of meth (0.01 and 0.03mg/kg) in a 90-min session. Experiment 2 was conducted to test higher meth doses (0.3-10mg/kg). In Experiment 3, separate sets of mice were pre-treated with various meth doses once or twice (one injection/week) prior to a locomotor challenge with a low meth dose. Finally, in Experiment 4, we tested whether locomotor activation would be affected by pretreatment with a low or moderate dose of meth one month prior to the low meth dose challenge. Results show that low doses of meth induce hypolocomotion whereas moderate to high doses induce hyperlocomotion. Prior exposure to either one moderate or high dose of meth or to two, low doses of meth attenuated the hypolocomotor effect of a low meth dose one week later. This effect was also attenuated in mice tested one month after administration of a moderate meth dose. These results show that low and high doses of meth can have opposing effects on locomotor activity. Further, prior exposure to the drug leads to tolerance, rather than sensitization, of the hypolocomotor response to low meth doses., (Published by Elsevier Inc.)

A 26 year-old lady presented with episodic breathlessness, chest tightness, recurrent nasal obstruction and excessive sneezing, mainly during change of season along with opacity of the right hemithorax on chest x-ray. Further detailed work-up including spirometry, high-resolution CT scan of the thorax and fibre-optic bronchoscopy confirmed complete right lung agenesis in patients with bronchial asthma and allergic rhinitis. Complete control of symptoms was achieved with formeterol 6 μg and mometasone 200 μg (via dry powder inhaler) and intranasal fluticasone 50 μg (nasal spray) 2 puffs twice daily and oral montelukast 10 mg with levocetirizine 5 mg once daily.

Background & Objectives: Accurate diagnosis of tuberculosis (TB) is crucial to facilitate early treatment of the patients, and to reduce its spread. Clinical presentation of Mycobacterium tuberculosis complex (MTBC) and non tuberculous mycobacteria (NTM) may or may not be the same, but the treatment regimen is always different for both the infections. Differentiation between MTBC and NTM by routine laboratory methods is time consuming and cumbersome. This study was aimed to evaluate an immunochromatographic test (ICT), based on mouse monoclonal anti-MPT64, for simple and rapid discrimination between MTBC and NTM in clinical isolates from extra-pulmonary tuberculosis cases., Methods: A total of 800 clinical samples were collected from patients suspected to have extra-pulmonary tuberculosis. Preliminary diagnosis has been done by direct Ziehl-Neelsen (ZN) staining followed by culture in BACTEC system. A total of 150 clinical isolates, which were found positive in BD 460 TB system during September 2009 to September 2010 were selected for the screening by ICT test. p-nitro-α-acetylamino- β-hydroxy propiophenone (NAP) test was performed for differentiation of MTBC and NTM. M. tuberculosis complex was further confirmed by IS6110 PCR of BACTEC culture positive isolates, this served as the reference method for MTBC identification and comparative evaluation of the ICT kit., Results: Of the 150 BACTEC culture positive isolates tested by ICT kit, 101 (67.3%) were found positive for MTBC and remaining 49 (32.7%) were considered as NTM. These results were further confirmed by IS6110 PCR that served as the reference method for detection of MTBC. H 37 Rv reference strain was taken as a control for ICT test and IS6110 PCR. The reference strain showed the presence of MPT64 antigen band in the ICT test. Similar bands were formed in 101 of 102 MTBC isolates tested, proving 99.1 per cent sensitivity and no bands were detected in 48 (100%) NTM isolates tested, proving 100 per cent specificity of the ICT kit., Interpretation & Conclusions: Our findings show that ICT test can be used on direct culture positive specimens. It does not require any special equipment, is simple and less time consuming. It can easily discriminate between MTBC and NTM and thus can help in appropriate management of tuberculosis.

Cyclosporine A, a potent immunosuppressive agent extensively used to prevent allograft rejections, is under scrutiny due to severe toxic effects. CsA therapy is often continued during pregnancy in conditions such as organ transplantations and autoimmune diseases. Herein, we investigated the effects of CsA on early morphogenesis of zebrafish and identified a spectrum of proteins whose expression was altered in the drug treated embryos. Time-lapse fluorescence imaging of germ-line double transgenic zebrafish embryos treated with CsA revealed severe blood regurgitation in heart chambers, absence of blood circulation in vessels, pericardial and yolk sac edema. We also observed lack of mature blood vessels and down-regulation of endothelial markers in CsA treated embryos. Proteomic analysis using 2D-DIGE followed by mass-spectrometry led to the identification of 37 proteins whose expression was significantly modulated in presence of the drug. These proteins were mostly associated with cytoskeletal/structural assembly, lipid-binding, stress response and metabolism. Furthermore, mRNA expression analysis of eight proteins and Western blotting of actin revealed consistency between the changes observed in protein expression and its corresponding mRNA levels. Our findings demonstrate that CsA administration during early morphogenesis in zebrafish modulates the expression of some proteins which are known to be involved in important physiological processes., (Copyright © 2011 Elsevier B.V. All rights reserved.)

When Eu(3+) ions occupy Ca(2+) sites of CaMoO(4), which has a body centered tetragonal structure with inversion symmetry, only the magnetic dipole transition ((5)D(0)→(7)F(1)) should be allowed according to Judd-Ofelt theory. Even if there are a few distortions in the Eu(3+) environment, its intensity should be more than that of the electric dipole transition ((5)D(0)→(7)F(2)). We report here the opposite effect experimentally and ascribe this to the polarizability effect of the MoO(4) tetrahedron, which is neighboring to EuO(8) (symmetric environment). The contribution of the energy transfer process from the Mo-O charge transfer band to Eu(3+) and the role of Eu(3+) over the surface of the particle could be distinguished when luminescence decay processes were measured at two different excitations (250 and 398 nm). Further, the luminescence intensities and lifetimes increase significantly with increasing heat-treatment temperature of the doped samples. This is attributed to the reduction of H(2)O from the surface of the particles and a non-radiative process after heat treatment.

In developing countries the diagnosis of extrapulmonary tuberculosis (EPTB) is a major burning challenge. EPTB encounters many problems like pauci-bacillary nature, inadequate specimen volume. All the limitations reflect in the poor contribution of conventional bacteriological technique in the establishment of diagnosis of EPTB. Nucleic acid amplification methods are rapid and sensitive has modified strategies for the detection of mycobacterial DNA. A fragment of DNA of 123 bp belonging to insertion sequence IS6110 based on specific gene of Mycobacterium tuberculosis complex was amplified by polymerase chain reaction (PCR) for the rapid diagnosis of EPTB. The present study was to comparative evaluation of IS6110 PCR via conventional methods in the rapid diagnosis of new and Previously treated cases of extra pulmonary tuberculosis. Four hundred fifty specimens were collected from suspected cases of EPTB were processed for Mycobacteria by Zeihl Neelson (ZN) staining and BACTEC culture for M. tuberculosis. All the specimens were also processed for IS6110 based PCR amplification with primers targeting 123 bp fragment of insertion element IS6110 of M. tuberculosis complex. We found significant difference was seen in sensitivities of different tests. Of these 450 specimens, 60 (13.4%) were positive for AFB by ZN staining, 202 (45%) for BACTEC culture and IS6110 PCR were positive for M. tuberculosis complex in 283 (63%) specimens (p< 0.05). However, there was no significant difference (p< 0.05) as far as specificity of different tests. We found that IS6110 PCR has higher sensitivity than smear microscopy and BACTEC culture in both cases of new cases as well as in previously treated cases. IS6110 PCR can be highly useful in diagnosis of new and treated cases of EPTB. It may facilitate therapeutic decisions for those with suspected of EPTB.

Garlic has been used medicinally since antiquity because of its antimicrobial activity, anticancer activity, antioxidant activity, ability to reduce cardiovascular diseases, improving immune functions, and antidiabetic activities and also in reducing cardiovascular diseases and improving immune functions. Recent studies identify that the wide variety of medicinal functions are attributed to the sulfur compounds present in garlic. Epidemiological observations and laboratory studies in animal models have also showed anticarcinogenic potential of organosulfur compounds of garlic. In this study, in silico analysis of organosulfur compounds is reported using the methods of theoretical chemistry to elucidate the molecular properties of garlic as it is more time and cost efficient, reduces the number of wet experiments, and offers the possibility of replacing some animal tests with suitable in silico models. The analysis of molecular descriptors defined by Lipinski has been done. The solubility of drug in water has been determined as it is of useful importance in the process of drug discovery from molecular design to pharmaceutical formulation and biopharmacy. All toxicities associated with candidate drug have been calculated. P-Glycoprotein expressed in normal tissues as a cause of drug pharmacokinetics and pharmacodynamics has been examined. Drug-plasma protein binding and volume of distribution have also been calculated. To avoid rejection of drugs, it is becoming more important to determine pK(a), absorption, polar surface area, and other physiochemical properties associated with a drug, before synthetic work is undertaken. The present in silico study is aimed at examining these compounds of garlic to evaluate its possible efficacy and toxicity under conditions of actual use in humans.

Introduction: The symptomatic central nervous system involvement is often seen in patients with miliary tuberculosis., Materials and Methods: In this study, we evaluated 60 consecutive miliary tuberculosis patients, who presented with some neurological manifestations. Evaluation included neurological examination, a battery of blood tests, HIV serology, sputum examination, cerebrospinal fluid (CSF) examination along with imaging of the brain and spinal cord. The patients were followed up after completion of 6 months of antituberculous treatment., Results: Patients ranged between 14 and 53 years in age. Three patients tested HIV positive. Forty-eight (80%) patients had tuberculous meningitis. In 12 (20%) patients, the CSF examination was normal. In 27 patients with tuberculous meningitis, neuroimaging revealed intracerebral tuberculoma. Fourteen patients showed multiple tuberculomas, while 7 had a solitary tuberculoma. In six patients, the tuberculomas were small and numerous. In two patients, neuroimaging revealed a spinal tuberculoma. For three patients with tuberculous brain masses, the CSF was normal. Nine (15%) patients presented with myelopathy. Three patients exhibited Pott's paraplegia. Three patients had transverse myelitis (with normal neuroimaging). In three patients, the spinal MRI revealed an intramedullary tuberculoma. On follow-up, 15 (25%) patients expired. Thirty-one (52%) patients showed significant improvement. Eight patients (13%) showed nil or partial recovery. Six of the patients with no improvement developed vision loss. Six (10%) patients were lost to follow up., Conclusion: A variety of neurological complications were noted in military tuberculosis patients, tuberculous meningitis and cerebral tuberculomas being the most frequent complications. However, a majority of patients improved following antituberculous treatment.

Treatments for cocaine abuse have been disappointingly ineffective, especially in comparison with those for some other abused substances. A new approach, using vaccination to elicit specific antibodies to block the access of cocaine to the brain, has shown considerable promise in animal models, and more recently in human trials. The mechanism of action for the antibody effect on cocaine is very likely to be the straightforward and intuitive result of the binding of the drug in circulation by antibodies, thereby reducing its entry into the central nervous system and thus its pharmacological effects. The effectiveness of such antibodies on drug pharmacodynamics is a function of both the quantitative and the qualitative properties of the antibodies, and this combination will determine the success of the clinical applications of anti-cocaine vaccines in helping addicts discontinue cocaine abuse. This review will discuss these issues and present the current developmental status of cocaine conjugate vaccines.

The structure and relative energies of the tautomers of dichloroacetate are predicted using Hartree Fock method. The infrared spectra of two dominant tautomers are calculated in the density functional theory. Good agreement between calculated (DFT) and experimental harmonic vibrational frequencies is found. Assuming Cs point symmetry, vibrational assignments for the observed frequencies have been proposed. The spectra exhibit distinct features originating from low frequency vibrational modes caused by inter-molecular motion. Energy is minimum for DCA-2 tautomer. Dipole moment is large for DCA-1 tautomer and polar surface area is somewhat larger in caso of DCA-2. Local ionization potential map and lowest-unoccupied molecular orbital (LUMO) map has been drawn and analyzed. Linear regression data for both the tautomers has also been calculated, so clearly DCA1 is more correlated to experimental wavenumbers.

Conventional substance-abuse treatments have only had limited success for drugs such as cocaine, nicotine, methamphetamine, and phencyclidine. New approaches, including vaccination to block the effects of these drugs on the brain, are in advanced stages of development. Although several potential mechanisms for the effects of antidrug vaccines have been suggested, the most straightforward and intuitive mechanism involves binding of the drug by antibodies in the bloodstream, thereby blocking entry and/or reducing the rate of entry of the drug into the central nervous system. The benefits of such antibodies on drug pharmacodynamics will be influenced by both the quantitative and the qualitative properties of the antibodies. The sum of these effects will determine the success of the clinical applications of antidrug vaccines in addiction medicine. This review will discuss these issues and present the current status of vaccine development for nicotine, cocaine, methamphetamine, phencyclidine, and morphine.

The opioidergic hypothesis suggests an association between genetic variations at the opioid receptor mu 1 (OPRM1) gene locus and opiate addiction. The OPRM1 gene, which encodes for mu opioid receptor, contains several single nucleotide polymorphisms (SNPs) in exon I. Two of these, C17T and A118G, have been reported to be associated with substance abuse. The present study aims to delineate the frequency of these variants in the subjects of Indian origin and study their association with the phenotype of opioid dependence. A118G (rs 1799971) and C17T (rs 1799972) were genotyped using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. For 118G allele, the control subjects (n = 156) showed a frequency of 0.12 while the opioid dependents (n = 126) had an approximately 2.5-fold higher frequency of 0.31 (Odds Ratio 3.501; CI(95%) 2.212-5.555; p < 0.0001). For C17T polymorphism, the controls (n = 57) showed a frequency of 0.89 for C allele versus 0.83 seen in dependents (n = 123; odds ratio of 0.555; CI(95%) 0.264-1.147; p = 0.121). A significant association was observed between the 118G allele and no association was seen with C17T polymorphism and opioid dependence.

Conventional substance abuse treatments have had only limited success. As a result, new approaches, including vaccination to block the effects of drugs such as cocaine, nicotine, methamphetamine, and phencyclidine, are in development. Although a number of possible rationales for the effects of antidrug vaccines have been suggested, the most straightforward and intuitive mechanism would involve binding of the drug by antibodies in the bloodstream, thereby blocking entry or reducing the rate of entry of the drug into the central nervous system. The theoretical parameters that would influence vaccine-induced drug pharmacodynamics are presented in this review, along with the current status on vaccine development for nicotine, cocaine, methamphetamine, and phencyclidine.

Opiates are known to induce immunosuppression in their users (addicts). Evidences supporting their role in suppressing a variety of immunological end points in addicts have been reported by several investigators. In the present study, we investigated the changes in serum immunoglobulin (Ig) levels and their correlation with Mu opiate receptor (MOR) genotypes. Eighty-seven users and forty-five non-users were recruited for the study. Genomic DNA, isolated from the peripheral blood, was used for genotyping for C17T and A118G polymorphism using PCR-RFLP method. The frequency of A and G alleles in non-users was 89% and 11% respectively, whereas in addicts, it was 67% and 33% respectively. Case control analysis between groups revealed that 118G allele was associated with opioid dependence [Chi square (chi2) = 13.56, odds ratio (OR) = 3.90, confidence interval 95% (CI 95%) = 1.80-8.67, p = 0.000231]. C17T polymorphism showed no association with opioid dependence [(chi2) = 0.9, OR = 2.49, CI 95% = 0.528-16.12, p = 0.343]. Mean Ig levels, both IgG (student's t-test = 2.2738, p = 0.007) and IgA (student's t-test = 2.848, p = 0.0051) differed between opiate users and nonusers. IgG and IgA levels were also significantly different in individuals with different MOR genotypes. Immunosuppression was observed in AA genotype-bearing individuals, while no suppression was seen in AG and GG genotypes bearing individuals. In case of C17T polymorphism, both CC and CT genotypes bearing individuals showed immunosuppression, as judged by circulating Ig levels.

Hepatitis C virus (HCV) is the major pathogen of chronic hepatitis and liver disease, but currently there are no prophylactic HCV vaccines available. The HCV core protein-encoding sequence is among the most conserved genes in the HCV genome, making it a prime candidate for a component of a vaccine. The core protein localizes to the endoplasmic reticulum (ER) through a C-terminal hydrophobic region that is cotranslationally inserted into the ER membrane. Here we show that removal of the C-terminal hydrophobic region confers nuclear localization and enhances proteasomal degradation of the core protein in mammalian cells. This efficient protein proteolysis induces enhanced core-specific CD8(+) T cell responses in BALB/c mice immunized with plasmids expressing C-terminal deletions of the HCV core protein. These results suggest that more potent HCV vaccines can be achieved by targeting the core protein for proteasomal degradation by deletion of its C-terminal hydrophobic domain.

A stable monolayer of N-octadecylaniline containing stearic acid at the air-water interface has been obtained. However, the Langmuir monolayer of pure poly(N-octadecylaniline) was not stable, but mixed Langmuir-Blodgett films of this polymer with stearic acid in different ratios as a spreading aid were stable. The electrical conductivity of these films increased by five orders of magnitude after doping with iodine as compared to that before iodine doping (5.8x10(-7) S cm(-1)). Temperature-dependent electrical conductivity suggested that these films were semiconducting in nature with low thermal activation energy. Impedance analyses of these films revealed that the equivalent circuit for poly(N-octadecylaniline) was (RQ) whereas that for mixed poly(N-octadecylaniline) with stearic acid was (RQ)(RQ).

Modest clinical outcomes of dendritic-cell (DC) vaccine trials call for the refinement of DC vaccine design. Although many potential antigens have been identified, development of methods to enhance antigen presentation by DCs has lagged. We have engineered a potent, drug-inducible CD40 (iCD40) receptor that permits temporally controlled, lymphoid-localized, DC-specific activation. iCD40 is comprised of a membrane-localized cytoplasmic domain of CD40 fused to drug-binding domains. This allows it to respond to a lipid-permeable, high-affinity dimerizer drug while circumventing ectodomain-dependent negative-feedback mechanisms. These modifications permit prolonged activation of iCD40-expressing DCs in vivo, resulting in more potent CD8(+) T-cell effector responses, including the eradication of previously established solid tumors, relative to activation of DCs ex vivo (P < 0.01), typical of most clinical DC protocols. In addition, iCD40-mediated DC activation exceeded that achieved by stimulating the full-length, endogenous CD40 receptor both in vitro and in vivo. Because iCD40 is insulated from the extracellular environment and can be activated within the context of an immunological synapse, iCD40-expressing DCs have a prolonged lifespan and should lead to more potent vaccines, perhaps even in immune-compromised patients.

Autoreactive T cells can be induced by altered peptide ligands to switch Th1 and Th2 phenotypes. The underlying molecular mechanism is critical for understanding of activation of autoreactive T cells and development of novel therapeutic strategies for autoimmune conditions. In this study, we demonstrated that analog peptides of an immunodominant epitope of myelin basic protein (residues 83-99) with alanine substitution at Val(86) and His(88) had a unique partial agonistic property in the induction of Th1 or Th2 deviation in MBP(83-99)-reactive T cell clones typical of Th0 phenotype. The observed phenotypic switch involved differential activation of ERK, p38, and JNK MAPKs. More specifically, Th1 deviation induced by peptide 86V-->A (86A) correlated with enhanced p38 and JNK activities, while Th2 deviation by peptide 88H-->A (88A) was associated with up-regulated ERK activity and a basal level of p38 and JNK activity. Further characterization revealed that a specific inhibitor for ERK selectively prevented Th2 deviation of MBP(83-99)-specific T cells. Conversely, specific inhibitors for p38 and JNK blocked Th1 deviation in the same T cell preparations induced by peptide 86A. The findings have important implications in our understanding of regulation of ERK, p38, and JNK by altered peptide ligands and their role in cytokine regulation and phenotype switch of autoreactive T cells.

Several gene-based vaccine approaches are being tested to drive multivalent cellular immune responses to control HIV-1 viral variants. To compare the utility of these approaches, HLA-A*0201 transgenic mice were genetically immunized with plasmids encoding wild-type (wt) gag-pol, codon-optimized (CO) gag-pol, and an expression library immunization (ELI) vaccine genetically re-engineered to express non-CO fragments of gag and pol fused to ubiquitin for proteasome targeting. Equimolar delivery of each vaccine into HLA-A*0201 transgenic mice generated CD8 T cell responses, with the ELI vaccine producing up to 10-fold higher responses than the wt or CO gag-pol plasmids against cognate and mutant epitopes. All three vaccines generated multivalent CD8 responses against varying numbers of epitopes after priming. However, when the animals were immunized again, the wt and CO gag-pol vaccines boosted only the responses against a subset of epitopes and attenuated the responses against all other Ags including epitopes from clade and drug-resistant viral variants. In contrast, the ELI vaccine boosted CD8 responses against all of the gag-pol Ags and against mutant epitopes from clade and drug-resistant variants. These data suggest that HIV-1 vaccines expressing structurally intact gag and pol proteins drive immunofocused CD8 responses that reduce the repertoire of T cell responses. In contrast, the genetically re-engineered ELI vaccine appears to better maintain the multivalent CD8 responses that may be required to control HIV-1 viral variants.

The information about Urban Mental Health Services has been nearly nonexistent in India, although the developed countries have been focusing on programmes for "Healthy Cities". The initiative taken as part of the WHO-ICMR Pilot Project on Urban Mental Health Services, with a public health perspective is being shared. The objectives of the Health Services Research (HSR) Arm of the project were to study the distribution and the availability of tertiary Mental Health Services, availability of human resources, average service load, mental health service gap, and perceptions of the users and the service providers, regarding the barriers in accessibility of mental health services, unmet service needs and strategies for improvement.The Research Methods involved Mapping Exercises with estimation of Service Loads and Qualitative Research Methods (QRM) like In-Depth Interviews (IDIs), Key Informant Interviews (KIIs), Free Listing and Focused Group Discussions (FGDs). The results indicate uneven availability of mental health services, human resource deficit specially for non-medical mental health professionals and mental health service gap (82% to96%). The average service load in the specialist mental health services is largely carried by the Govt. sector (half to two thirds), followed by the private sector (one third to half), with only a small portion by the NGO sector. The average mental health service load in the primary care general health services is largely carried by the private sector, with significant contribution from the non-formal service providers. The barriers to access, unmet needs and possible strategies as perceived by the community, users and service providers have been identified. The findings are discussed in the context of the mental health programmes and the public policy issues. The implications of the conclusions which suggest that Urban Mental Health Services are far from complete are highlighted.

Genetic immunization is a novel method for vaccination in which DNA is delivered into the host to drive both cellular and humoral immune responses against its protein product. While genetic immunization can be potent, it requires that one have, in hand, a gene that encodes a protective protein antigen. Therefore, for many diseases, one cannot make a genetic vaccine because no protective antigen is known or no gene for this antigen is available. This lack of candidate antigens and their genes is a considerable bottleneck in developing new vaccines against old infectious agents, new emerging pathogens, and bioweapons. To address this limitation, we developed expression library immunization (ELI) as a high-throughput technology to discover vaccine candidate genes at will, by using the immune system to screen the entire genome of a pathogen for vaccine candidate. To date, ELI has discovered new vaccine candidates from a diverse set of bacterial, fungal, and parasitic pathogens. In addition, the process of applying ELI to the genome of pathogens allows one to genetically re-engineer these antigens to convert immunoevasive pathogen proteins into immunostimulatory vaccine antigens. Therefore, ELI is a potent technology to discover new vaccines and also generate genomic vaccines with amplified, multivalent immunostimulatory capacities.

HIV-1 is a fundamentally difficult target for vaccines because of its high mutation rate and its repertoire of immune evasion strategies. To address these difficulties, a multivalent genetic vaccine or "live genetic vaccine" was recently developed against HIV-1 using the expression library immunization (ELI) approach. In this HIV-1 vaccine, all open reading frames of HTLV-IIIb are expressed as protein fragments to retain all viral T cell epitopes, but destroy protein toxicity, inactivate immune escape functions, and reveal subdominant epitopes. In addition, each antigen fragment is fused to the ubiquitin protein to increase antigen expression and target these antigens to the proteasome to enhance cytotoxic T lymphocyte (CTL) responses. This multivalent vaccine also has the advantage of being incapable of generating infectious HIV-1 virus because of the segregation of the HIV genome into 32 separate plasmids. In this work, we demonstrate the ability of this genetic vaccine to provoke robust HLA-A*0201-restricted T cell responses in MHC class I humanized mice against gag, pol, env, and nef after a single round of immunization. In addition, this HTLV-IIIb-derived vaccine demonstrated cross-clade, envelope-specific, HLA-restricted CD8 responses against clades A, D, and E. HLA-restricted CD8 responses were generated against all 32 open reading frames encoded by the multi-plasmid genetic vaccine demonstrating that a broad repertoire of human relevant CD8 responses are provoked by this vaccine. This work supports this approach to generate multivalent T cell responses to control the highly mutable and immuno-evasive HIV-1 virus.

To counter highly mutable pathogens like HIV-1, a number of vaccines are being developed to deliver multiple mutant forms of viral Ags to provoke multivalent antiviral CTLs. However, it is uncertain whether such multiple mutant epitope vaccines will generate the diverse CTL responses desired or will instead create immune interference. To characterize the role of immune interference by mutant epitopes in this process, we have tested a "worst case" scenario in which the immunodominant epitope of OVA (SIINFEKL) and its in vitro TCR antagonist (SIINFEDL) have been used to genetically immunize C57BL/6 mice. We demonstrate here that sequential delivery of these mutant epitopes provokes original antigenic sin in CD8 T cells as demonstrated by attenuation of CTLs, intracellular IFN-gamma production, and MHC I peptide-tetramer staining. By contrast, simultaneous exposure of the immune system to this agonist/antagonist pair not only fails to generate T cell antagonism in vivo, but also avoids original antigenic sin. These observations suggest that simultaneous immunization with vaccines containing mutant epitopes, even T cell antagonists, can indeed generate a diverse array of T cell responses and that at least some immune interference can be avoided by delivering mutant Ags to the immune system simultaneously.

HIV-1 is a fundamentally difficult target for vaccines due to its high mutation rate and its repertoire of immunoevasive strategies. To address these difficulties, a multivalent, proteasome-targeted, live genetic vaccine was recently developed against HIV-1 using the expression library immunization approach. In this HIV-1 vaccine all open reading frames of HIV-1 are expressed from 32 plasmids as Ag fragments fused to the ubiquitin protein to increase Ag targeting to the proteasome to enhance CTL responses. In this work we demonstrate the ability of the HIV-1 library vaccine to simultaneously provoke robust HLA-A*0201-restricted T cell responses against all 32 HIV-1 library vaccine Ags after single immunization by gene gun. These CD8 T cell responses included HLA-A*0201-restricted CTL activity, CD8/IFN-gamma T cell responses, and HLA tetramer binding against defined immunodominant epitopes in gag, pol, env, and nef as well as potent CD8/IFN-gamma responses against undefined HLA-A*0201-restricted epitopes in all remaining Ags of the library. CD8 responses mediated by single gag, pol, env, and nef plasmids from the vaccine demonstrated little reduction in specific T cell responses when these plasmids were diluted into the context of the full 32-plasmid library, suggesting that Ag dominance or immune interference is not an overt problem to limit the efficacy of this complex vaccine. Therefore, this work demonstrates the ability of the HIV-1 library vaccine to generate robust multivalent genome-wide T cell responses as one approach to control the highly mutable and immunoevasive HIV-1 virus.

Two hundred and thirty men, being treated for ICD-10 diagnosed dependence on alcohol, opioids or both, were studied 2-4 weeks after the last use of alcohol or opioids. Alienation Scale, Sensation Seeking Scale and Muliphasic Personality Questionnaire (MPQ), and selected sociodemographic and family history data were studied. All three groups showed high alienation (more in opioid cases), high sensation seeking (more in alcohol cases, more for boredom susceptibility), and a disturbed MPQ profile. The dual dependence group was similar to opioid group for age, but closer to alcohol group in terms of personality profile. Only alcohol cases showed a significantly positive correlation between alienation and sensation seeking- in terms of total scale, and boredom susceptibility and disinhibition subscales only. Thus, substance specificity was not reflected prominently in the inter-relationships between alienation, sensation seeking and MPQ scores, and sociodemographic variables.

Pathways to psychiatric care were evaluated among three hundred and eighty four first-contact psychiatric patients from five socio-culturally different regions of India by a modified version of World Health Organization encounter form for 'pathways in psychiatric care'. Concerning first caregivers, out of 384 respondents 34.1% had chosen the psychiatrists, 29.4% the general practitioners and 26% had chosen faith healers and exorcists. Choice for the first caregiver was not influenced by gender differences, literacy status and family type. Mostly the subjects hailing from rural areas and those presenting with somatic symptoms chose psychiatrist as their first caregivers.

Multiple sclerosis (MS) is a demyelinating disease of presumed T cell autoimmunity against self myelin. We hypothesized that if myelin-reactive T cells are associated with the disease processes, they may undergo activation and expansion during acute exacerbation. In this study, we examined the precursor frequency, epitope recognition and cytokine profile of myelin-reactive T cells in 14 relapsing/remitting MS patients during exacerbation and remission. The study revealed that T cells recognizing the immunodominant peptides of candidate myelin antigens, including myelin basic protein (MBP), proteolipid protein and myelin oligodendrocyte glycoprotein, occurred at increased precursor frequency during acute exacerbation. The T cell responses to MBP focused on the immunodominant regions (residues 83-99 and 151-170) during exacerbation and shifted toward other epitopes of MBP at the time of remission. Furthermore, there was a marked increase in the production of T(h)1 cytokines among T cell lines obtained during exacerbation compared to those obtained during remission. The study demonstrated that myelin-reactive T cells underwent selective activation and expansion during acute MS exacerbation. In contrast, myelin-reactive T cells found during remission in the same patients generally resembled those identified in healthy controls with some discrepancies. The findings suggest potential association of aberrant myelin-reactive T cell responses with acute exacerbation in MS, which may reflect transient activation of myelin-reactive T cell populations of pathogenic potential.

Long term intravenous drug abuse is associated with recurrent femoral pseudoaneurysm in a 36-year-old man. The clinical features alongwith a suitable discussion is described in this case report.

The putative replication origin of Azotobacter vinelandii was cloned as an autonomously replicating fragment after ligation to an antibiotic resistance cartridge. The resulting plasmids could be isolated and labelled by Southern hybridisation with the antibiotic resistance cartridge as probe and also visualised by electron microscopy. These plasmids integrated into the chromosome after a few generations, even in the recA mutant of A. vinelandii. The integrated copy of the plasmid was re-isolated from the chromosome and the DNA and its subfragments were cloned in the plasmid vector pBR322. A 200-bp DNA fragment was sufficient to allow the replication of pBR322 in an Escherichia coli polA strain. Electron microscopic analysis of this plasmid showed that replication initiated mostly within the A. vinelandii DNA fragment. The nucleotide sequence of the putative replication origin and its flanking regions was determined. In the sequence of the 200-bp fragment many of the distinctive features found in other replication origins are lacking. A greater variation from the consensus DnaA binding sequence was observed in A. vinelandii. Direct sequencing of the relevant genomic fragment was also carried after amplifying it from A. vinelandii chromosomal DNA by PCR. This confirmed that no rearrangements had taken place while the cloned fragment was resident in E. coli. It was shown by hybridisation that the 200-bp chromosomal origin fragment of A. vinelandii was present in three other field strains of Azotobacter spp.

Th0 clones recognizing an immunodominant peptide of myelin basic protein (residues 83-99) were derived from patients with multiple sclerosis. We demonstrate that analogue peptides with alanine substitution at Val86 and His88 had a unique partial agonistic property in inducing Th0 -->Th1 and Th0 -->Th2 deviation of the myelin basic protein-reactive T cell clones, respectively. Th0 to Th1 deviation induced by peptide 86V-->A correlated with up-regulation of Fyn and ZAP-70 kinase activities. Conversely, Th0 to Th2 deviation induced by peptide 88H-->A was associated with complete failure to activate Fyn and ZAP-70 kinases. The observed Th1 and Th2 shift also correlated, to a lesser extent, with Lck kinase activity that was down-regulated with Th1 deviation and increased with Th2 deviation in some T cell clones. We demonstrated that the Th1 and Th2 shift induced by the analogue peptides was a reversible process, as the T cell clones previously exposed to either 86V-->A or 88H-->A peptide could revert to an opposite phenotype when rechallenged reciprocally with a different analogue peptide. The study has important implications in our understanding of regulation of TCR-associated tyrosine kinases by altered peptide ligands and its role in cytokine regulation of autoreactive T cells.

An inorganic analogue of polystyrene, poly(phenylphosphinoborane) represents the first high molecular weight, well-characterized polymer with a backbone of alternating phosphorus and boron atoms. It is accessible by metal-catalyzed "dehydrocoupling" of a primary phosphane-borane adduct (see scheme).

T cell responses to the immunodominant peptide (residues 83-99) of myelin basic protein are potentially associated with multiple sclerosis (MS). This study was undertaken to examine whether a common sequence motif(s) exists within the TCR complementarity-determining region (CDR)-3 of T cells recognizing the MBP83-99 peptide. Twenty MBP83-99-reactive T cell clones derived from patients with MS were analyzed for CDR3 sequences, which revealed several shared motifs. Some V beta 13.1 T cell clones derived from different patients with MS were found to contain an identical CDR3 motif, V beta 13.1-LGRAGLTY. Oligonucleotides complementary to the shared CDR3 motifs were used as specific probes to detect identical target CDR3 sequences in a large panel of T cell lines reactive to MBP83-99 and unprimed PBMC. The results revealed that, in contrast to other CDR3 motifs examined, the LGRAGLTY motif was common to T cells recognizing the MBP83-99 peptide, as evident by its expression in the majority of MBP83-99-reactive T cell lines (36/44) and PBMC specimens (15/48) obtained from randomly selected MS patients. The motif was also detected in lower expression in some PBMC specimens from healthy individuals, suggesting the presence of low precursor frequency of T cells expressing this motif in healthy individuals. This study provides new evidence indicating that the identified LGRAGLTY motif is preferentially expressed in MBP83-99-reactive T cells. The findings have important implications in monitoring and targeting MBP83-99-reactive T cells in MS.

Cisplatin (cis-dichlorodiammineplatinum II), a potent antitumour compound, stimulates immune responses by activating monocytes/macrophages and other cells of the immune system. However, the exact mechanism by which cisplatin activates these cells is poorly characterized and attempts are being made to understand this mechanism. Previous studies from this laboratory have shown that Lyn, a protein tyrosine kinase of the src family, and nuclear factor (NF)-kappaB are involved in cisplatin-induced macrophage activation. Recent studies suggest that the RAS and mitogen-activated protein (MAP) kinases function as a connecting link between activated lyn and NF-kB, which raises the possibility of their involvement in cisplatin-induced macrophage activation. Therefore, this study was undertaken to investigate the effect of cisplatin treatment on the expression/activation of RAS (a low molecular weight GTP-binding protein) and MAP kinases in murine peritoneal macrophages. The underlying mechanism of expression/activation of RAS and MAP kinases in cisplatin-treated macrophages was also investigated. Immunoblotting and immune-complex kinase assays revealed that cisplatin treatment of macrophages leads to increased expression/activation of RAS and MAP kinases, with optimal expression/activation at 15 min of treatment. Using a battery of specific inhibitor/modulators of different signalling molecules, this study shows that expression and activation of MAP kinases are two unrelated processes. It was also observed that kinase (protein tyrosine and protein kinase C) inhibitor and Ca2+/calmodulin antagonist inhibit expression/activation of RAS/MAP kinases in macrophages, whereas phosphatases (protein tyrosine and serine/threonine) inhibitor up-regulate these kinases.

Cisplatin [cis-dichlorodiammine platinum (II)], a potent anti-tumour compound, stimulates immune responses by activating macrophages and other cells of the immune system. The mechanism by which cisplatin activates these cells is poorly characterized. Present investigations were undertaken to study the mechanism of antigen presentation by cisplatin-treated macrophages. Cisplatin-treated macrophages showed a biphasic pattern of antigen presentation to keyhole limpet haemocyanin (KLH)-primed T cells. The second phase of antigen presentation was not due to the continuous presence of cisplatin in the culture medium; rather, it was induced by soluble factors released by cisplatin-treated macrophages. Co-incubation of macrophages with cisplatin and inhibitor of serine/threonine or protein tyrosine phosphatase resulted in an augmentation of cisplatin-induced antigen presentation. In contrast, treatment of macrophages with cisplatin and inhibitor of protein kinase C or protein tyrosine kinase inhibited cisplatin-induced antigen presentation. These observations suggest that antigen presentation by cisplatin-treated macrophages is regulated by reversible action of protein phosphatases and kinases. The antigen-presenting ability of cisplatin-treated macrophages was also inhibited by EGTA, nifedipine, TMB-8, W-7 and calmidazolium, suggesting the probable involvement of Ca2+, calmodulin and calmodulin-dependent kinases in this process.

Cisplatin [cis-dichlorodiammine platinum (II)], a potent chemoimmunotherapeutic drug, activates macrophages to tumoricidal state which is inhibited by protein tyrosine kinase(s) inhibitor. Cisplatin induces protein tyrosine phosphorylation of a number of cellular proteins suggesting the involvement of protein tyrosine kinase(s) in the activation process of macrophages. Therefore, the effect of cisplatin treatment on the expression and activation of lyn, a protein tyrosine kinase of src family, in macrophages was investigated. The underlying mechanism of lyn expression and activation was also analyzed. Cisplatin treatment increased lyn expression and activation in macrophages within 5 min of treatment. The expression and activation of lyn were observed to be biphasic processes in cisplatin-treated macrophages with the first peak appearing at 15 min and the second peak at 2 h of treatment. The appearance of second phase of lyn activation and second phase of lyn expression were two unrelated processes. The second peak of lyn activation was produced by the autocrine action of some soluble product(s) of cisplatin-treated macrophages, whereas the second phase of lyn expression was due to some intracellular factor. It was further observed that cisplatin-induced lyn expression and activation involves serine/threonine phosphatases 1/2A, protein tyrosine phosphatases, protein tyrosine kinase and protein kinase C. It was also observed that Ca2+/calmodulin and calmodulin-dependent kinases are involved in the regulation of cisplatin-induced lyn expression and activation in macrophages.

In murine peritoneal macrophages cisplatin (cis-dichlorodiammine platinum (II)), a potent chemoimmunotherapeutic drug modulates the expression of several cytokines which contain nuclear factor kappa B (NF-kappaB) binding site suggesting the involvement of NF-kappaB in the activation process of macrophages by cisplatin. Therefore, we analyzed the effect of cisplatin treatment on NF-kappaB expression and activation in macrophages. The underlying mechanism of NF-kappaB translocation was also investigated. Cisplatin treatment increased cellular NF-kappaB content in macrophages treated for 60 min. NF-kappaB translocation was biphasic. Cisplatin-induced translocation of NF-kappaB took place within 5 min and reached its optimum by 15 min. A second phase of nuclear transfer of NF-kappaB was observed at 3 h of cisplatin treatment which was dependent on H2O2 production in cisplatin-treated macrophages. It was observed that cisplatin-induced NF-kappaB translocation involves serine/threonine phosphatases 1/2A, protein tyrosine phosphatases and genestein sensitive protein tyrosine kinase activities. H-7 sensitive protein kinase C do not fall in the signaling pathway of cisplatin leading to the translocation of NF-kappaB. Both cytosolic and membrane-associated factors were required for the induction of NF-kappaB translocation by cisplatin in macrophages.

The role of leucocyte function associated antigen-1 (LFA-1) (CD11a/18) in the tumoricidal activity of cisplatin-treated macrophages was investigated. Anti-LFA-1 antibodies inhibited cisplatin-induced macrophage cytotoxicity towards three different tumour cell lines. The decrease in tumoricidal activity of cisplatin-treated macrophages was attributed to their decreased binding to tumour cells in the presence of anti-LFA-1 (CD11a/18) antibodies. Western blot analysis revealed that cisplatin treatment leads to the expression of LFA-1 on macrophages which otherwise remains non-detectable. Because there is no information regarding the mechanism of cisplatin-induced LFA-1 expression and tumour cell binding by macrophages, the role of various second messenger molecules in these processes was investigated. Results suggest that protein phosphatase 2A (PP2A) is not involved in these processes whereas protein tyrosine phosphatases (PTP) negatively regulate LFA-1 expression and tumour-cell binding of cisplatin-treated macrophages. Inhibitors of protein phosphatase 1 (PP1), protein kinase C (PKC), protein tyrosine kinase (PTK), calmodulin and calmodulin-dependent kinase-II (CamK II) prevented LFA-1 expression on cisplatin-treated macrophages. A comparison with earlier results indicated that LFA-expression follows a distinct signalling pathway which is separate from the signalling pathway involved in NO or tumour necrosis factor/interleukin-1 (TNF/IL-1) expression in cisplatin-stimulated macrophages.

In the present study it was investigated whether cisplatin-treated murine peritoneal macrophages produce oncostatin M (OSM) and what is the underlying mechanism. The culture supernatants of cisplatin-treated macrophages significantly inhibited the proliferation of OSM-sensitive cell line A375. Within 15 min of cisplatin treatment significant OSM was synthesized and secreted by macrophages. Inhibitors of serine/threonine and protein tyrosine phosphatases augmented cisplatin-induced OSM production of macrophages. The protein kinase C and protein tyrosine kinase inhibitors significantly inhibited OSM production of cisplatin-treated macrophages. The OSM production of cisplatin-treated macrophages was also inhibited in the presence of Ca2+ chelators, Ca2+ channel blocker and calmodulin/calmodulin-dependent kinase inhibitors. These data suggest that OSM production of cisplatin-treated macrophages is regulated by opposing actions of phosphatases and kinases. It is also suggested that OSM production of cisplatin-treated macrophages is dependent on Ca2+, calmodulin and calmodulin-dependent kinase.

Late recurrent primary biliary cirrhosis (PBC) following orthotopic liver transplant remains a controversial topic. The first documented case of recurrence occurring in 16 patients transplanted for PBC and followed at the authors' institution for longer than one year is presented. A 54-year-old man transplanted for PBC developed a cholestatic pattern of enzyme elevation on post-transplant day (PTD) 1305. Repeat antimitochondrial antibody was strongly positive (1:300 to 1:400). A liver biopsy revealed severe bile duct damage, lymphocytic cholangitis, focal periductal noncaseating granuloma and minimal endotheliitis. Recurrent PBC was diagnosed. At the time of orthotopic liver transplant this patient received induction immunosuppression with OKT3 crossed over to cyclosporine (CsA), azathioprine (AZA) and prednisone. AZA was discontinued early and maintenance CsA tapered to a target trough level of 150 to 200 ng/mL by PTD 365. Prednisone was withdrawn by PTD 664. CsA levels during PTDs 1225 to 1305 (before elevation of hepatobiliary enzymes) were below target at 114 to 166 ng/mL. Of the 16 patients, all but three were maintained on CsA, AZA and prednisone. One was on CsA (trough levels on target) and AZA; the other two, including the patient with recurrent PBC, were on CsA only. The trough CsA level of the patient without recurrent PBC has been within the target range. The authors speculate that the underlying defect in immunoregulation in PBC persists post-transplant and that in the patient without recurrent PBC this defect was unmasked by lowered maintenance immunosuppression--allowing recurrence of PBC in a previously stable liver allograft.

Buprenorphine was introduced as a potent analgesic with low abuse potential. Reports of buprenorphine abuse by opiate abusers have accumulated over the years, highlighting its use as a cheap alternative to heroin. The lower potency compared with heroin is being compensated by using a cocktail of buprenorphine with benzodiazepines or cyclizine. This study of 18 cases seen over 3 years broadly confirms these findings. Four cases reported haematemesis during acute withdrawal, a symptom not reported in earlier studies.

The purified brain glutamine synthetase of a teleostean fish, Clarias batrachus has been examined under the influence of divalent metal ions activation and in vitro studies using different amino acids and metabolic modulators. All observations of the enzyme activity are based on transferase reaction at optimum temperature and pH. The enzyme exhibits an absolute requirement for Mn2+ (most potent) Mg2+ and Co2+. The activity is markedly inhibited by leucine, asparagine, isoleucine, carbamyl phosphate, uridine monophosphate and glutamate and activated by alpha-ketoglutarate. The significance of these results has been discussed in brief with emphasis on its involvement in teleostean physiology and metabolism.

Liver arginase of Clarias batrachus has been purified to 56.3-fold employing ammonium sulphate fraction, DEAE-cellulose and CM-cellulose chromatography. Bidirectional polyacrylamide gel electrophoresis shows the presence of two isoenzymes of arginase. The enzyme has a molecular weight of about 87,000 and Km 15.38 mM for L-arginine, optimum pH 9.5 and temperature 37 degrees C. Ornithine and leucine as competitive whereas valine and isoleucine act as non-competitive inhibitors with respect to L-arginine as substrate.

Glutamine synthetase (GS) from brain of Clarias batrachus is purified to about 42-fold and characterized at optimum pH and temperature with respect to its kinetic parameters. Values for apparent Michaelis constant of the enzyme for L-glutamine, hydroxylamine and ADP are 50, 62.5 and 0.833 mM respectively. The very low apparent Km for ADP may be specially related to the expression of GS action under high energy bond and also be evidenced by the requirement of enzyme for a high ionic strength of the ADP. The study is extended by examining the effect of various amino acids and metabolites on GS activity in order to gain further understanding of the changes in kinetics and regulation. It reveals that whereas uridine monophosphate and glutamate act competitively with respect to L-glutamine, carbamylphosphate and asparagine act non-competitively. GS activity is markedly inhibited by leucine, aspartic acid and AMP but not by lysine. ATP and methionine sulfoximine behaved as potent inhibitors of the enzyme in vitro. It is suggested that the teleostean GS has most of the properties similar to those reported for mammalian and avian glutamine synthetases. However, it is proposed that kinetic regulation of this enzyme may play a significant role in ammonia detoxication and rate of formation of glutamine-derived neurotransmitters in fish brain.

The composition of reaction mixture of glutamine synthetase (GS) assay system was perfected and utilized to determine the activity of this enzyme spectrophotometrically in selected tissues of the freshwater teleostean fish, Clarias batrachus. Of these tissues, brain was found to contain comparatively a very high activity representing a rapid role of GS in ammonia detoxication and synthesis of essential neurotransmitter substance in this tissue. Of other tissues, liver, kidney and gill were found to contain significant activities in the order representing their relative metabolic activities. The study was extended by examining the brain (neural) and liver (non-neural) GS system in more detail with a view to see the alterations (if any). GS activity of both, neural and non-neural tissues was found to be the same and also in the range reported for other Vertebrates. Observations regarding kinetic, physical and chemical properties of the enzyme are reported. Maximum enzyme activity was observed at pH 7.2 to 7.4 and temperature 35 degrees C. The enzyme was found to be more stable at 25 degrees C while activity decreased at higher temperatures (above 40 degrees C) and showed no activity at 60 degrees C (liver) and 70 degrees C (brain). A comparison and possible physiological roles of the enzyme for the concept of ammonia excretion, protein synthesis and nitrogen metabolism in teleostean fish tissues are discussed.

AGNIHOTRA is a simple vedic ritual of lightening a pyramid of fire in a small copper pot and giving offering of Ghee & Rice on this fire at the time of sunset and sunrise with enchanting of two mantras. It is reported to enhance the state of tranquility of mind and is reported to be of benefit to those addicted to various types of intoxicants. We used Agnihotra in a young smack addict who was poorly motivated and resisted all efforts to help him even when he got over the physical withdrawal features. The results were encouraging. Over a period of 4 weeks, we found him to be a totally changed man who could go back to his work without any drugs. Follow up for more than a year, revealed no recurrence and positive achievements in his work performance. The paper presents practice of Agnihotra, also known as homa therapy, and discusses its role in drug addiction.