HIV protease inhibitors and dyslipidemia

Highly active antiretroviral therapy (HAART) significantly prolongs the lives of HIV-infected patients. Current regimens may consist of a protease inhibitor (PI) combined with at least two or more other antiretroviral drugs. PI administration has been shown to be associated with alterations in plasma lipids (i.e. prompt and sustained increases in total cholesterol, low-density lipoprotein cholesterol, and triglycerides) and insulin levels that place PI-treated patients at risk for coronary heart disease (CHD). Because PI-associated dyslipidemia is generally asymptomatic and occurs in patients who are often younger than those traditionally at risk for CHD, the need for primary prevention of acute coronary events in these patients is often unappreciated. Statins form a significant component of pharmacotherapy for PI-associated dyslipidemia. However, because PIs and all statins except pravastatin are metabolized by the cytochrome P450 (CYP) system, co-administration of these agents produces a significant risk of drug interactions and statin-induced hepatotoxicity and myopathy. This risk can be greatly reduced by administering a statin not metabolized by CYP. The need for lipid reduction therapy may be minimized with the use of new PIs that are comparable in efficacy to current PIs but do not negatively affect lipid levels.     

Cross-reactive protection against enterohemorrhagic Escherichia coli infection by enteropathogenic E. coli in a mouse model

Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E. coli (EHEC) are related attaching and effacing (A/E) pathogens. The genes responsible for the A/E pathology are carried on a chromosomal pathogenicity island termed the locus of enterocyte effacement (LEE). Both pathogens share a high degree of homology in the LEE and additional O islands. EHEC prevalence is much lower in areas where EPEC is endemic. This may be due to the development of antibodies against common EPEC and EHEC antigens. This study investigated the hypothesis that EPEC infections may protect against EHEC infections. We used a mouse model to inoculate BALB/c mice intragastrically, first with EPEC and then with EHEC (E. coli O157:H7). Four control groups received either a nonpathogenic E. coli (NPEC) strain followed by EHEC (NPEC/EHEC), phosphate-buffered saline (PBS) followed by EHEC (PBS/EHEC), EPEC/PBS, or PBS/PBS. Mice were monitored for weight loss and symptoms. EPEC colonized the intestine after challenge, and mice developed serum antibodies to intimin and E. coli secreted protein B (encoded in the LEE). Prechallenge with an EPEC strain had a protective effect after EHEC infection, as only a few mice developed mild symptoms, from which they recovered. These mice had an increase in body weight similar to that in control animals, and tissue morphology exhibited mild intestinal changes and normal renal histology. All mice that were not prechallenged with the EPEC strain developed mild to severe symptoms after EHEC infection, with weight loss as well as intestinal and renal histopathological changes. These data suggest that EPEC may protect against EHEC infection in this mouse model.     

HIV protease inhibitors impact on apoptosis

HIV protease inhibitors are the backbone of HIV therapy. In addition to blocking intracellular HIV protease and dramatically decreasing viral burden, the protease inhibitors also regulate apoptosis. A growing body of data has confirmed the immunomodulatory effects of HIV protease inhibitors which block CD4+ and CD8+ T cell death in models of HIV infection. The mechanism of this apoptosis inhibition is still under active investigation and supported by several proposed hypothesis for how they alter the fate of the cell. More recently, the anti-apoptotic effects of the HIV protease inhibitors has been extended to the non-HIV, non-immune cell, whereby protease inhibitors prevent apoptosis, and disease, in animal models of sepsis, hepatitis and stroke. Interestingly, when HIV protease inhibitors are used at supra-therapeutic concentrations, they exert pro-apoptotic effects. This has been demonstrated in a number of tumor models. Although it is unclear how HIV protease inhibitors can induce apoptosis at increased concentrations, future research will define the targets of the immunomodulation and reveal the full clinical potential of this intriguing class of drugs.     

Inhibition of natural killer cytotoxicity in vitro by clinical grade serine protease inhibitors

The effects of clinical grade serine protease inhibitors on natural killer (NK) activity were compared. Cytotoxicity was measured with the Calcein-AM release method, using K562, Raji as a target. There is a significant correlation between measurements of NK activity by the Calcein-AM method and the 51Cr release assay. Cytotoxicity was inhibited with a calcium chelating agent or a perform inhibitor. Although up to 65% of cytotoxicity was inhibited by nafamostat mesilate with an E/T ratio of 10:1, and by 55% by ulinastatin, neither gabexate mesilate nor antithrombin III inhibited any cytotoxicity. None of these agents inhibited lymphokine-activated killer cell activity. In clinical applications, it should be noted that some protease inhibitors have been proven to have immunosuppressive effects.     

Use of HIV protease inhibitors as pharmacoenhancers

Short- and long-term therapy with many protease inhibitors (PIs) at standard dosing may be limited by inconvenient dosing regimens, high tablet volumes, and variable drug exposure. Booster agents, such as low (or "baby") doses of ritonavir, and codosing with the nonnucleoside analogue delavirdine are increasingly used to maintain high PI trough exposures. Combinations of HIV PIs, either alone or coadministered with nucleoside analogues, have demonstrated substantial virologic and immunologic responses sustained over long periods of follow-up. This approach is rapidly becoming the standard of care with PI use. The advantages of the boosted PI approach include raising trough drug concentrations, diminishing interpatient variability, prolonging drug half-life to allow twice-daily and possibly once-daily dosing, and diminishing food requirements and tablet volume. In addition, increases in drug exposure may potentially enable inhibition of the virus in the presence of reduced sensitivity to PIs. Using a boosted PI, in the absence of comparative data on resistance profiles of boosted regimens, should be considered differently from dual-PI combinations, where there is exposure to 2 active PIs. Some physicians are now using a booster agent plus 2 PIs in the salvage therapy setting in an attempt to achieve effective, yet less toxic, concentrations of 2 PIs to overcome different resistant populations in a patient's viral quasispecies.     

Bacterial cell division protein FtsZ is stable against degradation by AAA family protease FtsH in Escherichia coli cells

We have found that FtsH protease of Escherichia coli could degrade E. coli cell division protein FtsZ in an ATP- and Zn(2+)-dependent manner in vitro and that the degradation did not show specificity for the N-terminus or C-terminus of FtsZ, like in the case of degradation of its conventional substrate sigma(32) protein. In continuation of these observations, in the present study, we examined whether FtsH would affect the stability and turnover of FtsZ in vivo. We found that FtsZ levels were not elevated in E. coli AR754 (ftsH1 ts) cells at nonpermissive temperature as compared to the levels in an FtsH-active isogenic AR753 strain. Neither did FtsH degrade ectopically expressed FtsZ in AR754 strain nor did ectopic expression of FtsH reduced FtsZ levels in E. coli AR5090 ftsH null strain (ftsH::kan, sfhC21). Pulse chase experiments in AR754 and AR5090 strains showed that there were no compensatory changes in FtsZ turnover, in case FtsZ degradation had occurred. Even under cell division arrested conditions, wherein FtsZ was not required, FtsH protease did not degrade unutilized FtsZ. These experiments demonstrate that either FtsH protease may not have a role in regulating the levels of FtsZ in vivo under the conditions tested or that some cellular component(s) might be stabilising FtsZ against FtsH protease.     

Tolerability and safety of HIV protease inhibitors in adults

Antiretroviral drugs are associated with both short-term and long-term adverse events. Like other HIV drugs, protease inhibitors (PIs) may affect metabolic processes influencing body shape and body tissue composition, appearance, bone integrity, and cardiovascular status. However, numerous confounding variables including age, cigarette smoking, body mass index (BMI), duration of HIV infection, degree of immunodeficiency, concomitant antiretroviral agents, extent of previous treatment, and duration of treatment all blur the relationship between PI use and adverse events. Recent data suggest that the early PIs appear to have greater effects on such surrogate markers of disease risk as insulin resistance and cholesterol and triglyceride levels than the recently developed PIs. These data also suggest that evaluation of PIs as a class should be reconsidered and that it is probably not appropriate to extrapolate safety data obtained from individuals treated with first-generation agents in the era of potent combination antiretroviral therapy to those treated with recently developed PIs. Because PIs remain a critical component of successful antiretroviral therapy, evaluation of potential long-term complications with prolonged PI use is essential, as is delineation of the significant differences in safety profiles among individual PIs.     

Depressive symptoms decline among persons on HIV protease inhibitors

OBJECTIVE: To ascertain whether initiation of protease inhibitors was associated with a change in depressive symptoms among persons infected with HIV. METHODS: Study subjects included men and women who were enrolled in the HIV/AIDS Drug Treatment Program and who had completed an annual participant survey before and after initiating triple combination therapy with a protease inhibitor. Depressive symptoms were assessed using the Centre for Epidemiologic Studies-Depression scale (CES-D). Statistical analyses to determine the change in CES-D total and subscale scores before and after protease inhibitor use were conducted using parametric and multivariate methods. RESULTS: Our analysis was restricted to 453 participants. Of these 234 (52%) were depressed at baseline (CES-D score > or = 16). Compared with nondepressed participants, depressed participants were slightly younger (p = .048), less likely to be employed (p < .001) and more likely to have an annual income less than $10,000 per annum (p < .001). After adjusting for CD4 count, employment status, income, age, and CES-D total or subscale score at baseline, we found a significant improvement in total scale score (p = .001) and depressive mood (p = .002), positive affects (p = .005), and somatic symptoms (p = .011) subscale scores at follow-up. There was no significant change in the interpersonal relations score over the study period. CONCLUSION: Our findings indicate that in addition to conferring impressive clinical benefits, protease inhibitor use is associated with a significant improvement in HIV-positive individuals' mental health.     

Vaccines against enterotoxigenic Escherichia coli

Enterotoxigenic Escherichia coli (ETEC) is a major cause of diarrhea among children less than 3 years of age in developing countries and in travelers to these areas. The key pathogenic mechanisms that contribute to the pathogenesis of ETEC are the production of colonization factors (CFs) and a heat-labile enterotoxin (LT) and/or a heat-stable enterotoxin. To provide broad-spectrum protection, an ETEC vaccine should, most likely, contain the most prevalent fimbrial antigens, that is, CF antigen I and CS1-CS6, and/or a LT toxoid. Different strategies have been taken to deliver ETEC fimbriae and toxin antigens to the human immune system to elicit strong mucosal, in particular, intestinal immune responses that are considered to be of prime importance for protection against ETEC disease. There has been some promise when testing different ETEC candidate vaccines for protection against diarrhea in adult travelers. However, no ETEC candidate vaccine has been shown to be effective in the most important target group, which is infants and young children in endemic areas. Against this background, intense efforts are in progress to try to improve the immunogenicity of different available candidate vaccines, as well as to develop new types of ETEC vaccines.     

Protection against HIV-envelope-induced neuronal cell destruction by HIV attachment inhibitors

We demonstrate that HIV attachment inhibitors (AIs) prevent HIV envelope-induced destruction of two neuronal cell lines (SH-SY5Y and BE(2)-M17) at low nanomolar concentrations. The fusion inhibitor enfuvirtide and the CCR5 inhibitors UK427,857 and TAK779 do not display protection activity, suggesting the involvement of Env/cell interaction site(s) distinct from the sites involved in the viral entry process. We surmise that by inducing conformation changes in the envelope, AIs likely obstruct novel interactions with a neuronal cell factor(s) required for induction of apoptosis. This antiretroviral class may therefore have the potential to inhibit HIV-induced neuron damage, thereby curtailing the increasing incidence of HIV-associated cognitive impairment.     

Screening for Escherichia coli O157:H7 in Illinois

Objective: To determine the incidence of infection with Escherichia coli O157:H7 in a tertiary referral center in Chicago, where a similar study had been performed in 1984, to evaluate cases of disease reported to the Illinois Department of Public Health (IDPH) in 1993, and to determine laboratory practices used to detect this infection throughout the state.
Methods: During a 6-month period in 1993, all stool specimens at Rush-Presbyterian-St Luke's Medical Center (RPSLMC) were tested for E. coli O157:H7. Reports of diagnosed E. coli O157:H7 cases investigated by IDPH were also reviewed. A survey of 73 hospitals in the Chicago area was performed to determine routine culturing practices, specifically, the selection of stool specimens for evaluation for this pathogen.
Results: In the RPSLMC survey, two cases were identified among 1985 samples (incidence 0.1%), similar to the 0.08% incidence detected in a similar study conducted at the same institution in 1984. Through passive surveillance, the IDPH received 44 reports of E. coli O157:H7 in 1993. The hospital survey revealed that, in the seven labs testing all stool specimens for E. coli O157:H7, an incidence of 16/8137 specimens (0.2%) was determined.
Conclusions: These data suggest that sporadic E. coli O157:H7 remains uncommon in Illinois and that the incidence may not have changed over a 9-year period. The low yield and substantial cost of culturing all stools suggest that only specimens from patients with bloody diarrhea should be evaluated routinely in areas of low endemicity.     

Gender-specific effects of HIV protease inhibitors on body mass in mice

Protease inhibitors, as part of highly active anti-retroviral therapy (HAART), have significantly increased the lifespan of human immunodeficiency virus (HIV) infected patients. Several deleterious side effects including dyslipidemia and lipodystrophy, however, have been observed with HAART. Women are at a higher risk of developing adipose tissue alterations and these alterations have different characteristics as compared to men. We have previously demonstrated that in mice the HIV protease inhibitor, ritonavir, caused a reduction in weight gain in females, but had no effect on male mice. In the present study, we examined the potential causes of this difference in weight gain. Low-density lipoprotein receptor (LDL-R) null mice or wild-type C57BL/6 mice, were administered 15 μg/ml ritonavir or vehicle (0.01% ethanol) in the drinking water for 6 weeks. The percent of total body weight gained during the treatment period was measured and confirmed that female LDL-R gained significantly less weight with ritonavir treatment than males. In wild type mice, however, there was no effect of ritonavir treatment in either sex. Despite the weight loss in LDL-R null mice, ritonavir increased food intake, but no difference was observed in gonadal fat weight. Serum leptin levels were significantly lower in females. Ritonavir further suppressed leptin levels in (p < 0.05). Ritonavir did not alter serum adiponectin levels in either gender. To determine the source of these differences, female mice were ovariectomized remove the gonadal sex hormones. Ovariectomy prevented the weight loss induced by ritonavir (p < 0.05). Furthermore, leptin levels were no longer suppressed by ritonavir (p < 0.05). This study demonstrates that gonadal factors in females influence the hormonal control of weight gain changes induced by HIV protease inhibitors in an environment of elevated cholesterol.     

High-performance liquid chromatography of HIV protease inhibitors in human biological matrices

Methods for HPLC analysis of protease inhibitors (PIs) in human biological matrices were reviewed. Assays have been developed for analysis of single PIs or for simultaneous measurement of multiple PIs in plasma-serum, saliva, cerebrospinal fluid and semen. Liquid-liquid extraction was most often applied for sample pretreatment, but solid-phase extraction and protein precipitation were used as well. Reversed-phase or ion-pair chromatography have been used to separate PIs. Detection of PIs should be sensitive enough for quantitation of plasma concentrations below trough levels of single PIs, or below proposed therapeutic thresholds for PIs. The large majority of assays employs UV detection. As the potential for interferences is large, the selectivity of every method should be evaluated properly. The available high-performance liquid chromatography (HPLC) methods have been applied in clinical pharmacokinetic studies and for therapeutic drug monitoring of PIs. Participation in an interlaboratory quality control program is recommended for every laboratory engaged in the bioanalysis of PIs.     

Heterologous protection against invasive Escherichia coli K1 disease in newborn rats by maternal immunization with purified mannose-sensitive pili.

Heterologous protection against Escherichia coli K1 bacteremia with antibody to purified mannose-sensitive (MS) pili was demonstrated in a neonatal rat model. The serological relatedness of purified MS pili from 17 E. coli K1 clinical isolates was examined by an enzyme-linked immunosorbent assay. Five pilus serogroups were identified, with the pili in each group showing 50% or greater cross-reactivity with the typing serum of the group. The MS pili from 12 of 17 (70%) strains belonged to just two serogroups. Pregnant Sprague-Dawley rats (dams) were immunized with purified pili, and their newborns (pups) were challenged with heterologous E. coli. Bacteremia was significantly reduced when the pili used for immunization were from the same serogroup as the pili expressed by the challenge bacteria. Thus, immunization with C94 pili and challenge with E03 (71% cross-reactivity) or E04 (50% cross-reactivity) resulted in bacteremia rates of 12 of 17 (17%) versus 51 of 79 (65%) in controls and 0 of 75 (0%) versus 28 of 70 (40%) in controls, respectively (P less than 0.001 for each comparison). With lower cross-reactivity, less protection was observed (P less than 0.05 for 22 to 37% pilus serological relatedness). No protection was seen in pups suckled by dams immunized with MS pili having only 5% serological relatedness to the pili on the challenge strain.