Thymoquinone supplementation ameliorates cisplatin-induced hepatic pathophysiology.

Hepatotoxicity is a major dose-limiting side effect of CP chemotherapy besides nephrotoxicity and gastrointestinal dysfunction. TQ, a principal Nigella sativa seed oil constituent, has been shown to improve hepatic functions in various in vivo models of acute hepatic injury. In view of this, the present study aimed to evaluate the effect of TQ against CP-induced hepatotoxicity. Rats were divided into four experimental groups; control, CP, CP+TQ and TQ. Animals in CP+TQ and TQ groups were administered TQ (1.5 mg/kg bwt, orally), with or without a single hepatotoxic dose of CP (6 mg/kg bwt, i.p.) respectively, for 14 days before and four days following the CP treatment. CP induced an upsurge in serum ALT and AST activities, indicating liver injury, as also confirmed by the histopathological findings. CP caused significant alterations in the activities of membrane marker enzymes, carbohydrate metabolic enzymes, and the enzymatic and nonenzymatic components of the antioxidant defense system. TQ supplementation ameliorated all these adverse biochemical and histological changes in CP-treated rats. Thus, TQ may have excellent scope for clinical applications in combating CP-induced hepatic pathophysiology.

Aspirin treatment failure: is this a real phenomenon? A review of the aetiology and how to treat it.

Multiple clinical trials have shown that aspirin can reduce all cardiovascular events in primary and secondary prevention and yet there is a large population in whom aspirin fails. This review brings together the evidence and controversies surrounding the definition of 'aspirin treatment failure', its clinical significance and the possible approaches to managing such patients. Several different assays have been developed to measure the biochemical action of aspirin. At present there is no 'gold standard' and there is massive disparity between methods. Studies thus far have shown inconsistent results and to date the treatment of aspirin therapy failure is left to the discretion of the leading physician.

Intra-aortic balloon pump counterpulsation: are we optimizing the management of cardiogenic shock?

Intra-aortic balloon pump counterpulsation (IABPC) has been used in various forms for decades. The change in physiology brought about by their use is conceptually appealing in managing cardiogenic shock and mechanical complications of myocardial infarction. A common myth is that this method of managing acute cardiological emergencies is to be limited to the realms of this specialist field. However, as medical physicians an appreciation and understanding of this novel therapy is essential not only as a lifesaving measure but also as a bridging therapy to more definitive management in the acute medical setting. IABPC is a safe and under-utilized technique despite featuring in all major international guidelines (ESC and ACC) for the management of cardiogenic shock secondary to acute coronary syndromes. Without awareness of this intervention we may be suboptimally managing patients in the first instance. To improve awareness we examine the evidence supporting the use of the IABPC therapy and the contraindications to their use. Complications and advances in technology are also addressed.

Dynamic changes in biomarker profiles associated with clinical and subclinical tuberculosis in a high transmission setting: a four-year follow-up study.

Mycobacterium tuberculosis (MTB) is a slow growing bacterium. Therefore, the immune responses associated with resolution of infection or development of disease post-exposure may take several months to evolve. We have carried out a prospective longitudinal study in a high TB transmission setting to determine the evolution of biomarkers in a recently exposed household contact (HC = 77) and their respective sputum positive index cases (TB = 17). Mycobacterium-induced cytokines [interferon-gamma (IFN-gamma), tumour necrosis factor-alpha, interleukin-6 (IL-6) and IL-10)] were assessed in whole blood cultures and immunoglobulin G (IgG1) antibodies in plasma. When compared with non-exposed community controls (endemic controls = 59) the HC group at intake showed changes in biomarkers commensurate with recent exposure. The HC group showed significant increases in IFN-gamma between 0 and 6 months (paired t-test; P = 0.001) and IL-0 between 6 and 12 months (P = 0.001), most likely reflecting the role of these cytokines in resolution and immune recovery from infection as this HC cohort remained symptom-free for 4 years without prophylactic treatment. When the TB group post-treatment was compared with the HC group, the best discriminators (ANOVA; repeated measures) were IL-10 responses at 0 (P = 0.004) and 6 months (P = 0.001) and IgG1 at 6 (P = 0.004) and 12 months (P = 0.014) with a 3-4 fold higher responses in the TB group. Therefore, within each group, biomarkers show unique profile of responses. These studies highlighted the importance of assessing multiple biomarkers in longitudinal studies for providing better understanding of protective biomarker profiles associated with resolution of clinical and subclinical infections in TB.

A postgenomic approach to identification of Mycobacterium leprae-specific peptides as T-cell reagents.

To identify Mycobacterium leprae-specific human T-cell epitopes, which could be used to distinguish exposure to M. leprae from exposure to Mycobacterium tuberculosis or to environmental mycobacteria or from immune responses following Mycobacterium bovis BCG vaccination, 15-mer synthetic peptides were synthesized based on data from the M. leprae genome, each peptide containing three or more predicted HLA-DR binding motifs. Eighty-one peptides from 33 genes were tested for their ability to induce T-cell responses, using peripheral blood mononuclear cells (PBMC) from tuberculoid leprosy patients (n = 59) and healthy leprosy contacts (n = 53) from Brazil, Ethiopia, Nepal, and Pakistan and 20 United Kingdom blood bank donors. Gamma interferon (IFN-gamma) secretion proved more sensitive for detection of PBMC responses to peptides than did lymphocyte proliferation. Many of the peptides giving the strongest responses in leprosy donors compared to subjects from the United Kingdom, where leprosy is not endemic, have identical, or almost identical, sequences in M. leprae and M. tuberculosis and would not be suitable as diagnostic tools. Most of the peptides recognized by United Kingdom donors showed promiscuous recognition by subjects expressing differing HLA-DR types. The majority of the novel T-cell epitopes identified came from proteins not previously recognized as immune targets, many of which are cytosolic enzymes. Fifteen of the tested peptides had > or =5 of 15 amino acid mismatches between the equivalent M. leprae and M. tuberculosis sequences; of these, eight gave specificities of > or =90% (percentage of United Kingdom donors who were nonresponders for IFN-gamma secretion), with sensitivities (percentage of responders) ranging from 19 to 47% for tuberculoid leprosy patients and 21 to 64% for healthy leprosy contacts. A pool of such peptides, formulated as a skin test reagent, could be used to monitor exposure to leprosy or as an aid to early diagnosis.

Diagnostic assays for leprosy based on T-cell epitopes.

To date, only a limited number of antigens have been described as specific for Mycobacterium leprae, and in many cases, homologues have subsequently been shown to exist in mycobacteria such as M. avium and M. intracellulare. A Leprosy Synthetic Peptide Skin Test Initiative was established by the Steering Committee on the Immunology of Mycobacteria of the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases, to investigate the potential of synthetic peptides that encode T-cell epitopes as diagnostic tools, which could be used to develop a skin-test reagent specific for leprosy. Such M. leprae-specific peptides should have unique amino acid sequences, or significant sequence-dissimilarity from those in other mycobacteria. Synthetic peptides, 15 amino acids long, were synthesised from 33 genes or open reading frames within the M. leprae genome. Tuberculoid leprosy patients from four leprosy-endemic countries, Brazil, Ethiopia, Nepal and Pakistan, were tested as subjects known to have been infected with M. leprae, and to make good T-cell responses to antigens of M. leprae; UK blood donors were used as non-exposed or non-infected subjects. Peptides inducing potentially specific responses in leprosy patients and not in UK controls, and those inducing cross-reaction responses, present in both leprosy patients and non-exposed, non-infected controls, were identified. A difference from the equivalent M. tuberculosis sequence of five or more amino acid residues did not, by itself, identify peptides that were M. leprae-specific, suggesting that many of these peptides may have homologues in environmental mycobacteria. To date, this approach has identified a number of peptides with greater than 90% specificity and 19-47% sensitivity, which are undergoing further specificity-testing. Such peptides would have great potential as T-cell reagents with which to monitor exposure to M. leprae within communities, formulated either as skin-test reagents, or as antigens for tests in vitro.

Leprosy patients with lepromatous disease recognize cross-reactive T cell epitopes in the Mycobacterium leprae 10-kD antigen.

T cell responses play a critical role in determining protective responses to leprosy. Patients with self-limiting tuberculoid leprosy show high T cell reactivity, while patients with disseminated lepromatous form of the disease show absent to low levels of T cell reactivity. Since the T cell reactivity of lepromatous patients to purified protein derivative (PPD), a highly cross-reactive antigen, is similar to that of tuberculoid patients, we queried if lepromatous patients could recognize cross-reactive epitopes in Mycobacterium leprae antigens as well. T cell responses were analysed to a recombinant antigen 10-kD (a heat shock cognate protein) which is available from both M. tuberculosis (MT) and M. leprae (ML) and displays 90% identity in its amino acid sequence. Lymphoproliferative responses were assessed to ML and MT 10 kD in newly diagnosed leprosy patients (lepromatous, n = 23; tuberculoid, n = 65). Lepromatous patients showed similar, but low, lymphoproliferative responses to ML and MT 10 kD, while tuberculoid patients showed much higher responses to ML 10 kD. This suggests that the tuberculoid patients may be recognizing both species-specific and cross-reactive epitopes in ML 10 kD, while lepromatous patients may be recognizing only cross-reactive epitopes. This was further supported by linear regression analysis. Lepromatous patients showed a high concordance in T cell responses between ML and MT 10 kD (r=0.658; P<0.0006) not observed in tuberculoid patients (r=0.203; P>0.1). Identification of cross-reactive T cell epitopes in M. leprae which could induce protective responses should prove valuable in designing second generation peptide-based vaccines.

Low serum HDL-cholesterol is associated with raised tumor necrosis factor-alpha during ENL reactions.

The concentrations of serum lipids and tumor necrosis factor (TNF) were measured in leprosy patients across the spectrum of the disease and in erythema nodosum leprosum (ENL) patients at the onset of the reaction and after the reaction had clinically subsided. Lepromatous/borderline lepromatous (LL/BL) patients had significantly higher serum triglyceride and lower HDL-cholesterol levels; there was no such change in the tuberculoid/borderline tuberculoid (TT/BT) patients. The household contacts (HC) of the LL/BL patients also had significantly lower serum HDL levels. ENL patients during the acute phase of the reaction had significantly lower total, LDL-, HDL-cholesterol levels compared to the stable LL/BL patients, and these changes were reversible to pre-ENL levels after the reaction had subsided. Serum TNF levels were significantly higher in household contacts and in LL/BL patients but were not statistically different in TT/BT patients. Serum TNF levels were also significantly higher during the acute phase of ENL, and declined after the clinical remission of the reaction to levels comparable with those of LL/BL patients. There was a significant negative correlation between serum TNF and HDL-cholesterol levels during and after ENL reaction. However, there was no such correlation between TNF and total or LDL-cholesterol levels in ENL patients. Our results suggest that the changes in HDL-cholesterol metabolism are a specific part of the host response to lepromatous leprosy and to the ENL reaction and may be mediated by increased TNF production.

Erythema nodosum leprosum is associated with up-regulation of polyclonal IgG1 antibody synthesis.

Erythema nodosum leprosum (ENL) is a serious complication of lepromatous (L) disease in leprosy. We have previously shown that of the four IgG subclasses, IgG1 and IgG3 Mycobacterium leprae-specific antibodies are significantly lower in leprosy patients during ENL reaction compared with untreated L patients. To see if this decrease results from a down-regulation of antibody synthesis during ENL, the frequency of antibody-secreting B cells (ABSC) in the blood compartment was determined by ELISPOT and related to serum immunoglobulin concentrations (microgram/ABSC). Control groups consisted of 16 patients with stable L disease and 32 healthy endemic controls (EC). Paired samples were analysed during acute ENLS (n = 13) and after the reaction had subsided to identify changes associated with ENL. Polyclonal (PC) IgG1 was elevated in L patients compared with EC (325 micrograms versus 180 micrograms). Interestingly, patients during acute ENL showed concentrations higher than L patients (419 micrograms), which decreased after the reaction had subsided (260 micrograms), indicating the transient nature of the antibody response. IgG2 antibodies showed the reverse trend and were lower during ENL and increased after the reaction had subsided. The mean concentrations for PC IgG3 and IgG4 antibodies were similar during ENL and after the reaction had subsided. Thus, decrease in M. leprae-specific IgG1 and IgG3 antibodies is not related to down-regulation of B cell responses. Identification of factors which regulate PC IgG1 antibody synthesis may provide additional insights into determinants of ENL reactions.

Induction of Th1 cytokine responses by mycobacterial antigens in leprosy.

Twelve mycobacterial antigens were compared for induction of gamma interferon (IFN-gamma) secretion by human blood mononuclear cells of patients with leprosy. Fractionated Mycobacterium leprae antigens containing cell wall proteins or cytosolic and membrane proteins induced good IFN-gamma responses in tuberculoid leprosy patients. Lipoarabinomannan from M. tuberculosis Erdman and M. leprae mycolylarabinogalactan peptidoglycan were the poorest IFN-gamma inducers.

Cross-modulation by transforming growth factor beta in human tuberculosis: suppression of antigen-driven blastogenesis and interferon gamma production.

In tuberculosis, Mycobacterium tuberculosis (MTB)-stimulated T-cell responses are depressed transiently, whereas antibody levels are increased. Lymphoproliferative responses of peripheral blood mononuclear cells (PBMCs) from Pakistani tuberculosis (TB) patients to both mycobacterial and candidal antigens were suppressed by approximately 50% when compared to healthy purified protein derivative (PPD)-positive household contacts. Production of interferon gamma (IFN-gamma) in response to PPD also was depressed by 78%. Stimulation with PPD and the 30-kDa alpha antigen of MTB (30-kDa antigen) induced greater secretion of transforming growth factor beta (TGF-beta), but not interleukin 10 (IL-10) or tumor necrosis factor alpha (TNF-alpha), by PBMCs from TB patients compared to healthy contacts. The degree of suppression correlated with the duration of treatment; patients treated for <1 month had significantly lower T-cell blastogenesis and IFN-gamma production and higher levels of TGF-beta than did patients treated for >1 month. Neutralizing antibody to TGF-beta normalized lymphocyte proliferation in response to PPD, partially restored blastogenesis to candidal antigen, and significantly increased PPD-stimulated production of IFN-gamma in TB patients but not in contacts. Neutralizing antibody to IL-10 augmented, but did not normalize, T-cell responses to both PPD and candida in TB patients and candidal antigen in contacts. TGF-beta, produced in response to MTB antigens, therefore plays a prominent role in down-regulating potentially protective host effector mechanisms and looms as an important mediator of immunosuppression in TB.

Resonance energy transfer between cysteine-34 and tryptophan-214 in human serum albumin. Distance measurements as a function of pH.

The single cysteine residue (Cys-34) of human serum albumin was modified with the organic mercurial [4-[p-(dimethylamino)phenyl]azo]phenyl]mercuric acetate. Introduction of this chromophore into the protein results in the quenching of the protein tryptophan fluorescence spectrum due to energy transfer from the tryptophan residue to the mercurial. Since human albumin contains only a single tryptophan, it was then possible to calculate distances between the mercurial bound at Cys-34 and Trp-214 under various conditions. This distance contracted during the course of the N leads to F transition, being 34-35 A in the N conformation (pH 6-7.5) and 29.9 A in the F conformation (pH 3.6). The distance increased substantially during the course of the F leads to E transition occurring between pH 3.6 and pH 1.9 and was found to be nearly 37 A at pH 1.9. The distance between Cys-34 and Trp-214 was found to undergo a slight contraction during the N leads to B transition occurring between pH 7.0 and pH 9.0. At pH 8.5-9 where the protein is predominately in the B form, the distance was found to be slightly more than 31 A.

The lanthanide-induced N = to F transition and acid expansion of serum albumin.

Serum albumin exists in the native or N conformation between pH 5 and 7. As the pH is lowered from 5 to 3.5, the protein undergoes a conformational change resulting in expansion, known as the N = to F (partially acid expanded) transition. As the pH is lowered still further to 2, the protein continues to expand. In the present study, using the techniques of circular dichroism, fluorescence, and UV difference spectroscopy, lanthanide ions at concentrations between 1-30 mM have been shown to produce both changes in the albumin structure analogous to the N = to F transition and acid expansion of bovine serum albumin at a constant pH near 6.