Background Ending the global tuberculosis (TB) epidemic requires a focus on treating individuals with latent TB infection (LTBI) to prevent future cases. Promising trials of shorter regimens have shown them to be effective as preventative TB treatment, however there is a paucity of data on self-administered treatment completion rates. This pilot trial assessed treatment completion, adherence, safety and the feasibility of treating LTBI in the UK using a weekly rifapentine and isoniazid regimen versus daily rifampicin and isoniazid, both self-administered for 12 weeks. Methods An open label, randomised, multi-site pilot trial was conducted in London, UK, between March 2015 and January 2017. Adults between 16 and 65 years with LTBI at two TB clinics who were eligible for and agreed to preventative therapy were consented and randomised 1:1 to receive either a weekly combination of rifapentine/isoniazid (‘intervention’) or a daily combination of rifampicin/isoniazid (‘standard’), with both regimens taken for twelve weeks; treatment was self-administered in both arms. The primary outcome, completion of treatment, was self-reported, defined as taking more than 90% of prescribed doses and corroborated by pill counts and urine testing. Adverse events were recorded. Results Fifty-two patients were successfully enrolled. In the intervention arm 21 of 27 patients completed treatment (77.8, 95% confidence interval [CI] 57.7–91.4), compared with 19 of 25 (76.0%, CI 54.9–90.6) in the standard of care arm. There was a similar adverse effect profile between the two arms. Conclusion In this pilot trial, treatment completion was comparable between the weekly rifapentine/isoniazid and the daily rifampicin/isoniazid regimens. Additionally, the adverse event profile was similar between the two arms. We conclude that it is safe and feasible to undertake a fully powered trial to determine whether self-administered weekly treatment is superior/non-inferior compared to current treatment. Trial registration The trial was funded by the NIHR, UK and registered with ISRCTN ( 26/02/2013-No.04379941 ).

Background Emergency Department (ED) presentations could present a valuable opportunity to identify high-risk individuals with latent tuberculosis infection (LTBI) and active tuberculosis (TB) disease. We aimed to investigate the prevalence and cost-effectiveness of screening for LTBI and pulmonary TB disease among migrants and under-served groups attending London EDs. Methods We recruited adults attending EDs (18/7/2013–25/5/2017) who were either recent entrants from high TB-incidence countries; or had a history of homelessness, imprisonment, or substance use disorder. We assessed the yield of screening for LTBI (using an interferon-gamma release assay; IGRA) and active pulmonary TB (with a symptom screen, followed by chest radiograph and sputum Xpert MTB/RIF if positive). A cost-effectiveness analysis was performed by calculating cost per true-positive LTBI test. Results A total of 1420 participants were screened for LTBI. Median age was 45 years (IQR 34–60), and the majority were born outside the UK (1,162/1,420; 81.8%). A total of 241/1,420 participants (17.0%) had a history of either substance use disorder, homelessness or imprisonment. Of those with available IGRA results, 214/1,246 (17.2%) were positive. Male sex, age >35 years, non-UK country of birth, and previous TB contact were independent risk factors for being IGRA-positive. Of the 214 with a positive IGRA, 120 (56%) were followed-up by health record review for evidence of progression to TB disease; 5/120 (4.2%) were diagnosed with TB disease (median interval to TB 162 days (IQR 55–108.5)). In the economic analysis, the average cost per patient recruited of correctly diagnosing an LTBI case was £834 (compared to an estimated £1007 per LTBI case diagnosed through primary care). Only 14/513 (2.7%) participants screened for active TB had a positive symptom screen so were eligible for further testing. No cases of active TB were diagnosed. Conclusions Targeted LTBI screening in EDs could be considered to complement the primary care LTBI screening programme. In contrast, active TB screening in EDs using our symptom screen-led algorithm should not be pursued as the yield is likely to be minimal. Other active TB screening models (e.g. routine, automated reviews of ED chest radiographs) could be considered as a focus of future research.

In healthy individuals, prolonged intensive physical exercise leads to an activation of blood coagulation that results in the formation of thrombin and fibrin. This study investigated whether oxidative stress during intensive physical exercise induces tissue factor (TF) via activation of the redox-responsive transcription factor nuclear factor-kappaB (NF-kappaB). Twelve young men performed a standardized 1-h maximal run on a treadmill that gave rise to significant increases of markers of thrombin and fibrin formation. The ratio of intracellular reduced to oxidized glutathione as measured by HPLC decreased from 23.3 +/- 10.7 to 14.2 +/- 6.5 (P < 0.05), indicating the generation of free radicals during exercise. Electrophoretic mobility shift assays from nuclear extracts of peripheral blood mononuclear cells revealed that exercise testing increased NF-kappaB (p50/p65) binding activity to a NF-kappaB consensus sequence by 105 +/- 68% (P < 0.01) but did not affect NF-kappaB (p65/c-Rel) binding to a nonconsensus-kappaB-like site present in the TF promoter. Consistently, there was no exercise-induced increase in TF expression as demonstrated by TF-specific immunofluorescence staining and ELISA. Thus selective activation of NF-kappaB (p50/p65) during intensive physical exercise does not result in the expression of TF, suggesting that the TF-dependent pathway in peripheral blood mononuclear cells does not account for exercise-induced formation of thrombin and fibrin.

Abnormally low intramuscular glutamate and glutathione (GSH) levels and/or a decreased muscular uptake of glutamate by the skeletal muscle tissue have previously been found in malignant diseases and simian immunodeficiency virus (SIV) infection and may contribute to the development of cachexia. We tested the hypothesis that an impaired mitochondrial energy metabolism may compromise the Na+-dependent glutamate transport. A randomized double-blind clinical trial was designed to study the effects of L -carnitine, i.e. an agent known to enhance mitochondrial integrity and function, on the glutamate transport and plasma glutamate level of cancer patients. The effect of carnitine on the intramuscular glutamate and GSH levels was examined in complementary experiments with tumour-bearing mice. In the mice, L -carnitine treatment ameliorated indeed the tumour-induced decrease in muscular glutamate and GSH levels and the increase in plasma glutamate levels. The carnitine-treated group in the randomized clinical study showed also a significant decrease in the plasma glutamate levels but only a moderate and statistically not significant increase in the relative glutamate uptake in the lower extremities. Further studies may be warranted to determine the effect of L -carnitine on the intramuscular GSH levels in cancer patients. © 2000 Cancer Research Campaign

. To determine the therapeutic effect of sulfur amino acid supplementation in HIV infection we randomized 40 patients with antiretroviral therapy (ART; study 1) and 29 patients without ART (study 2) to treatment for 7 months with N -acetyl-cysteine or placebo at an individually adjusted dose according to a defined scheme. The main outcome measures were the change in immunological parameters including natural killer (NK) cell and T cell functions and the viral load. Both studies showed consistently that N-acetyl-cysteine causes a marked increase in immunological functions and plasma albumin concentrations. The effect of N -acetyl-cysteine on the viral load, in contrast, was not consistent and may warrant further studies. Our findings suggest that the impairment of immunological functions in HIV + patients results at least partly from cysteine deficiency. Because immune reconstitution is a widely accepted aim of HIV treatment, N -acetyl-cysteine treatment may be recommended for patients with and without ART. Our previous report on the massive loss of sulfur in HIV-infected subjects and the present demonstration of the immunoreconstituting effect of cysteine supplementation indicate that the HIV-induced cysteine depletion is a novel mechanism by which a virus destroys the immune defense of the host and escapes immune elimination.

This chapter reviews the role of antioxidants in senescence and wasting. The hallmarks of senescence as well as wasting include the massive loss of body cell mass (bcm) and muscle function, decreased resistance to infections, frailty (increased probability of disability), and organ failure. Wasting is a common phenomenon in malignancies, sepsis, trauma, and certain infectious diseases, including HIV infection. In the treatment of cancer patients, wasting is often a limiting factor that prevents the application of aggressive chemotherapy. In old age, the loss of skeletal muscle mass is associated with a compromised physical and social function. An increasingly popular hypothesis states that senescence may result from the accumulation of oxidative damage and that dietary antioxidants may slow the degenerative process. In line with this hypothesis, vitamin E is shown to ameliorate age-related health problems, and certain age-related degenerative processes are even found to be reversed by treatment with antioxidants.

698 The nuclear factor kappa B (NF-κB) plays a role in the transcriptional regulation of tissue factor, the cellular initiator of the coagulation cascade. The aim of this study was to evaluate the impact of physical exercise on the activation of NF-κB. Twelve young men (age: 24 ± 5 years, VO2max: 68 ± 5 ml/kg/min [mean ± SD]) were subjected to a 1 hour run on a treadmill at a velocity corresponding to 75 - 80% of VO2max. Binding activity for NF-κB consensus probes was determined by electrophoretic mobility shift assay in nuclear extracts of mononuclear cells before and immediately after exercise. Since the activation of NF-κB is redox-regulated, we also assessed the intracellular glutathione status as an important antioxidative defense mechanism measuring concentrations of reduced (GSH) and oxidized glutathione (GSSG) by high-pressure liquid chromatograpy (HPLC) in monocytes. One hour of exhaustive running lead to an increase of NF-κB binding activity by 105 ± 68% (P<0.01). The ratio of GSH/GSSG decreased significantly from 23.3 ± 10.7 to 14.2 ± 6.5 (P<0.01) thus indicating the generation of free radicals during exercise. In conclusion, our data demonstrate that intensive physical exercise affects the transcriptional regulation by activation of NF-κB which may be linked to oxidative stress.

Signaling by insulin requires autophosphorylation of the insulin receptor kinase (IRK) at Tyr1158, Tyr1162, and Tyr1163. Earlier experiments with 32P‐γ‐ATP indicated that the nonphosphorylated IRK (IRK‐0P) is relatively inactive, and crystallographic data indicated that the ATP binding site of IRK‐0P is blocked by its activation loop. We now show that phosphocreatine (PCr) in combination with hydrogen peroxide serves as an alternative phosphate donor and that ATP and PCr use distinct binding sites. Whereas phosphorylation of the IRK by ATP is inhibited by the nonhydrolyzable competitor adenylyl‐imidodiphosphate, phosphorylation by PCr is enhanced. The IRK mutant Tyr1158Phe showed no phosphorylation with PCr but almost normal phosphorylation with ATP, whereas Tyr1162Phe was phosphorylated well with PCr but less then normal with ATP. 3‐Dimensional models of IRK‐0P revealed that the conversion of any of the four cysteine residues 1056, 1138, 1234, and 1245 into sulfenic acid produces structural changes that bring Tyr1158 into close contact with Asp1083 and render the well‐known catalytic site at Asp1132 and Tyr1162 accessible from a direction that differs from the known ATP binding site. The mutant Cys1138Ala, in contrast, showed relatively inaccessible catalytic sites and weak catalytic activity in functional experiments. Taken together, these findings indicate that ‘redox priming’ of the IRK facilitates its autophosphorylation by PCr in the activation loop.—Schmid, E., Hotz‐Wagenblatt, A., Hack, V., Droge, W. Phosphorylation of the insulin receptor kinase by phosphocreatine in combination with hydrogen peroxide: the structural basis of redox priming. FASEB J. 13, 1491–1500 (1999)

The mechanism of wasting, as it occurs in malignant diseases and various etiologically unrelated conditions, is still poorly understood. We have, therefore, studied putative cause/effect relationships in a murine model of cancer cachexia, C57BL/6 mice bearing the fibrosarcoma MCA-105. The plasma of these mice showed decreased albumin and increased glutamate levels, which are typically found in practically all catabolic conditions. Skeletal muscles from tumor-bearing mice were found to have an abnormally low mitochondrial respiratory chain activity (mito.RCA) and significantly decreased glutathione (GSH) levels. The decrease in mito.RCA was correlated with an increase in the i.m. GSH disulfide/GSH ratio, the plasma cystine/thiol ratio, and the GSH disulfide/GSH ratio in the bile. This is indicative of a generalized shift in the redox state extending through different body fluids. Treatment of tumor-bearing mice with ornithine, a precursor of the radical scavenger spermine, reversed both the decrease in mito.RCA and the change in the redox state, whereas treatment with cysteine, a GSH precursor, normalized only the redox state. Treatment of normal mice with difluoromethyl-ornithine, a specific inhibitor of ornithine decarboxylase and spermine biosynthesis, inhibited the mito.RCA in the skeletal muscle tissue, thus illustrating the importance of the putrescine/spermine pathway in the maintenance of mito.RCA. Ornithine, cysteine, and N-acetyl-cysteine (NAC) also reconstituted the abnormally low concentrations of the GSH precursor glutamate in the skeletal muscle tissue of tumor-bearing mice. Higher doses, however, enhanced tumor growth and increased the plasma glucose level in normal mice. In the latter, cysteine and NAC also decreased i.m. catalase and GSH peroxidase activities. Taken together, our studies on the effects of ornithine, cysteine, and NAC illuminate some of the mechanistic pathways involved in cachexia and suggest targets for therapeutic intervention.

Wilson's disease can result in fulminant liver failure due to hepatic copper overload. The CD95 system mediates apoptosis and has been demonstrated to be involved in liver disease. In this study CD95 mediated apoptosis was investigated in patients with fulminant hepatic failure in the course of Wilson's disease and in an in vitro model of copper treated human hepatoma cells. In patients, hepatic expression of CD95 and CD95L mRNA and apoptosis were detected. Copper overload in vitro resulted in hepatocytic apoptosis which could be reduced with a neutralizing anti-CD95L antibody. Copper treatment of hepatocytes results in activation of the CD9S system and induction of apoptosis which is operative during the course of hepatic failure in acute Wilson's disease.

Background-Reactive oxygen species contribute to tissue injury in inflammatory bowel disease (IBD). The tripeptide glutathione (GSH) is the most important intracellular antioxidant.Aims-To investigate constituent amino acid plasma levels and the GSH redox status in different compartments in IBD with emphasis on intestinal GSH synthesis in Crohn's disease.Methods-Precursor amino acid levels were analysed in plasma and intestinal mucosa. Reduced (rGSH) and oxidised glutathione (GSSG) were determined enzymatically in peripheral blood mononuclear cells (PBMC), red blood cells (RBC), muscle, and in non-inflamed and inflamed ileum mucosa. Mucosal enzyme activity of gamma-glutamylcysteine synthetase (gamma GCS) and gamma-glutamyl transferase (gamma GT) was analysed. Blood of healthy subjects and normal mucosa from a bowel segment resected for tumour growth were used as controls.Results-Abnormally low plasma cysteine and cystine levels were associated with inflammation in IBD (p<10(-4)). Decreased rGSH levels were demonstrated in noninflamed mucosa (p<0.01) and inflamed mucosa (p=10(-6)) in patients with IBD, while GSSG increased with inflammation (p=0.007) compared with controls. Enzyme activity of gamma GCS was reduced in non-inflamed mucosa (p<0.01) and, along with gamma GT, in inflamed mucosa (p<10(-4)). The GSH content was unchanged in PBMC, RBC, and muscle.Conclusions-Decreased activity of key enzymes involved in GSH synthesis accompanied by a decreased availability of cyst(e)ine for GSH synthesis contribute to mucosal GSH deficiency in IBD. As the impaired mucosal antioxidative capacity may further promote oxidative damage, GSH deficiency might be a target for therapeutic intervention in IBD.

The loss of body cell mass (bcm) in senescence and wasting is poorly understood. We now show that the plasma cystine/acid soluble thiol ratio, ie, an indicator of the redox state, is increased in old age and cancer patients and correlated with a decrease in bcm and plasma albumin. A cause/effect relationship was suggested by two independent studies with N-acetyl-cysteine (NAC). NAC caused an increase in the bcm of healthy persons with high plasma cystine/thiol ratios, and treatment of cancer patients with NAC plus interleukin-2 caused an increase in bcm, plasma albumin, and functional capacity. Albumin levels below 680 micromol/L were associated with an increase in body water. Our studies suggest that the shift in the redox state may contribute to the loss of bcm and may provide a quantitative guideline for therapeutic intervention. Treatment of cancer patients with thiol-containing antioxidants may improve the quality of life.

Loss of skeletal muscle tissue (cachexia) is one of the hallmarks of HIV infection. It has been found (1) that creatine kinase, i.e., an enzyme of pivotal importance in muscular mitochondrial energy metabolism, is inhibited by oxidative glutathiolation, and (2) that reduced glutathione (GSH) is decreased in skeletal muscle of SIV-infected rhesus monkeys. We, therefore, have studied the phosphocreatine (P-Cr) levels. Muscle tissue from SIV-infected macaques showed significantly decreased P-Cr but normal creatine (Cr), ATP, and ADP when compared with uninfected macaques. Individual P-Cr levels were significantly correlated with GSH. Our findings may explain the dysregulation of energy metabolism in cachexia.

Several diseases of varying etiology that are commonly associated with the loss of skeletal muscle mass were found to be associated with a decrease in muscular glutamate and glutathione levels and in glutamate uptake in the postabsorptive state. In view of the Na+ dependency and insulin responsiveness of glutamate transport we studied the postabsorptive glutamate exchange in more detail. Our study demonstrates a linkage between glutamate uptake and the export of other amino acids, suggesting that protein catabolism and the resulting coexport of amino acids plus Na+ substitute for insulin as a driving force for the Na+ gradient in the postabsorptive state. The regression function of the correlation between relative glutamate exchange and cumulative amino acid exchange in cancer patients was lower than that in non-tumor-bearing subjects, suggesting that cancer patients must release more amino acids to achieve the same glutamate uptake. In addition, cancer patients had a lower average cumulative amino acid exchange rate than non-tumor-bearing subjects, suggesting that the abnormally low relative glutamate exchange capacity of cancer patients results mainly from inadequate postabsorptive protein catabolism in the skeletal muscle tissue. Both cancer patients and non-tumor-bearing elderly subjects had higher arterial glutamate levels and alanine release than young subjects, indicative of a substantial glycolytic activity in the skeletal muscle. However, elderly non-tumor-bearing subjects showed, in contrast to cancer patients, in the postabsorptive state a stronger cumulative amino acid release and postabsorptive glutamate uptake than healthy young subjects. These changes are discussed in view of the age-related loss of skeletal muscle mass.

© 1997 Federation of European Biochemical Societies.

Glutamin ist das Schlüssel-Substrat zur Energiegewinnung in Enterocyten [1] und damit unentbehrlich für die Proliferation und den Turnover von Mucosazellen. Der Dünndarm ist der Hauptkonsument von Glutamin, wo Glutamin durch die Wirkung der Glutaminase zu Glutamat umgewandelt wird. Der Darm entzieht dem zirkulierenden Blut mit jeder Passage bis zu 30% dieser Aminosäure, während die Muskulatur das Speicherorgan für Glutamin darstellt [2]. Obgleich Glutamin die quantitativ bedeutenste Aminosäure im Körper ist, kommt es in katabolen Krankheitszuständen und nach Langzeitparenteralernährung zu einer Glutamin-Verarmung des Organismus [3]. Folge ist eine Störung der Funktion und Integrität der Darmmucosa, welche durch Substitution von Glutamin wiederhergestellt werden kann [4–6]. Bei der chronisch entzündlichen Darmerkrankung ist der Darm auf eine gesteigerte Glutamin-Zufuhr zur Behebung des Entzündungsschadens angewiesen. Da zur Bedeutung von Glutamin bei Morbus Crohn (MC) keinerlei Daten vorliegen, untersuchten wir den Glutamin-Metabolismus dieser Patienten, um die Notwendigkeit einer Glutamin-Substitution bei MC zu evaluieren.

Oxidative stress has been associated with the induction of programmed cell death. The CD95 ligand/receptor system is a specific mediator of apoptosis. We have used the model of drug-induced apoptosis to assess whether the CD95 ligand mRNA is induced by reactive oxygen intermediates. Treatment of HepG2 hepatoma cells with bleomycin induced the production of reactive oxygen intermediates and, as an additional parameter of oxidative stress, resulted in glutathione (GSH) depletion. In parallel, CD95 ligand mRNA expression was induced. In a similar fashion CD95 ligand mRNA expression increased after treatment with H2O2. Additional treatment with the antioxidant and GSH precursor N-acetylcysteine resulted in partial restoration of intracellular GSH levels and in reduced induction of CD95 ligand mRNA. Induction of CD95 ligand mRNA by bleomycin was further reduced by combined treatment withN-acetylcysteine and deferoxamine. These data suggest a direct role of oxygen radicals in the induction of the CD95 ligand.

The purpose of the present study was to investigate the role of plasma amino acids and glutathione (GSH) on the absolute number of leukocyte and lymphocyte subpopulations in response to different training programs. Healthy untrained subjects were randomly assigned to an 8-wk aerobic (AET) or anaerobic (ANT) exercise training program. Absolute number of cell counts did not significantly change in AET, whereas a decrease of CD4+ T cell counts (P < 0.05), a fall in cells expressing CD45RA+ antigen (P < 0.05), and a marked increase in CD8+ T cell numbers (P < 0.01) were noted in ANT at the end of the training period compared with baseline values. Furthermore, ANT demonstrated a marked rise (P < 0.001) in plasma glutamate from 27.6 +/- 2.8 to 49.8 +/- 5.2 microM and a considerable reduction (P < 0.001) of the plasma glutamine pool from 713 +/- 22 to 601 +/- 30 microM after 8 wk of training. The decrease in glutamine showed a strong positive correlation to the individual loss of CD4+ T cells (r = 0.67, P < 0.001). AET demonstrated a rise (P < 0.05) in GSH from 20.7 +/- 2.5 to 28.1 +/- 1.5 nmol/mg protein at terminal examination. In conclusion, our data indicate impairment of the number and activity of CD4+ T cells in response to 8 wk of ANT, which might be linked to metabolic factors such as glutamine.

Patients with skeletal muscle catabolism (cachexia) fail to conserve the skeletal muscle protein and release large amounts of nitrogen as urea. Previous studies suggest that the threshold for the conversion of amino acids into other forms of chemical energy and the concomitant production of urea are regulated by the plasma cystine level and hepatic cysteine catabolism. Studies of plasma amino acid exchange rates in the lower extremities now show that healthy young subjects regulate their plasma cystine level in a process that may be described as controlled constructive catabolism. The term controlled describes the fact that the release of cystine and other amino acids from the peripheral tissue is negatively correlated with (certain) plasma amino acid levels. The term constructive describes the fact that the release of cystine is correlated with an increase of the plasma cystine level. The regulation of the plasma cystine level is disturbed in conditions with progressive skeletal muscle catabolism including cancer, HIV infection, and old age. These conditions show also a low plasma glutamine:cystine ratio indicative of an impaired hepatic cystine catabolism. In HIV+ patients and SIV-infected macaques, a decrease of the plasma cystine level was found to coincide with the decrease of CD4+ T cells.