Antibiotic Resistance Patterns of Uropathogens in Pediatric Emergency Department Patients

OBJECTIVES: To evaluate the prevalence of resistance of the various urinary tract infection (UTI) pathogens obtained from patients in an urban pediatric emergency department (PED), and to identify risk factors for infection with resistant strains. METHODS: The data were collected retrospectively in an urban, academic PED in northeastern Florida. The microbiology-computerized database was used to identify all positive urine cultures from October 1999 through June 2000. All patients aged 17 years or less, whose urine specimen was collected in the ED and grew cultures with greater than 10,000 colony forming units (CFU) per milliliter of a single organism on Maconkey or blood agar, were included. The medical records of the patients were reviewed and selective demographic and clinical data were collected. Patients were excluded if their charts were unavailable for review or if the pathogen that grew in culture was a suspected contaminant. All patients lacking clinical symptoms of UTI (frequency, dysuria, abdominal pain, fever, or urgency) and whose urine was collected by clean-catch were excluded if their culture grew between 10,000 and 100,000 CFU. Resistance to trimethoprim-sulfamethoxazole (T-S) was estimated for the subset of gram-negative pathogens. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated to compare rates of resistance among patients with and without the following risk factors: age greater than 4 years; current or recent antibiotic use; day care attendance; and previous UTI. RESULTS: A total of 126 urine cultures were identified for inclusion. Of these, 45 patients were excluded, leaving 81 who met the study criteria. The majority of isolated organisms were Escherichia coli, accounting for 89% of the patients (n = 72). Other organisms identified were Klebsiella 3.7%, Proteus 1.2%, Citrobacter 1.2%, Staphylococcus 1.2%, and Enterococcus 3.7% (all in children < 4 years old). The resistance to T-S was 6.5% (95% CI = 0.9% to 12.1%) for gram-negative pathogens. Overall, 48% of gram-negative isolates were resistant to one or more antibiotics, any resistance (95% CI = 36.5% to 59.5%). T-S resistance was nominally higher for older children and for those with a history of antibiotic use, although not to a significant degree. Children less than age 4 were more likely to have any resistance (OR 2.6; 95% CI = 1.0 to 6.7). CONCLUSIONS: The resistance to T-S in this study was 6.7% for gram-negative pathogens. These rates are lower than rates reported in adult populations, international pediatric studies, and the authors' hospital antibiograms, demonstrating the importance of local, population-specific data in selecting antibiotics. This study did not identify any statistically significant risk factors for resistance to T-S, but suggests that those with a recent history of antibiotic use may be at highest risk. While children less than 4 years old with gram-negative pathogens have nominally lower rates of T-S resistance, they are at higher risk for resistance to one or more antibiotics (any resistance) and are at risk for UTI caused by enterococcus (uniformly nonsusceptible to T-S). Prospective studies are needed to validate these results and to identify predisposing factors for urinary pathogens with antibiotic resistance.     

Peritoneal Protein Clearance Rather than Faster Transport Status Determines Outcomes in Peritoneal Dialysis Patients

Faster peritoneal transport has been associated with an increased risk of therapy failure and patient mortality. However, faster transport can the result of many factors. Peritoneal protein clearance (PPC) has been proposed to distinguish faster peritoneal transport attributable to inflammatory conditions, as protein clearance reflects large-pore flow, which increases during inflammation. We followed a cohort of 300 peritoneal dialysis patients, and after adjustments for age and comorbidity, higher PPC was associated with increased risk of death (hazard ratio: 1.81; 95% confidence interval: 1.11 to 2.95), even after patients underwent transplantation or transferred to hemodialysis.     

The Inactivation of Intrinsic Antibiotic Resistance Determinants Widens the Mutant Selection Window for Quinolones in Stenotrophomonas maltophilia

We have determined that the mutational inactivation of the SmeDEF efflux pump and the SmQnr quinolone resistance protein widens the mutant selection windows for ofloxacin and ciprofloxacin of Stenotrophomonas maltophilia by reducing their MICs. Resistant mutants arising from a strain lacking SmeDEF and SmQnr presented levels of susceptibility similar to those of the wild-type strain. This indicates that inactivation of intrinsic resistance determinants might increase the chances for selecting resistant mutants at low antibiotic concentrations.     

河源市新生儿期遗传代谢缺陷病的选择性筛查分析

目的明确河源市新生儿期遗传代谢缺陷病的选择性筛查结果。方法选取2011年1月1日~2014年1日期间河源市新出生儿出生3天时的足跟血滤纸干血斑标本,检测G6PD活性筛查G6PD缺乏症,检测甲状腺素筛查先天性甲状腺功能低下,检测苯丙氨酸筛查苯丙酮尿症,对筛查为阳性患儿进行确诊和治疗。比较筛查结果为阳性患儿和阴性患儿的智力发育情况。结果研究3年期间每年的筛查的医院和筛查的产儿覆盖率分别为77.69%、91.13%、90.63%和65.22%、81.58%、71.05%,G6PD缺乏症缺陷、先天性甲状腺功能低下、苯丙酮尿症和总缺陷检出率分别为30.08%、0.31%、0.03%和30.56%。筛查结果为阳性和阴性患儿智力正常、低于正常和智力低下患儿比例分别为86.84%、12.49%、4.47%和98.56%、1.27%和0.17%,筛查结果为阴性患儿中智力正常的患儿比例高于筛查结果为阳性患儿,智力低于正常和智力低下患儿比例则低于于筛查结果为阳性患儿(P0.05)。结论河源市新生儿G6PD缺乏症缺陷、先天性甲状腺功能低下和苯丙酮尿症的检出率较高,遗传代谢缺陷可影响患儿的智力水平,及早进行遗传代谢缺陷病的筛查可提高出生人口素质。     

Antibiotic combinations significantly more active than monotherapy in an in vitro infection model of Stenotrophomonas maltophilia

The goal of this study was to investigate clinical doses of trimethoprim-sulfamethoxazole (TMP-SMX) alone and in combination against Stenotrophomonas maltophilia in an in vitro pharmacodynamic infection model. A 1-compartment model was established using 4 clinical isolates of S. maltophilia susceptible to TMP-SMX and susceptible or intermediately susceptible to at least one other agent (ie, ceftazidime, ciprofloxacin, gentamicin, tobramycin). Antibiotics alone and in combination were tested by simulating unbound serum concentration profiles achieved with multiple-dose regimens in humans. Despite susceptible minimum inhibitory concentrations, TMP-SMX alone was bacteriostatic at best against all isolates. All antibiotic combinations were more active than monotherapy as determined by bacterial reductions at both 24 and 48 h (P < 0.0001). Significant benefit was observed even with agents inactive alone or only intermediately susceptible based on minimum inhibitory concentrations. These preclinical data support further investigation of antibiotic combinations in the management of serious S. maltophilia infections.     

Selective digestive decontamination in patients in intensive care. The Dutch Working Group on Antibiotic Policy

Selective digestive decontamination (SDD) is the most extensively studied method for the prevention of infection in patients in intensive care units (ICUs). Despite 27 prospective randomized studies and six meta-analyses, routine use of SDD is still controversial. In this review, we summarize the available scientific information on effectiveness of SDD in ICU patients. The effects of SDD have been studied in different combinations of the concept, using different antibiotics. Comparison of the individual studies, therefore, is difficult. In most studies, SDD resulted in significant reductions in the number of diagnoses of ventilator-associated pneumonia. However, incidences of ventilator-associated pneumonia in control groups ranged from 5% to 85%. Moreover, these reductions in incidences of ventilator-associated pneumonia in individual studies were not associated with improved patient survival, reductions of duration of ventilation or ICU stay, or reductions in antibiotic use. The numbers of patients studied are too small to determine effects on patient survival. Although two meta-analyses suggested a 20% mortality reduction when using the full concept of SDD (topical and systemic prophylaxis) these results should be interpreted with caution. Formal cost-benefit analyses of SDD have not been performed. SDD is associated with the selection of microorganisms that are intrinsically resistant to the antibiotics used. However, the studies are too small and too short to investigate whether SDD will lead to development of antibiotic resistance. As long as the benefits of SDD (better patient survival, reduction in antibiotic use or improved cost-effectiveness) have not been firmly established, the routine use of SDD for mechanically ventilated patients is not advised.     

Metabolic Fate of [14C]Cefamandole, a Parenteral Cephalosporin Antibiotic, in Rats and Dogs

The biotransformation of the parenterally effective cephalosporin antibiotic cefamandole nafate (I) has been studied in rats and dogs. After rapid in vivo hydrolysis of the nafate pharmaceutical form to cefamandole (II), the antibiotic was found to be very resistant to metabolic degradation in both species. In dogs, cefamandole escaped metabolism and was eliminated as unaltered antibiotic almost exclusively by renal excretion. In rats, cefamandole was somewhat labile to metabolism; however, a major portion of the administered antibiotic was eliminated unchanged principally by renal excretion.     

A Simulation Study Reveals Lack of Pharmacokinetic/Pharmacodynamic Target Attainment in De-escalated Antibiotic Therapy in Critically Ill Patients

De-escalation of empirical antibiotic therapy is often included in antimicrobial stewardship programs in critically ill patients, but differences in target attainment when antibiotics are switched are rarely considered. The primary objective of this study was to compare the fractional target attainments of contemporary dosing of empirical broad-spectrum β-lactam antibiotics and narrower-spectrum antibiotics for a number pathogens for which de-escalation may be considered. The secondary objective was to determine whether alternative dosing strategies improve target attainment. We performed a simulation study using published population pharmacokinetic (PK) studies in critically ill patients for a number of broad-spectrum β-lactam antibiotics and narrower-spectrum antibiotics. Simulations were undertaken using a data set obtained from critically ill patients with sepsis without absolute renal failure (n = 49). The probability of target attainment of antibiotic therapy for different microorganisms for which de-escalation was applied was analyzed. EUCAST MIC distribution data were used to calculate fractional target attainment. The probability that therapeutic exposure will be achieved was lower for the narrower-spectrum antibiotics with conventional dosing than for the broad-spectrum alternatives and could drastically be improved with higher dosages and different modes of administrations. For a selection of microorganisms, the probability that therapeutic exposure will be achieved was overall lower for the narrower-spectrum antibiotics using conventional dosing than for the broad-spectrum antibiotics.     

Thr202Ala in thyA Is a Marker for the Latin American Mediterranean Lineage of the Mycobacterium tuberculosis Complex Rather than Para-Aminosalicylic Acid Resistance

Single nucleotide polymorphisms (SNPs) involved in the development of resistance represent powerful markers for the rapid detection of first- and second-line resistance in clinical Mycobacterium tuberculosis complex (MTBC) isolates. However, the association between particular mutations and phenotypic resistance is not always clear-cut, and phylogenetic SNPs have been misclassified as resistance markers in the past. In the present study, we investigated the utility of a specific polymorphism in thyA (Thr202Ala) as a marker for resistance to para-aminosalicyclic acid (PAS). Sixty-three PAS-susceptible MTBC strains comprising all major phylogenetic lineages, reference strain H37Rv, and 135 multidrug-resistant (MDR) strains from Germany (comprising 8 PAS-resistant isolates) were investigated for the presence of Thr202Ala. In both strain collections, the Thr202Ala SNP was found exclusively in strains of the Latin American Mediterranean (LAM) lineage irrespective of PAS resistance. Furthermore, PAS MICs (0.5 mg/liter) for selected LAM strains (all containing the SNP) and non-LAM strains (not containing the SNP), as well as the results of growth curve analyses performed in liquid 7H9 medium in the presence of increasing PAS concentrations (0 to 2.0 mg/liter), were identical. In conclusion, our data demonstrate that the Thr202Ala polymorphism in thyA is not a valid marker for PAS resistance but, instead, represents a phylogenetic marker for the LAM lineage of the M. tuberculosis complex. These findings challenge some of the previous understanding of PAS resistance and, as a consequence, warrant further in-depth investigations of the genetic variation in PAS-resistant clinical isolates and spontaneous mutants.para-Aminosalicylic acid (PAS) was one of the first drugs used in the treatment of tuberculosis (TB) but initially caused severe side effects, resulting in poor patient compliance. Thanks to a new formulation of the drug leading to improved gastrointestinal tolerance (5), PAS has since then become one of the components in second-line drug treatment to which even highly drug-resistant isolates are susceptible (3, 12, 16).Rengarajan et al. (15) first reported that PAS targets the folate pathway: selection for PAS resistance using transposon mutagenesis led to the isolation of strains with transposon-disrupted thyA (Rv2764c) which exhibited a reduced thymidylate synthase activity. Unlike the wild-type thyA from Mycobacterium bovis BCG (MtbBCG), the expression of the mutated thyA gene failed to rescue a thyA-negative phenotype in Escherichia coli. Similarly, only the expression of the wild-type MtbBCG thyA in the same E. coli background restored sensitivity to trimethoprim (TMP) (15).Complementing these results, two mutations in thyA (Thr202Ala in strains R1 and R3 and Arg222Gly in strain R2) were identified in clinical PAS-resistant Mycobacterium tuberculosis strains. Both alleles failed to fully restore sensitivity to TMP in the thy-negative E. coli background described above. Lastly, the effect of expressing the wild-type thyA gene as well as both clinical alleles in one of their original transposon-disrupted thyA MtbBCG mutants was investigated. Again, only the expression of the wild-type gene completely restored susceptibility to PAS (15).The involvement of thyA in PAS resistance has also been supported by a study of clinical strains from China. Of the 51 PAS-sensitive and 44 PAS-resistant strains investigated, only the resistant strains showed mutations in thyA. However, these accounted for only 36% of the phenotypic resistance, pointing to one or several unknown resistance mechanisms (20).In the attempt to identify further the mechanism leading to PAS resistance, six genes of the folate and thymidine biosynthetic pathways have been analyzed by Mathys et al. (11). Although some mutations were identified in these genes, they did not appear to be relevant for PAS resistance. Instead, the detection of 25 distinct mutations in thyA reaffirmed the role of this gene. Notably, the Thr202Ala mutation discussed earlier was the mutation most frequently identified in 23 clinical isolates and in 1 spontaneous mutant. It was predicted to have a structurally destabilizing effect (11).Our initial analysis of all previously sequenced M. tuberculosis complex (MTBC) strains revealed that this single nucleotide polymorphism (SNP) was present in the following strains: 98-R604, F11, GM 1503, KZN 506, KZN 605, KZN 1435, KZN 2475, and KZN 4207. Given that these eight genomes encompass different subfamilies of the Latin American Mediterranean (LAM) genotype, on the basis of their spoligotype patterns, which were found to accurately define this group (1), this indicated that thyA Thr202Ala might be a specific mutation or even a marker for this lineage.To investigate this striking finding and to define the actual role of thyA Thr202Ala in PAS resistance development, we carried out a more systematic investigation. Sequence analyses of a fragment of the thyA gene of MTBC strains of two different collections, one comprising 63 PAS-susceptible MTBC strains of all major phylogenetic lineages and reference strain H37Rv and the other comprising 135 multidrug-resistant (MDR) strains from Germany, 8 of which were PAS resistant, was carried out. In addition, PAS MIC determinations and growth curve analyses in 7H9 medium with increasing PAS concentrations were performed for selected strains with and without the mutation.     

Lysyl-Phosphatidylglycerol Attenuates Membrane Perturbation Rather than Surface Association of the Cationic Antimicrobial Peptide 6W-RP-1 in a Model Membrane System: Implications for Daptomycin Resistance

The presence of the cationic phospholipid lysyl-phosphatidylglycerol (lysyl-PG) in staphylococcal cytoplasmic membranes has been linked to increased resistance to cationic compounds, including antibiotics such as daptomycin as well as host defense antimicrobial peptides. We investigated the effects of lysyl-PG on binding of 6W-RP-1, a synthetic antimicrobial peptide, to lipid vesicles and on peptide-induced membrane permeabilization. Unexpectedly, physiological lysyl-PG concentrations only minimally reduced membrane binding of 6W-RP-1. In contrast, 6W-RP-1-induced dye leakage was severely inhibited by lysyl-PG, suggesting that lysyl-PG primarily impacts membrane defect formation.Aminoacylated phosphatidylglycerols are common lipids in the cytoplasmic membranes of Gram-positive bacteria, most notably in the genera Staphylococcus, Bacillus, Clostridium, Lactobacillus, Listeria, and Streptococcus (7, 11, 15). Esterification of the phosphatidylglycerol (PG) headgroup occurs with either lysine (lysyl-PG), alanine (alanyl-PG), or, less commonly, ornithine (ornithyl-PG) or arginine (arginyl-PG). Such modifications may serve to modulate the cytoplasmic membrane charge, as the adduct converts the anionic PG into a net cationic or zwitterionic lipid, depending on the amino acid involved (13). In Staphylococcus aureus, this reaction is mediated by the multiple peptide resistance factor F (MprF), an integral membrane protein that catalyzes the transfer of the aminoacyl group from Lys-tRNA^Lys to the sn-3 position of the PG headgroup (14).In S. aureus, resistance to killing by mammalian cationic antimicrobial peptides (CAPs) has been attributed to reduced peptide binding to the bacterial cell surface (16). Furthermore, resistance to daptomycin, a calcium-dependent, cationic lipopeptide, has been suggested to have coevolved with reduced susceptibility to host defense CAPs (8, 9). The presence of lysyl-PG in the bacterial membrane has been clearly linked to these phenomena in S. aureus and Listeria (8, 17). For instance, reduced levels of lysyl-PG were found to increase the susceptibility of S. aureus to many antibiotics, including some CAPs (10, 18, 19), and mutant S. aureus strains that do not produce lysyl-PG exhibit reduced virulence in a variety of in vivo models (18). Of note, the observed correlation between PG lysinylation and reduced susceptibility to cationic antibiotics (e.g., daptomycin) and CAPs has been attributed to a relative decrease in the negative surface charge of the target bacterial cytoplasmic membrane as a consequence of PG modification (8, 10, 12, 13, 18). However, no detailed analyses of the mechanism(s) by which lysyl-PG protects the organism from cationic agent-induced killing have been carried out in either whole bacterial cells or model membranes. For this reason, we employed model membrane systems composed of various PG-to-lysyl-PG ratios and the synthetic peptide 6W-RP-1 to further probe this interaction. 6W-RP-1 is a fluorescent derivative of RP-1, a synthetic peptide modeled in part on the C-terminal helical and microbicidal domain of the mammalian platelet factor 4 (PF-4) family of molecules (20).The peptide 6W-RP-1 (ALYKKWKKKLLKSLKRLG), a tryptophan-substituted congener of the parent peptide, RP-1 (20), was synthesized and authenticated as described previously (20). Large unilamellar vesicles (LUVs) were prepared by mixing chloroform solutions of lipids in the appropriate proportions (1). To mimic the cytoplasmic membrane of S. aureus, a 70:30 mixture of 1-palmitoyl-2-oleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG) and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was used instead of pure POPG to increase vesicle stability. The impact of lysyl-DOPG on peptide activity was investigated by replacing various fractions of POPG with 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(3-lysyl(1-glycerol))] (lysyl-DOPG). For lipid vesicles used in the binding studies, the fluorescent lipid 7MC-POPE (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine [POPE], derivatized with 7MC through an amide bond to the amino group of the ethanolamine headgroup) was synthesized as previously described (5) and added in a chloroform solution to the unlabeled lipids.The association kinetics of 6W-RP-1 with lipid vesicles were measured by Förster resonance energy transfer (FRET) from the Trp residue in the peptide to the acceptor fluorophore, 7MC-POPE, embedded in the bilayer at a concentration of 2 mol% (5). Peptide binding leads to an increase in FRET and can thus be monitored through the increased fluorescence emission from the acceptor. The reaction was followed in a stopped-flow fluorimeter (SX.18MV; Applied Photophysics) as previously described (1). The peptide concentration was held constant at 0.5 μM in all FRET experiments, and the lipid concentration was varied between 25 and 200 μM.If the initial peptide binding to lipid vesicles is well separated in time from other bilayer-perturbing events, peptide binding is described by the simple equilibrium equation where P stands for free peptide in solution, L represents lipid, PL represents lipid-bound peptides, and k_on and k_off are the rate constants for peptide association and dissociation, respectively (1). The coupled differential equations that describe this equilibrium are d[P]/dt = −k_on [L] [P] + k_off [PL] d[PL]/dt = k_on [L] [P] − k_off [PL].As the lipid concentration does not change throughout the experiment, the product of k_on and [L] remains constant. Hence, the kinetics of peptide binding follow a pseudo-first-order rate law and are characterized by an apparent rate constant, k_app, equal to (k_on [L] + k_off), which can be obtained from a fit of the experimental traces to an exponential function (Fig. ​(Fig.11 A). Since the magnitude of k_app depends on the lipid concentration, a plot of k_app against [L] will be linear (Fig. ​(Fig.1B).1B). The molecular rate constants of peptide binding, k_on values, were obtained from the slope of a linear regression to the data points and k_off from the y intercept. The dissociation constant, K_D, was then obtained from the k_off/k_on ratio.Open in a separate windowFIG. 1.Binding of 6W-RP-1 to vesicles composed of POPG and POPC (70:30 ratio). (A) Experimental binding kinetics recorded using 50 μM lipid and 0.5 μM peptide. The experimental data are the averages of five individual traces to improve the signal-to-noise ratio; the solid line is a single exponential fit to the data. (B) The apparent rate constant (k_app) was plotted against the lipid concentration to obtain k_on and k_off, as explained in the text. The error bars represent the standard deviations from five sets of experiments.We determined k_app as a function of lysyl-DOPG content and found that at a given lipid concentration, k_app varied only slightly with a lysyl-DOPG content between 0 and 30 mol% but decreased significantly at lysyl-DOPG concentrations exceeding ∼30 mol% (Fig. ​(Fig.22 A). By using the dependence of k_app on lipid concentration to obtain the molecular rate constants k_on and k_off for each lysyl-DOPG concentration, we were able to show that this effect is caused primarily by a change in k_off. The rate constant for 6W-RP-1 binding to lipid vesicles varied linearly with lysyl-DOPG content (Fig. ​(Fig.2B).2B). However, the dependence was relatively weak, dropping to about 60% of its original value at 40% lysyl-DOPG. In contrast, k_off was found to remain essentially constant up to an overall lysyl-DOPG content of ∼30 mol% (Fig. ​(Fig.2C),2C), which encompasses the lysinylation profile of most clinical S. aureus isolates (6, 12, 13). Only at a lysyl-DOPG concentration of >30 mol% did k_off become notably faster, leading to a significantly reduced net association and a correspondingly higher K_D (Fig. ​(Fig.2D).2D). We propose that this observed lack of correlation between the lysyl-DOPG concentration and peptide binding until the lysyl-DOPG content exceeds 30 mol% is related to peptide-induced lipid domain formation that results in the separation of anionic PG from the cationic lysyl-DOPG and the remaining membrane lipids. In a homogeneous bilayer, the Gibbs free energy (ΔG^o) of binding can be expected to be largely a linear function of the lysyl-DOPG fraction. In that case, we would predict the dissociation constant, K_D, to depend exponentially on the fraction of lysyl-DOPG in the mixture, according to the equation K_D = exp(ΔG^o/RT), which it clearly did not in our studies (Fig. ​(Fig.2D2D).Open in a separate windowFIG. 2.6W-RP-1 interaction with lipid vesicles as a function of the lysyl-DOPG content. The vesicle composition is based on a POPG:POPC ratio of 70:30, in which a fraction of the POPG was replaced by lysyl-DOPG. (A) The apparent rate constant, k_app, as a function of lysyl-PG content, measured at a lipid concentration of 100 μM. (B to D) The molecular rate constant for peptide binding, k_on (B), the molecular rate constant for 6W-RP-1 desorption, k_off (C), and the dissociation constant, K_D (D). The peptide concentration was 0.5 μM in all experiments. The error bars are the standard deviations for a minimum of two and usually three sets of experiments.In contrast to the data on 6W-RP-1 binding, the extent of peptide-induced membrane permeabilization of lipid vesicles was strongly inhibited as a function of lysyl-DOPG content, even at low lysyl-DOPG concentrations. Dye leakage was assessed by encapsulating a self-quenching concentration of carboxyfluorescein (CF) in LUVs by methods described previously (1). A 5 μM peptide solution was allowed to interact with a lipid suspension, and the rate and extent of dye leakage were monitored. The kinetics of CF leakage, measured by the relief of fluorescence self-quenching, were recorded in a stopped-flow fluorimeter to ensure rapid and complete mixing of the reactants. We found that inclusion of 20 mol% lysyl-DOPG decreased both the extent and rate of dye leakage, and at 30 mol%, at which peptide binding is still largely unaffected, dye leakage was essentially abolished (Fig. ​(Fig.3).3). Although unanticipated, this finding is consistent with a biological function of lysyl-DOPG that derives in part from the inhibition of membrane perturbation and leakage, rather than the inhibition of CAP binding. Thus, while daptomycin-resistant clinical S. aureus strains often exhibit increased lysyl-PG membrane content and an enhanced surface positive charge (8), the current findings suggest that such phenotypes are more likely to stabilize membrane integrity than repel cationic antimicrobial agents.Open in a separate windowFIG. 3.Carboxyfluorescein leakage induced by 6W-RP-1 (5 μM) from vesicles (50 μM lipid) composed of POPG:POPC:lysyl-DOPG (70:30:0; dashed line), POPG:POPC:lysyl-DOPG (50:30:20; dotted line), POPG:POPC:lysyl-DOPG (40:30:30; dash-dotted line), and POPG:POPC:lysyl-DOPG (30:30:40; solid line). All curves are normalized to the maximum dye release, which was determined by adding Triton X-100 to the vesicles at a final concentration of 1% to dissolve the vesicles (1).Figure ​Figure44 represents a working model that interprets our experimental findings. In this view, binding of 6W-RP-1 occurs from an unstructured state in solution to a bilayer in which the three lipid types (PG, lysyl-PG, and PC) are homogeneously distributed. The preferential interaction of the peptide with anionic PG (Fig. ​(Fig.4,4, red lipids) leads to the formation of lipid domains enriched in PG (Fig. ​(Fig.4,4, right) and the exclusion of cationic lysyl-PG (Fig. ​(Fig.4,4, blue lipids). We postulate that at a high lysyl-PG content, the remaining fraction of PG becomes limiting and the peptides no longer bind efficiently. It is not clear why this process per se should lead to an inhibition of dye leakage from vesicles; it is conceivable that the presence of the positively charged lysyl-PG leads to an overall stabilization of a membrane rich in anionic PG. The possibility of PG domain formation in mixed lipid vesicles induced by the binding of a cationic peptide has been suggested previously (2-4).Open in a separate windowFIG. 4.Cartoon showing peptide binding to a homogeneous bilayer (left) and desorption from a bilayer with PG domains induced by 6W-RP-1 binding (right). Red headgroups represent unmodified PG; blue headgroups represent lysyl-PG; white headgroups represent PC. In the left panel, peptides are drawn as gray, random structures above the bilayer, representing the unfolded state in solution; in the right panel, they are shown as gray rods to indicate their more ordered conformation in association with the bilayer-bound state.     

Myelin Basic Protein–specific T Helper 2 (Th2) Cells Cause Experimental Autoimmune Encephalomyelitis in Immunodeficient Hosts Rather than Protect Them from the Disease

Chronic inflammatory autoimmune diseases such as multiple sclerosis, diabetes, and rheumatoid arthritis are caused by CD4⁺ Th1 cells. Because Th2 cells antagonize Th1 cell functions in several ways, it is believed that immune deviation towards Th2 can prevent or cure autoimmune diseases. Experimental autoimmune encephalomyelitis (EAE) is a demyelinating disease used as a model for multiple sclerosis. Using an adoptive transfer system we assessed the role of Th1 and Th2 cells in EAE. In vitro generated Th1 and Th2 cells from myelin basic protein (MBP)-specific TCR transgenic mice were transferred into normal and immunodeficient mice. Th1 cells caused EAE in all recipients after a brief preclinical phase. Surprisingly, Th2 cells also caused EAE in RAG-1 KO mice and in αβ T cell–deficient mice, albeit after a longer preclinical phase. Normal or γδ T cell–deficient mice were resistant to EAE induced by Th2 cells. The histopathological features of this disease resembled those of an allergic process. In addition, disease induction by Th1 cells was not altered by coadmininstration of Th2 cells in any of the recipients. These findings indicate that MBP-specific Th2 cells have the potential to induce EAE and that the disease induced by previously activated Th1 cells cannot be prevented by normal lymphocytes nor by previously activated Th2 cells.     

Outcome of End-stage Renal Disease in Patients with Rare Causes of Renal Failure. I. Inherited and Metabolic Disorders

SUMMARY This study represents the first national multi-center evaluationof treatment for end-stage renal disease outcome in patientswith rare metabolic and inherited disorders in the United States.Because of the small number of such patients at single centersonly a co-operative study could provide adequate data on enoughpatients for meaningful conclusions. By co-ordinating such data,the Network Forum provides a model for the newly organized U.S.Renal Data System and demonstrates the potential for usefulspecial studies in the future. It is clear from these data thatdespite the systemic nature of these illnesses, treatment forESRD can be applied to achieve excellent survival rates.