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1.  Human N-acetyltransferase 1 (NAT1) *10 and *11 alleles increase protein expression via distinct mechanisms and associate with sulfamethoxazole-induced hypersensitivity 
Pharmacogenetics and genomics  2011;21(10):652-664.
Objectives
N-acetyltransferase 1 (NAT1) metabolizes drugs and environmental carcinogens. NAT1 alleles *10 and *11 have been proposed to alter protein level or enzyme activity compared to wild-type NAT1 *4 and to confer cancer risk, via uncertain pathways. This study characterizes regulatory polymorphisms and underlying mechanisms of NAT1 expression.
Methods
We measured allelic NAT1 mRNA expression and translation, as a function of multiple transcription start sites, alternative splicing, and three 3′-polyadenylation sites in human livers (one of which discovered in this study), B lymphocytes, and transfected cells. In a clinical study of 469 HIV/AIDS patients treated with the NAT1/NAT2 substrate sulfamethoxazole (SMX), associations were tested between SMX induced hypersensitivity and NAT1 *10 and *11 genotypes, together with known NAT2 polymorphisms.
Results
NAT1*10 and *11 were determined to act as common regulatory alleles accounting for most NAT1 expression variability, both leading to increased translation into active protein. NAT1*11 (2.4% minor allele frequency) affected 3′polyadenylation site usage, thereby increasing formation of NAT1 mRNA with intermediate length 3′UTR (major isoform) at the expense of the short isoform, resulting in more efficient protein translation. NAT1 *10 (19% minor allele frequency) increased translation efficiency without affecting 3′-UTR polyadenylation site usage. Livers and B-lymphocytes with *11/*4 and *10/*10 genotypes displayed higher NAT1 immunoreactivity and NAT1 enzyme activity than the reference genotype *4/*4. Patients who carry *10/*10 and *11/*4 (‘fast NAT1 acetylators’) were less likely to develop hypersensitivity to SMX, but this was observed only in subjects also carrying a slow NAT2 acetylator genotype.
Conclusion
NAT1 *10 and *11 significantly increase NAT1 protein level/enzyme activity, enabling the classification of carriers into reference and rapid acetylators. Rapid NAT1 acetylator status appears to protect against SMX toxicity by compensating for slow NAT2 acetylator status.
doi:10.1097/FPC.0b013e3283498ee9
PMCID: PMC3172334  PMID: 21878835
N-acetyltransferase; NAT1; polyadenylation; allelic expression imbalance; sulfamethoxazole; cotrimoxazole; protein translation; acetylator phenotype; idiosyncratic drug reactions
2.  Combined ascorbate and glutathione deficiency leads to decreased cytochrome b5 expression and impaired reduction of sulfamethoxazole hydroxylamine 
Archives of toxicology  2010;84(8):597-607.
Sulfonamide antimicrobials such as sulfamethoxazole (SMX) have been associated with drug hypersensitivity reactions, particularly in patients with AIDS. A reactive oxidative metabolite, sulfamethoxazole-nitroso (SMX-NO), forms drug-tissue adducts that elicit a T cell response. Antioxidants such as ascorbic acid (AA) and glutathione (GSH) reduce SMX-NO to the less reactive hydroxylamine metabolite (SMX-HA), which is further reduced to the non-immunogenic parent compound by cytochrome b5 (b5) and its reductase (b5R). We hypothesized that deficiencies in AA and GSH would enhance drug-tissue adduct formation and immunogenicity towards SMX-NO, and that these antioxidant deficiencies might also impair the activity of the b5/b5R pathway. We tested these hypotheses in guinea pigs fed either a normal or AA-restricted diet, followed by buthionine sulfoximine treatment (250 mg/kg SC daily, or vehicle); and SMX-NO (1 mg/kg IP 4 days per week, or vehicle), for 2 weeks. Guinea pigs did not show any biochemical or histopathologic evidence of SMX-NO related toxicity. Combined AA and GSH deficiency in this model did not significantly increase tissue drug-adduct formation, or splenocyte proliferation in response to SMX-NO. However, combined antioxidant deficiency was associated with decreased mRNA and protein expression of cytochrome b5, as well as significant decreases in SMX-HA reduction in SMX-NO treated pigs. These results suggest that SMX-HA detoxification may be down-regulated in combined AA and GSH deficiency. This mechanism could contribute to the higher risk of SMX hypersensitivity in AIDS patients with antioxidant depletion.
doi:10.1007/s00204-010-0530-z
PMCID: PMC2910208  PMID: 20221587
Guinea pig; antioxidants; drug hypersensitivity; NADH hydroxylamine reductase
3.  Immune response to sulfamethoxazole in patients with AIDS. 
Antibody- and cell-mediated responses to sulfamethoxazole (SMX) were analyzed in AIDS patients with or without a history of hypersensitivity and in negative controls. In 20 of 20 (P < 0.01) human immunodeficiency virus (HIV)-seropositive patients with skin reactions to cotrimoxazole, we found SMX-specific antibodies, while only 9 of 20 and 17 of 20 HIV-seropositive patients without a history of hypersensitivity to cotrimoxazole had SMX-specific immunoglobulin M (IgM) and IgG, respectively. The levels of specific IgM and IgG were higher in patients with skin reactions than in patients without reactions (IgM, 1.0 +/- 0.19 versus 0.47 +/- 0.23 [P < 0.001]; IgG, 0.68 +/- 0.15 versus 0.47 +/- 0.14 [P < 0.001] [mean optical density values +/- standard deviations]). Seronegative controls with no history of exposure to sulfa compounds did not have SMX-specific IgG or IgM antibodies, and controls with a history of intake of SMX with or without reactions had low levels of IgG and IgM. The SMX-specific IgG subclasses were exclusively IgG1 and IgG3. None of the patients had detectable SMX-specific IgE or IgA antibodies nor did they exhibit a cell-mediated response as measured by a lymphocyte proliferation assay. Antibodies to SMX recognized N-acetyl-sulfonamide, N-(2-thiazolyl)-sulfanilamide, sulfadiazine, and sulfisoxazole but did not recognize sulfanilamide or 3-amino-5-methyl isoxazole in an inhibition assay. It is not known whether the SMX-specific antibodies associated with hypersensitivity reactions to SMX in HIV-seropositive patients have a pathogenic role in these reactions. Sulfanilamide or 3-amino-5-methyl isoxazole, on the other hand, could be potential alternative therapies in HIV-seropositive patients with a history of skin reactions to SMX.
PMCID: PMC170127  PMID: 7697529
4.  Cytochrome b5 and NADH cytochrome b5 reductase: genotype-phenotype correlations for hydroxylamine reduction 
Pharmacogenetics and genomics  2010;20(1):26-37.
Objectives
NADH cytochrome b5 reductase (b5R) and cytochrome b5 (b5) catalyze the reduction of sulfamethoxazole hydroxylamine (SMX-HA), which can contribute to sulfonamide hypersensitivity, to the parent drug sulfamethoxazole. Variability in hydroxylamine reduction could thus play a role in adverse drug reactions. The aim of this study was to characterize variability in SMX-HA reduction in 111 human livers, and investigate its association with single nucleotide polymorphisms (SNPs) in b5 and b5R cDNA.
Methods
Liver microsomes were assayed for SMX-HA reduction activity, and b5 and b5R expression was semi-quantified by immunoblotting. The coding regions of the b5 (CYB5A) and b5R (CYB5R3) genes were resequenced.
Results
Hepatic SMX-HA reduction displayed a 19-fold range of individual variability (0.06–1.11 nmol/min/mg protein), and a 17-fold range in efficiency (Vmax/Km) among outliers. SMX-HA reduction was positively correlated with b5 and b5R protein content (p < 0.0001, r = 0.42; p = 0.01, r = 0.23, respectively), and expression of both proteins correlated with one another (p < 0.0001; r = 0.74). A novel cSNP in CYB5A (S5A) was associated with very low activity and protein expression. Two novel CYB5R3 SNPs, R59H and R297H, displayed atypical SMX-HA reduction kinetics and decreased SMX-HA reduction efficiency.
Conclusion
These studies indicate that while novel cSNPs in CYB5A and CYB5R3 are associated with significantly altered protein expression and/or hydroxylamine reduction activities, these low frequency cSNPs only appear to minimally impact overall observed phenotypic variability. Work is underway to characterize polymorphisms in other regions of these genes to further account for individual variability in hydroxylamine reduction.
doi:10.1097/FPC.0b013e3283343296
PMCID: PMC2905818  PMID: 19997042
sulfamethoxazole; sulfonamide hypersensitivity; cytochrome b5; NADH cytochromeb5 reductase; single nucleotide polymorphism
5.  Variants in the Glutamate-Cysteine-Ligase Gene Are Associated with Cystic Fibrosis Lung Disease 
Background: Chronic progressive lung disease is the most serious complication of cystic fibrosis (CF). Glutathione plays an important role in the protection of the CF lung against oxidant-induced lung injury.
Objectives: We hypothesized that a polymorphism in a novel candidate gene that regulates glutathione synthesis might influence CF lung disease.
Methods: In a cross-sectional study, subjects were recruited from CF clinics in Seattle and multiple centers in Canada. We tested for an association between CF lung disease and a functional polymorphism in the glutamate-cysteine ligase catalytic subunit (GCLC) gene. Multiple linear regression was used to test for association between polymorphisms of GCLC and severity of CF lung disease while adjusting for age, Pseudomonas aeruginosa infection, and cystic fibrosis transmembrane conductance regulator (CFTR) genotype. Analysis was repeated for patients with CF stratified by CFTR genotype.
Measurements and Main Results: A total of 440 subjects with CF participated in the study (51% male; mean [± SD] age, 26 ± 11 yr; mean FEV1, 62 ± 28% predicted). In the total population, there was a trend toward an association between GCLC genotypes and CF lung disease (linear regression coefficient [SEM], 1.68 [1.0]; p = 0.097). In the stratified analysis, there was a highly significant association between GCLC genotype and CF lung function in subjects with a milder CFTR genotype (linear regression coefficient [SEM], 5.5 (1.7); p = 0.001).
Conclusions: In patients with CF with a milder CFTR genotype, there is a strong association between functional polymorphisms of the GCLC gene and CF lung disease severity.
doi:10.1164/rccm.200508-1281OC
PMCID: PMC2648118  PMID: 16690975
CFTR genotype; glutathione; modifier genes
6.  Trimethoprim-sulfamethoxazole-induced aseptic meningitis. 
We present a 46-year-old African-American man with AIDS who was admitted on two different occasions within three weeks for signs and symptoms of meningitis after using trimethoprim/sulfamethoxazole (TMP/SMX). TMP/SMX is primarily used for the treatment of pneumocystis carinii pneumonia prophylaxis in AIDS patients. Drug-induced aseptic meningitis (DIAM) is commonly seen with nonsteroidal anti-inflammatory drugs (NSAIDS), antibiotics (with TMP/SMX being the most frequently implicated), intravenous immunoglobulins and OKT3 antibodies. However, the implication of TMP/SMX inducing aseptic meningitis has been underreported to FDA/MEDWATCH program. This might be due to the fact that it has also been used to treat bacterial meningitis from organisms like Listeria monocytogenes, which is a common pathogen in the elderly and in infants. We reviewed the literature in an attempt to characterize the pattern and predictors of TMP/SMX-induced aseptic meningitis.
PMCID: PMC2640752  PMID: 16396068
7.  A comparison of trimethorprim-sulfamethoxazole with sulfamethoxazole alone in infections localized to the kidneys. 
Canadian Medical Association Journal  1975;112(13 Spec No):9-12.
Ninety patients with urinary tract infections were treated in a randomized double-blind study with either a combination of trimethoprim and sulfamethoxazole (TMP-SMX) or sulfamethoxazole alone (SMX). Thirty of 42 patients treated with TMP-SMX were cured by the time of follow-up compared with 26 of 48 treated with SMX alone. Of the 29 patients infected with SMX-resistent organisms, the combination TMP-SMX cured 12 of 17, whereas SMX alone cured 2 of 12. Of the 61 patients infected with SMX-sensitive organisms, TMP-SMX cured 18 of 25; SMX alone cured 24 of 36. In 50 women the infection was found localized to The upper urinary tract by the use of the Fairley bladder washout technique. TMPsmx cured 16 or 24 of these patients with proved upper tract infections and SMX alone cured 11 of 26. Although none of these differences were significant, TMP-SMX appears to be an effective drug combination for the therapy of proved upper tract infection and is also effective in eradicating sulfonamide-resistant organisms.
PMCID: PMC1956456  PMID: 1093658
8.  Influence of NAT2 Polymorphisms on Sulfamethoxazole Pharmacokinetics in Renal Transplant Recipients 
The sulfamethoxazole (SMX)-trimethoprim drug combination is routinely used as prophylaxis against Pneumocystis pneumonia during the first 3 to 6 months after renal transplantation. The objective of this study was to examine the impact of N-acetyltransferase 2 (NAT2) and CYP2C9 polymorphisms on the pharmacokinetics of SMX in 118 renal transplant recipients. Starting on day 14 after renal transplantation, patients were administered 400 mg/day-80 mg/day of SMX-trimethoprim orally once daily. On day 14 after the beginning of SMX therapy, plasma SMX concentrations were determined by a high-performance liquid chromatography method. The SMX area under the concentration-time curve from 0 to 24 h (AUC0-24) for 15 recipients with the NAT2 slow acetylator genotype (NAT2*5/*6, -*6/*6, -*6/*7, and -*7/*7) was significantly greater than that for 56 recipients with the NAT2 rapid acetylator genotype (homozygous for NAT2*4) (766.4 ± 432.3 versus 537.2 ± 257.5 μg-h/ml, respectively; P = 0.0430), whereas there were no significant differences in the SMX AUC0-24 between the CYP2C9*1/*1 and -*1/*3 groups. In a multiple regression analysis, the SMX AUC0-24 was associated with NAT2 slow acetylator polymorphisms (P = 0.0095) and with creatinine clearance (P = 0.0499). Hepatic dysfunction in NAT2 slow acetylator recipient patients during the 6-month period after SMX administration was not observed. SMX plasma concentrations were affected by NAT2 polymorphisms and renal dysfunction. Although standard SMX administration to patients with NAT2 slow acetylator polymorphisms should be accompanied by monitoring for side effects and drug interaction effects from the inhibition of CYP2C9, SMX administration at a low dose (400 mg) as prophylaxis may not provide drug concentrations that reach the level necessary for the expression of side effects. Further studies with a larger sample size should be able to clarify the relationship between SMX plasma concentration and side effects.
doi:10.1128/AAC.05037-11
PMCID: PMC3264276  PMID: 22106207
9.  Genetic interaction of GSH metabolic pathway genes in cystic fibrosis 
BMC Medical Genetics  2013;14:60.
Background
Cystic fibrosis (CF) is a monogenic disease caused by CFTR gene mutations, with clinical expression similar to complex disease, influenced by genetic and environmental factors. Among the possible modifier genes, those associated to metabolic pathways of glutathione (GSH) have been considered as potential modulators of CF clinical severity. In this way it is of pivotal importance investigate gene polymorphisms at Glutamate-Cysteine Ligase, Catalytic Subunit (GCLC), Glutathione S-transferase Mu 1 (GSTM1), Glutathione S-transferase Theta 1 (GSTT1), and Glutathione S-transferase P1 (GSTP1), which have been associated to the GSH metabolic pathway and CF clinical severity.
Method
A total of 180 CF’s patients were included in this study, which investigated polymorphisms in GCLC and GST genes (GCLC -129C>T and -3506A>G; GSTM1 and GSTT1 genes deletion, and GSTP1*+313A>G) by PCR and PCR-RFLP associating to clinical variables of CF severity, including variables of sex, clinical scores [Shwachman-Kulczycki, Kanga e Bhalla (BS)], body mass index, patient age, age for diagnosis, first clinical symptoms, first colonization by Pseudomonas aeruginosa, sputum’s microorganisms, hemoglobin oxygen saturation in the blood, spirometry and comorbidities. The CFTR genotype was investigated in all patients, and the genetic interaction was performed using MDR2.0 and MDRPT0.4.7 software.
Results
The analysis of multiple genes in metabolic pathways in diseases with variable clinical expression, as CF disease, enables understanding of phenotypic diversity. Our data show evidence of interaction between the GSTM1 and GSTT1 genes deletion, and GSTP1*+313A>G polymorphism with CFTR gene mutation classes, and BS (Balance testing accuracy= 0.6824, p= 0.008), which measures the commitment of bronchopulmonary segments by tomography.
Conclusion
Polymorphisms in genes associated with metabolism of GSH act on the CF’s severity.
doi:10.1186/1471-2350-14-60
PMCID: PMC3685592  PMID: 23758905
10.  Nrf1 and Nrf2 Regulate Rat Glutamate-Cysteine Ligase Catalytic Subunit Transcription Indirectly via NF-κB and AP-1 
Molecular and Cellular Biology  2005;25(14):5933-5946.
Glutamate-cysteine ligase catalytic subunit (GCLC) is regulated transcriptionally by Nrf1 and Nrf2. tert-Butylhydroquinone (TBH) induces human GCLC via Nrf2-mediated trans activation of the antioxidant-responsive element (ARE). Interestingly, TBH also induces rat GCLC, but the rat GCLC promoter lacks ARE. This study examined the role of Nrf1 and Nrf2 in the transcriptional regulation of rat GCLC. The baseline and TBH-mediated increase in GCLC mRNA levels and rat GCLC promoter activity were lower in Nrf1 and Nrf2 null (F1 and F2) fibroblasts than in wild-type cells. The basal protein and mRNA levels and nuclear binding activities of c-Jun, c-Fos, p50, and p65 were lower in F1 and F2 cells and exhibited a blunted response to TBH. Lower c-Jun and p65 expression also occurs in Nrf2 null livers. Levels of other AP-1 and NF-κB family members were either unaffected (i.e., JunB) or increased (i.e., Fra-1). Overexpression of Nrf1 and Nrf2 in respective cells restored the rat GCLC promoter activity and response to TBH but not if the AP-1 and NF-κB binding sites were mutated. Fra-1 overexpression lowered endogenous GCLC expression and rat GCLC promoter activity, while Fra-1 antisense had the opposite effects. In conclusion, Nrf1 and Nrf2 regulate rat GCLC promoter by modulating the expression of key AP-1 and NF-κB family members.
doi:10.1128/MCB.25.14.5933-5946.2005
PMCID: PMC1168815  PMID: 15988009
11.  Trimethoprim-sulfamethoxazole-induced Steven Johnson syndrome in an HIV-infected patient 
Indian Journal of Pharmacology  2012;44(4):533-535.
Trimethoprim-sulfamethoxazole (TMP/SMX) is a widely prescribed antimicrobial for the management of several uncomplicated infections. It is commonly used for the treatment and prophylaxis of Pneumocystis jirovecii pneumonia (PCP) in the HIV-infected population. The adverse reaction to TMP/SMX is more frequent and severe in HIV-infected patients as compared to the general population. Here, we report a case of Stevens-Johnson syndrome (SJS) secondary to TMP/SMX. The patient had a generalized cutaneous reaction with involvement of the eyes, oral cavity, and genitals. He had elevated hepatic alanine aminotransferase and aspartate aminotransferase enzyme. TMP/SMX therapy was stopped and supportive treatment was started. His condition improved after eight days of stopping TMP/SMX therapy.
doi:10.4103/0253-7613.99346
PMCID: PMC3469966  PMID: 23087524
Desensitization; HIV infection; glutathione enzyme; P. jirovecii pneumonia; Steven Johnson syndrome; trimethoprim-sulfamethoxazole
12.  Trimethoprim-Sulfamethoxazole Prophylaxis and Antibiotic Nonsusceptibility in Invasive Pneumococcal Disease 
Among 5,043 invasive pneumococcal disease (IPD) isolates identified through South African national surveillance from 2003 to 2007, we estimated the effect of trimethoprim-sulfamethoxazole (TMP-SMX) prophylaxis on antimicrobial resistance. Patients on TMP-SMX prophylaxis were more likely to have a pneumococcal isolate nonsusceptible to TMP-SMX, penicillin, and rifampin. TMP-SMX nonsusceptibility was associated with nonsusceptibility to penicillin, erythromycin, and rifampin and multidrug resistance. This study informs empirical treatment of suspected IPD in patients with a history of TMP-SMX use.
doi:10.1128/AAC.05813-11
PMCID: PMC3294938  PMID: 22232291
13.  Prevalence and risk factor analysis of resistant Escherichia coli urinary tract infections in the emergency department  
Pharmacy Practice  2013;11(2):96-101.
Background
Escherichia coli (E. coli) is a frequent uropathogen in urinary tract infections (UTI). Widespread resistance to sulfamethoxazole-trimethoprim (SMX-TMP) and increasing resistance to fluoroquinolones amongst these isolates has been recognized. There are limited data demonstrating risk factors for resistance to both SMX-TMP and fluoroquinolones.
Objective
This study was conducted to assess for the prevalence of community resistance amongst E. coli isolates to SMX-TMP and levofloxacin in ambulatory patients discharged from the emergency department (ED).
Methods
Adults presenting for evaluation and discharged from the ED with a diagnosis of an E. coli UTI were retrospectively reviewed. Utilizing demographic and clinical data the prevalence of E. coli resistance and risk factors associated with SMX-TMP- and fluoroquinolone-resistant infection were determined.
Results
Among the 222 patients, the mean rates of E. coli susceptibility to levofloxacin and SMX-TMP were 82.4% and 72.5%, respectively. Significant risk factors for resistance to SMX-TMP included prior antibiotic use (p=0.04) and prior diagnosis of UTI (p= 0.01). Significant risk factors for resistance to levofloxacin included: male gender, age, presence of hypertension, diabetes, chronic respiratory disease, nursing home resident, previous antibiotic use, previous diagnosis of UTI, existence of renal or genitourinary abnormalities, and prior surgical procedures (p <0.05 for all comparisons). The number of hospital days prior to initial ED evaluation (p<0.001) was determined to be a predictive factor in hospital and ED readmission.
Conclusions
These results suggest that conventional approaches to monitoring for patterns of susceptibility may be inadequate. It is imperative that practitioners develop novel approaches to identifying patients with risk factors for resistance. Identification of risk factors from this evaluation should prompt providers to scrutinize the use of these agents in the setting of patients presenting with an uncomplicated UTI in the ED.
PMCID: PMC3798174  PMID: 24155856
Drug Resistance, Bacterial; Risk Factors; Urinary Tract Infections; Uropathogenic Escherichia coli; Trimethoprim-Sulfamethoxazole Combination; Fluoroquinolones; Emergency Service, Hospital; United States
14.  HIV Nonnucleoside Reverse Transcriptase Inhibitors and Trimethoprim-Sulfamethoxazole Inhibit Plasmodium Liver Stages 
The Journal of Infectious Diseases  2012;206(11):1706-1714.
Background. Although nonnucleoside reverse transcriptase inhibitors (NNRTIs) are usually part of first-line treatment regimens for human immunodeficiency virus (HIV), their activity on Plasmodium liver stages remains unexplored. Additionally, trimethoprim-sulfamethoxazole (TMP-SMX), used for opportunistic infection prophylaxis in HIV-exposed infants and HIV-infected patients, reduces clinical episodes of malaria; however, TMP-SMX effect on Plasmodium liver stages requires further study.
Methods. We characterized NNRTI and TMP-SMX effects on Plasmodium liver stages in vivo using Plasmodium yoelii. On the basis of these results, we conducted in vitro studies assessing TMP-SMX effects on the rodent parasites P. yoelii and Plasmodium berghei and on the human malaria parasite Plasmodium falciparum.
Results. Our data showed NNRTI treatment modestly reduced P. yoelii liver stage parasite burden and minimally extended prepatent period. TMP-SMX administration significantly reduced liver stage parasite burden, preventing development of patent parasitemia in vivo. TMP-SMX inhibited development of rodent and P. falciparum liver stage parasites in vitro.
Conclusions. NNRTIs modestly affect liver stage Plasmodium parasites, whereas TMP-SMX prevents patent parasitemia. Because drugs that inhibit liver stages target parasites when they are present in lower numbers, these results may have implications for eradication efforts. Understanding HIV drug effects on Plasmodium liver stages will aid in optimizing treatment regimens for HIV-exposed and HIV-infected infected patients in malaria-endemic areas.
doi:10.1093/infdis/jis602
PMCID: PMC3488198  PMID: 23125449
15.  Randomized Trial of Trimethoprim-Sulfamethoxazole versus Pyrimethamine-Sulfadiazine for Therapy of Toxoplasmic Encephalitis in Patients with AIDS 
The aim of the present pilot study was to compare the efficacy and safety of trimethoprim (TMP) and sulfamethoxazole (SMX) with those of the standard therapy pyrimethamine (P)-sulfadiazine (S) for the treatment of toxoplasmic encephalitis in patients with AIDS. This was a pilot, multicenter, randomized, and prospective study. Patients were randomly assigned to receive TMP (10 mg/kg of body weight/day) and SMX (50 mg/kg/day) or P (50 mg daily) and S (60 mg/kg/day) as acute therapy (for 4 weeks) and then as maintenance therapy for 3 months at half of the original dosage. Seventy-seven patients were enrolled and randomized to the study: 40 patients were treated with TMP-SMX and 37 were treated with P-S. There was no statistically significant difference in clinical efficacy during acute therapy. In contrast, patients randomized to TMP-SMX appeared more likely to achieve a complete radiologic response after acute therapy. Adverse reactions were significantly more frequent in patients treated with P-S, and skin rash was the most common adverse event noted in these patients. In conclusion, the results of the study suggest that TMP-SMX appears to be a valuable alternative to P-S, in particular in patients with opportunistic bacterial infections.
PMCID: PMC105601  PMID: 9624473
16.  Enzymatic defects underlying hereditary glutamate cysteine ligase deficiency are mitigated by association of the catalytic and regulatory subunits 
Biochemistry  2011;50(29):6508-6517.
Glutamate cysteine ligase (GCL) deficiency is a rare autosomal recessive trait that compromises production of glutathione, a critical redox buffer and enzymatic cofactor. Patients have markedly reduced levels of erythrocyte glutathione, leading to hemolytic anemia and in some cases, impaired neurological function. Human glutamate cysteine ligase is a heterodimer comprised of a catalytic (GCLC) and a regulatory subunit (GCLM), which catalyzes the initial rate limiting step in glutathione production. Four clinical missense mutations have been identified within GCLC: Arg127Cys, Pro158Leu, His370Leu, and Pro414Leu. Here, we have evaluated the impacts of these mutations on enzymatic function in vivo and in vitro to gain further insights into the pathology. Embryonic fibroblasts from GCLC null mice were transiently transfected with wild-type or mutant GCLC and cellular glutathione levels were determined. The four mutant transfectants each had significantly lower levels of glutathione relative to wild-type, with the Pro414Leu mutant being most compromised. The contributions of the regulatory subunit to GCL activity were investigated using an S. cerevisiae model system. Mutant GCLC alone could not complement a glutathione-deficient strain and required the concurrent addition of GCLM to restore growth. Kinetic characterizations of the recombinant GCLC mutants indicated that the Arg127Cys, His370Leu, and Pro414Leu mutants have compromised enzymatic activity that can largely be rescued by the addition of GCLM. Interestingly, the Pro158Leu mutant has kinetic constants comparable to wild-type GCLC, suggesting that heterodimer formation is needed for stability in vivo. Strategies that promote heterodimer formation and persistence would be effective therapeutics for the treatment of GCL deficiency.
doi:10.1021/bi200708w
PMCID: PMC3142541  PMID: 21657237
17.  Dose of Trimethoprim-Sulfamethoxazole To Treat Skin and Skin Structure Infections Caused by Methicillin-Resistant Staphylococcus aureus ▿ 
Antimicrobial Agents and Chemotherapy  2011;55(12):5430-5432.
We undertook this study to investigate whether treatment with a higher dose of trimethoprim-sulfamethoxazole (TMP/SMX) led to greater clinical resolution in patients with skin and soft tissue infections (SSTIs) caused by methicillin-resistant Staphylococcus aureus (MRSA). A prospective, observational cohort with nested case-control study was performed at a public tertiary health system. Among patients with MRSA SSTIs during the period from May 2008 to September 2008 who received oral monotherapy with TMP/SMX and whose clinical outcome was known, the clinical characteristics and outcomes were compared between patients treated with a high dose of TMP/SMX (320 mg/1,600 mg twice daily) for 7 to 15 days and patients treated with the standard dose of TMP/SMX (160 mg/800 mg twice daily) for 7 to 15 days. In patients with MRSA SSTIs, those treated with the high dose of TMP/SMX (n = 121) had clinical characteristics similar to those of patients treated with the standard dose of TMP/SMX (n = 170). The only exception was a higher proportion of patients with a history of trauma upon admission among the patients treated with the higher dose. The proportion of patients with clinical resolution of infection was not different in the two groups (88/121 [73%] versus 127/170 [75%]; P = 0.79). The lack of significance remained in patients with abscess upon stratified analysis by whether surgical drainage was performed. The study found that patients with MRSA SSTIs treated with the higher dose of TMP/SMX (320/1,600 mg twice daily) for 7 to 15 days had a similar rate of clinical resolution as patients treated with the standard dose of TMP/SMX (160/800 mg twice daily) for 7 to 15 days.
doi:10.1128/AAC.00706-11
PMCID: PMC3232808  PMID: 21930870
18.  Trimethoprim-Sulfamethoxazole Resistance Among Urinary Coliform Isolates 
OBJECTIVE
A large majority of urinary tract infections are caused by coliform organisms. Trimethoprim-sulfamethoxazole (TMP-SMX) resistance among uropathogens is increasing in many areas. The objective of this study was to determine risk factors for TMP-SMX-resistant coliforms in patients with urinary tract infections.
DESIGN
Retrospective case-control study.
SETTING
Emergency department of a tertiary care university hospital.
PATIENTS
We studied 448 emergency department patients aged 14 years or older with a urinary tract infection caused by a coliform organism. Cases consisted of all patients with a culture-documented urinary tract infection caused by a TMPSMX- resistant coliform, while control patients were those with a TMP-SMX-sensitive organism.
MEASUREMENTS AND MAIN RESULTS
A univariate analysis of clinical variables associated with TMP-SMX resistance was performed. Multiple logistic regression was performed to determine independent predictors of TMP-SMX resistance. Resistance to TMP-SMX was seen in 15% of isolates. Numerous variables were associated with TMP-SMX resistance on the univariate screen. Independent predictors of resistance were diabetes (odds ratio [OR] 3.1; 95% confidence interval [CI] 1.2, 8.4), recent hospitalization (OR 2.5; 95% CI 1.1, 5.7), current use of antibiotics (OR 4.5; 95% CI 2.0, 10.2), and recent use of TMP-SMX (OR 5.1; 95% CI 2.2, 11.5). When those with recent hospitalization were excluded from analysis, independent predictors were current use of any antibiotic (OR 3.5; 95% CI 1.4, 8.4) and recent use of TMP-SMX (OR 5.9; 95% CI 2.4, 14.3).
CONCLUSIONS
Coliforms resistant to TMP-SMX are common in our emergency department. Diabetes, recent hospitalization, and the use of antibiotics, particularly the use of TMPSMX, are independent risk factors for TMP-SMX resistance. Clinicians should consider these findings when deciding on antimicrobial therapy for patients with urinary tract infections.
doi:10.1046/j.1525-1497.1999.10128.x
PMCID: PMC1496756  PMID: 10571705
trimethoprim-sulfamethoxazole resistance;; urinary tract infection;; coliform organisms; emergency department.
19.  Determinants of Quinolone versus Trimethoprim-Sulfamethoxazole Use for Outpatient Urinary Tract Infection 
Quinolones are increasingly favored over trimethoprim-sulfamethoxazole (TMP-SMX) for empirical treatment of uncomplicated urinary tract infection (UTI). This is associated with increasing resistance toward this broad-spectrum group of antibiotics. Our objective is to describe the prescribing patterns and identify determinants of the choice between TMP-SMX and quinolones for outpatient UTI treatment in Switzerland. An ongoing national Sentinel surveillance system was used to study 11,799 antibiotic prescriptions for UTI in adult outpatients and associated physician and patient factors between 2006 and 2008, to compare the prescription of quinolones versus that of TMP-SMX for treatment of UTI. Most UTI episodes were diagnosed as cystitis (90%). TMP-SMX was prescribed for one-fifth (22%) of UTIs. Independent predictors for prescribing quinolones were pyelonephritis and physicians with low thresholds for prescribing antibiotics for upper respiratory tract infections (“high prescribers”), whereas female patients were more likely to receive TMP-SMX. High-prescribing physicians also more often cared for patients who themselves favor antibiotic treatment (P < 0.001). Quinolones are commonly prescribed to outpatients with UTI. Nonclinical factors influence the choice of quinolones versus TMP-SMX, which may provide opportunities for interventions to improve prescribing patterns and control quinolone resistance.
doi:10.1128/AAC.05321-11
PMCID: PMC3294920  PMID: 22232276
20.  Therapeutic Potential of Caspofungin Combined with Trimethoprim-Sulfamethoxazole for Pneumocystis Pneumonia: A Pilot Study in Mice 
PLoS ONE  2013;8(8):e70619.
Pneumocystis pneumonia (PcP) is a major cause of mortality and morbidity in immunocompromised patients. There are limited alternative therapeutic choices to trimethoprim-sulfamethoxazole (TMP-SMX) which is the standard first line therapy/prophylaxis for PcP. The efficacy of low doses of caspofungin and caspofungin in association with TMP-SMX standard-prophylactic dose was evaluated in an experimental model of Pneumocystis. Susceptibility of Pneumocystis spp. to low doses of caspofungin and caspofungin/TMP-SMX was evaluated in Balb/c immunosuppressed mice, infected intranasally with P. murina. Caspofungin was administered once daily at 0.1 mg/kg, 0.05 mg/kg, and 0.001 mg/kg and TMP-SMX was administered by oral gavage (12.25 mg/62.5 mg/day), for 21 days. Efficacy was calculated based on the reduction in organism burden determined through quantitative fluorescent-based real-time PCR (qPCR). Serum β-1,3-D-glucan was measured as an additional marker of infection. The present data showed that caspofungin demonstrated anti-Pneumomocystis effect. However, the doses administrated were too low to achieve Pneumocystis eradication, which suggests that echinocandin treatment should not be administrated as mono-therapy. After 21 days of treatment, P. murina was not detected in the lungs of mice with either TMP-SMX or caspofungin/TMP-SMX. The results showed that, even at the lowest concentrations tested, the efficacy of caspofungin in association with TMP-SMX was higher than the efficacy of either drug used alone. The administration of caspofungin/TMP-SMX was at least 1.4 times more effective against P. murina infection than TMP-SMX used alone. The most promising result was achieved with the combination of caspofungin 0.05 mg/kg/day with TMP-SMX 12.5 mg–62.5 mg/day, which reduced the parasite burden to undetectable levels immediately at the 14th day of treatment, showing a highly marked anti-Pneumomocystis effect. These data suggest that the administration of low doses of caspofungin in combination with low doses of TMP-SMX may provide an improved treatment protocol for Pneumocystis infection clearance.
doi:10.1371/journal.pone.0070619
PMCID: PMC3734247  PMID: 23940606
21.  Overexpression of glutamate–cysteine ligase protects human COV434 granulosa tumour cells against oxidative and γ-radiation-induced cell death 
Mutagenesis  2009;24(3):211-224.
Ionizing radiation is toxic to ovarian follicles and can cause infertility. Generation of reactive oxygen species (ROS) has been implicated in the toxicity of ionizing radiation in several cell types. We have shown that depletion of the antioxidant glutathione (GSH) sensitizes follicles and granulosa cells to toxicant-induced apoptosis and that supplementation of GSH is protective. The rate-limiting reaction in GSH biosynthesis is catalysed by glutamate–cysteine ligase (GCL), which consists of a catalytic subunit (GCLC) and a regulatory subunit (GCLM). We hypothesized that overexpression of Gclc or Gclm to increase GSH synthesis would protect granulosa cells against oxidant- and radiation-induced cell death. The COV434 line of human granulosa tumour cells was stably transfected with vectors designed for the constitutive expression of Gclc, Gclm, both Gclc and Gclm or empty vector. GCL protein and enzymatic activity and total GSH levels were significantly increased in the GCL subunit-transfected cells. GCL-transfected cells were resistant to cell killing by treatment with hydrogen peroxide compared to control cells. Cell viability declined less in all the GCL subunit-transfected cell lines 1–8 h after 0.5 mM hydrogen peroxide treatment than in control cells. We next examined the effects of GCL overexpression on responses to ionizing radiation. ROS were measured using a redox-sensitive fluorogenic dye in cells irradiated with 0, 1 or 5 Gy of γ-rays. There was a dose-dependent increase in ROS within 30 min in all cell lines, an effect that was significantly attenuated in Gcl-transfected cells. Apoptosis, assessed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling and activated caspase-3 immunoblotting, was significantly decreased in irradiated Gclc-transfected cells compared to irradiated control cells. Suppression of GSH synthesis in Gclc-transfected cells reversed resistance to radiation. These findings show that overexpression of GCL in granulosa cells can augment GSH synthesis and ameliorate various sequelae associated with exposure to oxidative stress and irradiation.
doi:10.1093/mutage/gen073
PMCID: PMC2674984  PMID: 19153097
22.  Laboratory Abnormalities Among HIV-Exposed, Uninfected Infants: IMPAACT Protocol P1025 
Background.
Infant laboratory abnormalities have been associated with exposure to antiretrovirals and to trimethoprim/sulfamethoxazole (TMP/SMX).
Methods.
We analyzed data from International Maternal Pediatric Adolescent AIDS Clinical Trials Group (IMPAACT) Protocol P1025, a prospective cohort study of human immunodeficiency virus type 1 (HIV)–infected women and their infants. Live-born, singleton, HIV-uninfected infants with at least 6 months of follow-up who represented the first pregnancy on study of HIV-infected mothers with at least 1 prenatal visit, CD4 count, and viral load during pregnancy and who used at least 1 antiretroviral during pregnancy were eligible for inclusion in this analysis.
Results.
The study population comprised 1524 infants. During the first 6 months of life, 7.4% of laboratory serious adverse events (SAEs) were related to glucose, 7.2% were related to hemoglobin, 8.7% were related to absolute neutrophil count, and 4.0% were related to total lymphocyte count. The likelihood of laboratory SAEs decreased with increasing age for hemoglobin, absolute neutrophil count, and glucose. Infant preterm birth and current receipt of antiretroviral(s) were the factors with the strongest associations with laboratory SAEs.
Conclusions.
The overall frequency of laboratory SAEs was low and decreased with age. Preterm infants are at higher risk of hemoglobin- and total lymphocyte count–related SAEs.
doi:10.1093/jpids/pis036
PMCID: PMC3656554  PMID: 23687574
23.  A Case of Recurrent Pancreatitis Induced by Trimethoprim-Sulfamethoxazole Re-Exposure 
Gut and Liver  2010;4(2):250-252.
A 32-year-old man presented with epigastric pain. He had a previous episode of acute pancreatitis of undetermined cause 2 years earlier. The patient had taken trimethoprim (80 mg) and sulfamethoxazole (400 mg) twice daily because of acute urethritis 3 days prior to admission. No definite cause of acute pancreatitis could be identified on baseline studies. A thorough history-taking revealed that the patient had an episode of acute pancreatitis while taking trimethoprim (80 mg) and sulfamethoxazole (400 mg) twice daily for 2 weeks for prostatitis prior to the previous admission. Therefore, a cause-and-effect relationship between trimethoprim-sulfamethoxazole (TMP-SMX) and repeated episodes of pancreatitis was highly suggested. The patient was presumably diagnosed as TMP-SMX-induced pancreatitis. The final diagnosis was TMP-SMX-induced pancreatitis. Since drugs are rare causes of acute pancreatitis and the diagnosis of drug-induced pancreatitis is difficult to establish, we report this interesting case along with a review of medical literature.
doi:10.5009/gnl.2010.4.2.250
PMCID: PMC2886936  PMID: 20559530
Pancreatitis; Drug; Ttrimethoprim-sulfamethoxazole
24.  The Risk of Overanticoagulation with Antibiotic Use in Outpatients on Stable Warfarin Regimens 
BACKGROUND
Medication interactions account for a significant proportion of overanticoagulation in warfarin users. However, little is known about the incidence or degree of interaction with commonly used oral antibiotics.
OBJECTIVE
To investigate the incidence and degree of overanticoagulation associated with commonly used oral antibiotics.
DESIGN
Retrospective cohort study of patients using warfarin who initiated an antibiotic (azithromycin, levofloxacin, or trimethoprim/sulfamethoxazole (TMP/SMX)) or terazosin for clinical indications between January 1998 and December 2002. The incidence of international normalized ratio (INR) elevation and the degree of change and bleeding events after institution of either medication type was recorded.
SUBJECTS
Patients at a university-affiliated Veteran's Affairs Medical Center.
RESULTS
The mean change in INR was −0.15 for terazosin, 0.51 for azithromycin, 0.85 for levofloxacin, and 1.76 for TMP/SMX. These mean INR changes in the antibiotic groups were all statistically different from the terazosin group. The incidence of supratherapeutic INR was 5% for terazosin, 31% for azithromycin, 33% for levofloxacin, and 69% for TMP/SMX. The incidence of absolute INR >4.0 was 0% for terazosin, 16% for azithromycin, 19% for levofloxacin, and 44% for TMP/SMX.
CONCLUSIONS
Among acutely ill outpatients, oral antibiotics (azithromycin, levofloxacin, and TMP/SMX) increase the incidence and degree of overanticoagulation.
doi:10.1111/j.1525-1497.2005.0136.x
PMCID: PMC1490169  PMID: 16050864
warfarin; overanticoagulation; drug interaction; azithromycin; levofloxacin; trimethoprim/sulfamethoxazole
25.  Heterozygosity in the glutathione synthesis gene Gclm increases sensitivity to diesel exhaust particulate induced lung inflammation in mice 
Inhalation Toxicology  2011;23(12):724-735.
Context
Inhalation of ambient fine particulate matter (PM2.5) is associated with adverse respiratory and cardiovascular effects. A major fraction of PM2.5 in urban settings is diesel exhaust particulate (DEP), and DEP-induced lung inflammation is likely a critical event mediating many of its adverse health effects. Oxidative stress has been proposed to be an important factor in PM2.5-induced lung inflammation, and the balance between pro- and antioxidants is an important regulator of this inflammation. An important intracellular antioxidant is the tripeptide thiol glutathione (GSH). Glutamate cysteine ligase (GCL) carries out the first step in GSH synthesis. In humans, relatively common genetic polymorphisms in both the catalytic (Gclc) and modifier (Gclm) subunits of GCL have been associated with increased risk for lung and cardiovascular diseases.
Objective
This study was aimed to determine the effects of Gclm expression on lung inflammation following DEP exposure in mice.
Materials and methods
We exposed Gclm wild type, heterozygous, and null mice to DEP via intranasal instillation and assessed lung inflammation as determined by neutrophils and inflammatory cytokines in lung lavage, inflammatory cytokine mRNA levels in lung tissue, as well as total lung GSH, Gclc, and Gclm protein levels.
Results
The Gclm heterozygosity was associated with a significant increase in DEP-induced lung inflammation when compared to that of wild type mice.
Discussion and conclusion
This finding indicates that GSH synthesis can mediate DEP-induced lung inflammation and suggests that polymorphisms in Gclm may be an important factor in determining adverse health outcomes in humans following inhalation of PM2.5.
doi:10.3109/08958378.2011.608095
PMCID: PMC3337699  PMID: 21967497
Diesel exhaust particulate; lung inflammation; glutathione; oxidative stress; glutamate cysteine ligase

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