Subcellular localization, stoichiometry, and protein levels of 26 S proteasome subunits in yeast.

The 26 S proteasome of eukaryotes is responsible for the degradation of proteins targeted for proteolysis by the ubiquitin system. Yeast has been an important model organism for understanding eukaryotic proteasome structure and function. Toward a quantitative characterization of the proteasome, we have determined the localization, cellular levels, and stoichiometry of proteasome subunits. The subcellular localization of two ATPase components of the regulatory complex of the proteasome, Sug2/Rpt4 and Sug1/Rpt6, and a subunit of the 20 S proteasome, Pre1, were determined by immunofluorescence. In contrast to findings in multicellular organisms, these proteins are localized almost exclusively to the nucleus throughout the cell cycle. We have also determined the cellular abundance and stoichiometry of these proteasome subunits. Sug1/Rpt6, Sug2/Rpt4, and Pre1 are present in roughly equal stoichiometry with an abundance of 15,000-30,000 molecules/cell, corresponding to a concentration of 13-26 microM in the nucleus. Also, in contrast to mammalian cells, we find no evidence of a p27-containing "modulator" of the proteasome in yeast. This information will be useful in comparing and contrasting the yeast and mammalian proteasomes and should contribute to a mechanistic understanding of how this complex functions.

Influenza vaccination of human immunodeficiency virus (HIV)-infected adults: impact on plasma levels of HIV type 1 RNA and determinants of antibody response.

We assessed the effect of influenza vaccination on plasma levels of human immunodeficiency virus type 1 (HIV-1) RNA and the impact of age, plasma HIV-1 RNA level, CD4 cell count, and anti-HIV therapy on immune response. Forty-nine adults (mean age, 38.7 years; mean CD4 cell count +/- SD, 190 +/- 169/mL; mean plasma HIV-1 RNA level +/- SD, 154,616 +/- 317,192 copies/mL) were immunized. Elevations of > or = 0.48 log in plasma HIV-1 RNA levels occurred in two (4%) of 49 subjects within 4 weeks of vaccination. A fourfold or greater increase in antibody titer occurred in 13 (45%) of 29 subjects, correlating directly with CD4 cell count (P = .002) and inversely with plasma HIV-1 RNA level (P = .034). By multivariate analysis, CD4 cell count was a stronger predictor of antibody response than was plasma HIV-1 RNA level. We conclude that increases in plasma HIV-1 RNA levels following influenza vaccination are rare and transient and that antibody response is impaired with CD4 cell counts of < 100/mL and plasma HIV-1 RNA levels of > 100,000 copies/mL. Prospective trials are needed to evaluate the impact of highly active therapy on immune response after vaccination.

Provider attitudes regarding participation of women and persons of color in AIDS clinical trials.

Provider attitudes and perceptions that may influence recruitment and enrollment of diverse patients into AIDS clinical trials were examined by conducting a cross-sectional survey of all HIV/AIDS providers at a municipal teaching hospital. Providers were less likely to feel confident explaining trials to non-English-speaking patients (p < .05). Providers also reported being more confident of their ability to give an overview of clinical trials in culturally appropriate terms to white patients than to patients of other races/ethnicities (p < .05). Many providers perceived the interest in clinical trials by African American (25%), Latino (14%), and Haitian patients (30%) to be lower; and primarily cited suspicions about clinical research as the reason. Some providers (13%) perceived that women with HIV/AIDS are less interested in clinical trials. Despite these perceptions, all providers reported that they are just as likely to inform women and African Americans about available clinical trials; a small proportion reported that they were less likely to inform Latinos (6%) and Haitians (11%). None of these findings differed significantly by provider race, gender, HIV experience, languages spoken, or specialty. Underrepresentation of minorities and women in AIDS Clinical Trials may partially result from attitudes and perceptions of providers.

HIV/AIDS patients' perspectives on adhering to regimens containing protease inhibitors.

OBJECTIVE: To gather qualitative data regarding HIV/AIDS patients' perspectives about HIV-1 protease inhibitors (PIs), and about their experiences taking and adhering to regimens containing PIs. DESIGN: Six focus groups of persons under care for HIV were conducted between September and November 1996 regarding participants' knowledge, awareness, experiences when taking, and adherence to antiretroviral regimens containing PIs. An identical discussion guide was used to facilitate all six groups. Focus group proceedings were audiotaped, transcribed, coded for themes, and analyzed qualitatively. SETTING: HIV/AIDS practices of three teaching hospitals and two community health centers. PATIENTS/PARTICIPANTS: Fifty-six patients with HIV disease: 28 men and 28 women. MEASUREMENTS AND MAIN RESULTS: Knowledge and positive impressions of PIs were prevalent among this diverse group of persons with HIV, and did not differ by race/ethnicity or gender. Most knew that these were new, potent medications for treating HIV/AIDS. Networks of persons with HIV and medical providers were the most important information sources. Those taking PIs were aware that adherence to the regimen is important, and most were using special strategies to maximize their own adherence, but expressed considerable frustration about the central role these medication regimens had assumed in their life. A subset who did not believe they would adhere to these regimens had declined treatment with them. Motivating factors for taking and adhering to these complex regimens were improving CD4 counts and viral loads and the patient-provider relationship. CONCLUSIONS: Among those with HIV/AIDS, awareness of PIs and their effectiveness is substantial, owing to the impact of informal networks and medical providers. This early positive "reputation" of PIs may enhance motivation for adherence. Those who are taking PIs invest substantial effort adhering to these complex regimens, but resent the need to make medications the focus of their lives.

Ventilator-associated bacterial pneumonia: challenges in diagnosis, treatment, and prevention.

Ventilator-associated pneumonia (VAP) is a common infection in intensive care unit patients that results in high mortality and morbidity and increased duration of hospital stay. Clinical diagnostic methods are sensitive, but lack specificity. Quantitative analysis of specimens from the lower respiratory tract increases specificity. Bacteria causing VAP may originate from the patient's endogenous flora, other patients or hospital personnel, or from environmental sources. Aspiration or direct inoculation are the major routes of bacterial entry into the lower respiratory tract. The bacterial inoculum and host response in the lung are important factors for pathogenesis. Late-onset nosocomial pneumonia is often caused by Pseudomonas aeruginosa, Acinetobacter species, and Staphylococcus aureus. Streptococcus pneumoniae and Haemophilus influenzae, however, are the more common pathogens in early-onset disease. Oropharyngeal and gastric colonization with bacteria, cross-infection, as well as the indiscriminate use of antibiotics or invasive devices substantially increase the risk of VAP. An understanding of the epidemiology and pathogenesis of VAP, along with implementation of appropriate preventive measures, are needed to decrease the incidence, morbidity, and mortality associated with VAP.

Response of lymphoepithelial parotid cysts to antiretroviral treatment in HIV-infected adults.

BACKGROUND: Surgical resection has been the usual therapy for HIV-infected patients with lymphoepithelial parotid cysts. OBJECTIVE: To study antiretroviral therapy for lymphoepithelial parotid cysts. DESIGN: Case series. SETTING: HIV outpatient clinics. PATIENTS: HIV-infected patients with lymphoepithelial parotid cysts. INTERVENTION: Antiretroviral therapy. MEASUREMENTS: Change in size of the parotid cyst, CD4 lymphocyte count, and HIV viral load. RESULTS: Nine HIV-infected adults presented with chronic, large parotid cysts, eight of which were bilateral. In at least seven patients, the cysts were the initial sign of HIV infection. In six patients, the cysts resolved completely with combination antiretroviral therapy. Four of these patients also received prednisone. Three patients who did not comply with antiretroviral therapy had partial responses followed by relapses. CONCLUSIONS: Parotid cysts are an unrecognized sign of early HIV infection. These cysts respond to combination antiretroviral therapy, with or without corticosteroids. Surgical resection should be reserved for patients in whom medical therapy has failed or those who refuse or are poorly compliant with medical therapy.

Occupational exposure to HIV: experience at a tertiary care center.

We examined our hospital-based occupational health clinic's experience with combination antiretroviral therapy for postexposure prophylaxis for human immunodeficiency virus (HIV). Over a 12-month period, 68 workers started postexposure prophylaxis: 23 with zidovudine and lamivudine and 45 with zidovudine, lamivudine, and indinavir. Fifty-one (75%) of the 68 workers starting postexposure prophylaxis reported one or more side effects. Side effects were more common among those taking three drugs. Many workers failed to complete the recommended 28-day regimen because of the side effects of the various treatments. The estimated mean cost for evaluations, prophylaxis, and monitoring of exposed workers was $669 per reported exposure. In our experience, major challenges in carrying out the current HIV postexposure prophylaxis guidelines include expeditious source testing, improved staff education and prevention measures, and scrupulous monitoring of workers taking combination antiretroviral drugs for postexposure prophylaxis, with consideration of alternate regimens for intolerant workers.

Hospital-acquired pneumonia: perspectives for the healthcare epidemiologist.

Nosocomial pneumonia is defined as an infection of lung parenchyma that was neither present nor incubating at the time of the patient's admission to the hospital. In the United States, hospital-acquired pneumonia is the second most common nosocomial infection and accounts for the most deaths from nosocomial infection. We describe how infection control personnel can use targeted surveillance to identify clusters of cases and to prevent pneumonia. We also discuss common pathogens that cause nosocomial pneumonia; ventilator-associated pneumonia; and strategies for prevention of hospital-acquired pneumonia.

Multidrug-resistant human sarcoma cells with a mutant P-glycoprotein, altered phenotype, and resistance to cyclosporins.

A variant of the multidrug-resistant human sarcoma cell line Dx5 was derived by co-selection with doxorubicin and the cyclosporin D analogue PSC 833, a potent inhibitor of the multidrug transporter P-glycoprotein. The variant DxP cells manifest an altered phenotype compared with Dx5, with decreased cross-resistance to Vinca alkaloids and no resistance to dactinomycin. Resistance to doxorubicin and paclitaxel is retained. The multidrug resistance phenotype of DxP cells is not modulated by 2 microM PSC 833 or cyclosporine. DxP cells manifest a decreased ability to transport [3H]cyclosporine. DNA heteroduplex analysis and sequencing reveal a mutant mdr1 gene (deletion of a phenylalanine at amino acid residue 335) in the DxP cell line. The mutant P-glycoprotein has a decreased affinity for PSC 833 and vinblastine and a decreased ability to transport rhodamine 123. Transfection of the mutant mdr1 gene into drug-sensitive MES-SA sarcoma cells confers resistance to both doxorubicin and PSC 833. Our study demonstrates that survival of cells exposed to doxorubicin and PSC 833 in a multistep selection occurred as a result of a P-glycoprotein mutation in transmembrane region 6. These data suggest that Phe335 is an important binding site on P-glycoprotein for substrates such as dactinomycin and vinblastine and for inhibitors such as cyclosporine and PSC 833.

Causes of fever in patients infected with human immunodeficiency virus who were admitted to Boston City Hospital.

We prospectively studied causes of fever in patients with human immunodeficiency virus (HIV) infection that required admission to a municipal hospital. A total of 168 HIV-infected persons were admitted for 220 episodes of fever: 72% were male, 80% were nonwhite, 65% reported prior injection drug use, and 74% had a baseline CD4 lymphocyte count of < 200/mm3. Bacterial infections, principally pneumonia, accounted for > 60% of the episodes; Streptococcus pneumoniae and Staphylococcus aureus were most commonly isolated. Pneumocystis carinii pneumonia (PCP) and disseminated infection with Mycobacterium avium complex (MAC) comprised 53% of the remaining sources of fever. In comparison with episodes of fever due to nonbacterial causes, those associated with common bacterial infections were significantly more likely to involve patients with a history of injection drug use (P = .02), higher admission leukocyte count (P < .004), shorter duration of fever (P = .003), shorter hospital stays (P = .0001), and a CD4 count of > 100/mm3 (P = .002). We conclude that bacterial infection, especially pneumonia, is a common cause of fever in HIV-infected patients admitted to our hospital. Patients with bacterial infections are more likely to report a history of injection drug use and have CD4 counts of > 100/mm3, shorter duration of fever, decreased length of hospitalization, and lower mortality than patients with fever due to PCP, disseminated MAC infection, or other causes.

Nosocomial colonization and infection in persons infected with human immunodeficiency virus.

Nosocomial infections appear to be increased in patients with acquired immunodeficiency syndrome (AIDS), compared to individuals with asymptomatic infection due to human immunodeficiency virus (HIV). Risk factors for bacterial colonization and infection include immunosuppression, prior treatment with some antibiotics, increased hospitalizations with longer lengths of stay, greater exposure to invasive devices such as indwelling intravenous or urinary catheters, and the degree of immunosuppression. Data suggest that other infectious agents such as Pneumocystis carinii, Mycobacterium tuberculosis, Mycobacterium avium complex, and Cryptosporidium may be acquired in healthcare facilities. Diagnosis and management of nosocomial infections in HIV-infected persons may be complicated by an atypical presentation, increased rates of relapse following treatment, presence of multiple infections, and early discharge from the inpatient setting. Accurate assessment of nosocomial infections and outbreaks in the hospital is complicated by limited data on the risk of transmission of both traditional and unusual pathogens in this population. Furthermore, some patients may acquire nosocomial pathogens during their initial hospitalization and present later with infections that normally would be classified as community acquired. Therefore, there probably is an underestimation of current nosocomial infection rates, and perhaps "hospital-associated" or "healthcare-facility-associated" might be more accurate terms for these infections.

Nosocomial pneumonia in mechanically ventilated adult patients: epidemiology and prevention in 1996.

Mechanically ventilated patients have a higher incidence of pneumonia and mortality than do nonventilated patients. Ventilator-associated pneumonia (VAP) is diagnosed clinically, by bronchoscopy or "blind" bronchoalveolar lavage (BAL) or protected specimen brush (PSB), and by quantitative endobronchial aspirates (QEA). VAP is usually caused by bacteria, but Legionella pneumophila, Mycobacterium tuberculosis, viruses, and fungi are also potential pathogens. Bacteria causing nosocomial pneumonia may be part of the patient's endogenous flora, originate from other patients, hospital personnel, or environmental sources. Pseudomonas aeruginosa, Acinetobacter spp, and Staphylococcus aureus are the most common causative agents in late-onset nosocomial pneumonia, and Streptococcus pneumoniae and Hemophilus influenzae are more commonly found in early-onset pneumonia. Aspiration appears to be the major route for the entry of bacteria into the lower respiratory tract. Host factors, oropharyngeal and gastric colonization, cross-infection, and complications from the use of antibiotics and nasogastric and endotracheal tubes increases the risk of bacterial VAP. A working knowledge of the epidemiology and strategies for prevention of VAP should reduce infection rates, morbidity, and mortality in critically ill patients.

Resistance mechanisms in human sarcoma mutants derived by single-step exposure to paclitaxel (Taxol).

A fluctuation analysis experiment was performed by exposing 15 expanded populations of MES-SA sarcoma cells to paclitaxel (Taxol) at a concentration of 10 nM for 7 days. The mutation rate was approximately 8 multiplied by 10(-7)/cell generation. ANOVA supports a stochastic cell survival mechanism of spontaneous mutation rather than induction of an adaptive response under these selection conditions. Surviving colonies were found in 12 populations, 9 of which had clones that remained resistant to paclitaxel after a 2-month period of propagation. Analysis of mdr1 gene expression by reverse transcription PCR demonstrated positive clones in 4 of the 9 populations with stable resistance. Accumulation of [(3)H]paclitaxel was decreased in these clones but not in the mdr1-negative clones compared with parental cells. A high degree of resistance to paclitaxel (36- to 93-fold) was selected by this single drug exposure in all 9 stably resistant mutants. Those with mdr1 activation demonstrated a broad cross-resistance to vinblastine, doxorubicin, and etoposide, whereas the other 6 mutants were cross-resistant only to the Vinca alkaloids. Because tubulins are the target molecules for paclitaxel cytotoxicity, we evaluated total tubulin content by immunoblotting and performed semiquantitative reverse transcription PCR analysis for expression of the alpha-tubulin isotypes B alpha 1, K alpha 1 and H alpha 44, the beta-tubulin isotypes M40, beta9, 5beta, beta2 and beta4, and gamma-tubulin. Total tubulin content was decreased significantly in one of the single-step mutants. All surviving clones, both resistant and sensitive to paclitaxel, displayed reduced expression of the 5beta and beta 4 beta-tubulin isotype transcripts in comparison with the parental cell line. These data suggest that stringent exposure to paclitaxel selected clones with reduced transcript levels of 5beta and beta4 beta-tubulin isotypes, but that these reduced levels were not directly involved in the resistance of the clones to paclitaxel. The results suggest an important role for non-multidrug-resistant mechanisms of resistance to paclitaxel. These mechanisms do not involve reduced drug accumulation and provide cross-resistance among both paclitaxel and tubulin depolymerizing agents.

Differential expression of tubulin isotypes during the cell cycle.

Microtubules play an essential role in cell division. Little is known about possible variations of total tubulin and tubulin isotype expression during the cell cycle. We analyzed the total tubulin content, tubulin polymerization status and tubulin isotype content in resting and dividing human K562 leukemic cells and human MES-SA sarcoma cells. Although the total cellular tubulin content increases as the cells progress toward mitosis, the total tubulin/total protein ratio is stable during the cell cycle. Reverse transcriptase-polymerase chain reaction was applied to analyze the levels of expression of alpha, beta, and gamma-tubulin isotypes. Whereas alpha-tubulin isotype and gamma-tubulin transcripts were found to be expressed at constant levels throughout the cell cycle, some of the beta-tubulin isotype transcripts were found to be more highly expressed in dividing then in resting cells. Both of the class IV beta-tubulin isotype transcripts (human 5 beta and beta 2, Class IVa and IVb, respectively) were expressed in dividing K562 and MES-SA cells at twice the levels found in resting cells. Increased expression of the class IV isotype proteins in dividing cells was confirmed by immunoblotting, both in K562 and in MES-SA cells. A larger fraction of total cell tubulin was found to be polymerized in dividing cells (36-40%) than in resting cells (27-30%). The degree of polymerization of class IV tubulin in dividing and resting cells was similar to that of total tubulin. These results show that total tubulin is expressed as constant levels throughout the cell cycle but that the degree of polymerization is increased as cells are committed to division. The relative overexpression of the two class IV beta-tubulin isotypes in dividing cells suggests functional specificity for these isotypes and a regulatory role of these isotypes on the microtubule network during mitosis.

Factitious HIV infection: the importance of documenting infection.

OBJECTIVE: To examine possible causes for factitious human immunodeficiency virus (HIV) infection among patients in an HIV clinic. DESIGN: Retrospective chart review, a case-control study, and a survey of local hospital practices for documenting HIV infection. SETTING: Clinical acquired immunodeficiency syndrome (AIDS) program at a municipal hospital. RESULTS: Seven patients with self-reported, undocumented HIV infection were identified as HIV seronegative after a mean of 9.2 months of care in our clinical AIDS program. The median CD4 count for these patients was 740 cells/mm3; 6 patients had a history of illicit narcotic use and clinical symptoms consistent with HIV disease. Compared with 70 randomly selected controls from HIV clinics, patients with factitious HIV infection had higher CD4 counts (difference, 519 cells/mm3; P < 0.001) and were more likely to have an HIV-infected sexual partner (odds ratio, 15.0; P = 0.005) and a history of a suicide attempt (odds ratio, 9.8; P = 0.02). Known cases of alleged HIV infection have occurred at 8 of the 10 other local hospitals surveyed. However, only 1 of the 10 hospitals routinely documented HIV infection in patients before initiating care. CONCLUSIONS: Limitations of the current serologic tests for HIV, the use of anonymous HIV testing, and recent reports of factitious HIV disease or immune deficiency syndromes that may mimic AIDS underscore the need for clear documentation of HIV infection before medical care is started.

Human immunodeficiency virus infection in pregnancy: epidemiology and prevention of vertical transmission.

The preponderance of AIDS cases in women during their reproductive years demands attention to primary prevention, early recognition, and appropriate interventions. Increases in the epidemic among injection drug users and heterosexually exposed persons have resulted in an increased impact of HIV/AIDS among women and children. It is clear that vertical transmission of HIV can occur early in pregnancy, during labor and delivery, or in the postpartum period. For these reasons, prevention strategies must be comprehensive and include primary prevention and appropriate intervention during pregnancy and in the peripartum and postpartum area. Stringent infection control techniques are imperative during delivery and in the postpartum period, even in the absence of clear data supporting this intervention. Recent data suggest that the cost of treating one adult patient with AIDS is approximately $100,000, making prevention and intervention imperative.

Selective decontamination of the digestive tract: risks outweigh benefits for intensive care unit patients.

Selective decontamination of the digestive tract (SDD) involves the administration of non-absorbable antibiotics (+/- a systemic antibiotic) to prevent colonization and infection in intensive care unit patients. The regimen is targeted at nosocomial gram-negative bacilli, some gram-positive bacteria and yeast. Although all studies of SDD have demonstrated reduced rates of bacterial colonization and most yielded lower respiratory tract infection, it is unclear if the major impact of SDD is on pneumonia or tracheobronchitis. SDD regimens utilizing a broad spectrum, systemic antibiotic appear to be more effective, suggesting that this constitutes early treatment rather than prophylaxis. To date, there is conflicting evidence that SDD significantly reduces length of stay, mortality, or hospital costs. Currently, there are concerns that SDD may result in increased colonization and infection with gram-positive organisms and multi-drug resistant pathogens, particularly in medical ICU patients or when used for extended periods of time.

Nutritional outcome and pneumonia in critical care patients randomized to gastric versus jejunal tube feedings. The Critical Care Research Team.

OBJECTIVE: To compare nutritional status, gastric colonization, and rates of nosocomial pneumonia in ICU patients randomized to gastric tube feeding vs. patients fed by an endoscopically placed jejunal tube. DESIGN: Randomized, prospective study. SETTING: Medical and surgical ICUs at Boston City Hospital; surgical ICU at University Hospital. PATIENTS: Of the 38 study patients, 19 were randomized to gastric tube feeding and 19 were randomized to an endoscopically placed jejunal tube. The two groups were similar in age, sex, race, underlying disease, and type of surgery. RESULTS: The two patient groups were similar in number of days fed, duration of ICU stay, duration of mechanical ventilation, days of antibiotic therapy, and days with fever. Compared with the gastric group, the jejunal group had more patients with circulatory shock on admission (79% vs. 68.4%), higher admission Acute Physiology Score (24.0 vs. 21.7), and fewer patients with pneumonia at randomization (26.3% vs. 31.6%). The jejunal group received a significantly higher percentage of their daily goal caloric intake (p = .05), and had greater increases in serum prealbumin concentrations (p < .05) than the patients with gastric tube feeding. Although the jejunal tube group had more days of diarrhea (3.3 +/- 6.6 vs. 1.8 +/- 2.9), this difference was not statistically significant. Nosocomial pneumonia was diagnosed clinically in two (10.5%) patients in the gastric tube group and in no patients in the jejunal tube group. CONCLUSIONS: Patients fed by jejunal tube received a significantly higher proportion of their daily goal caloric intake, had a significantly greater increase in serum prealbumin concentrations, and had a lower rate of pneumonia than patients fed by continuous gastric tube feeding.

Seroprevalence of human immunodeficiency virus in parturients at Boston City Hospital: implications for public health and obstetric practice.

OBJECTIVE: We measured the seroprevalence of human immunodeficiency virus in women seeking reproductive services. STUDY DESIGN: Demographic and risk behavior data from women were linked anonymously to human immunodeficiency virus antibody results. RESULTS: The overall human immunodeficiency virus seropositivity rate of cord blood was 22 per 1000. Crude seroprevalence rates were higher for black women versus white women (25/1000 vs 22/1000) but lower for black Americans versus white Americans (21/1000 vs 29/1000). Human immunodeficiency virus infection was significantly higher for those women who acknowledge intravenous drug use (odds ratio 12.9, 95% confidence interval 7.3 to 22.7), were born in Haiti (odds ratio 2.6, 95% confidence interval 1.6 to 4.1), lacked prenatal care (odds ratio 2.2, 95% confidence interval 1.1 to 4.2), or received prenatal care at the hospital clinic versus a neighborhood health center (odds ratio 3.0, 95% confidence interval 1.7 to 5.3). The seroprevalence rates were 18/1000 for women seeking abortion and 16/1000 for women seeking family-planning services. CONCLUSION: Intravenous drug use and country of origin are major risk factors for human immunodeficiency virus infection in women, which may explain differences in seroprevalence rates in various racial or ethnic groups. Hospital-specific data on human immunodeficiency virus infection may be useful for monitoring the epidemic and allocating resources for education, counseling, testing, and prevention.