Dimorphic HLA-B signal peptides differentially influence HLA-E- and natural killer cell-mediated cytolysis of HIV-1-infected target cells.

As a mechanism of self-protection, signal peptides cleaved from human leukocyte antigen (HLA) class I products bind to HLA-E before the complex interacts with the natural killer (NK) cell receptor CD94/NKG2A to inhibit NK-mediated cell lysis. Two types of the signal peptides differ in their position 2 (P2) anchor residue, with P2-methionine (P2-M) having higher HLA-E binding affinity than P2-threonine (P2-T). All HLA-A and HLA-C molecules carry P2-M, whereas HLA-B products have either P2-M or P2-T. Epidemiological evidence suggests that P2-M is unfavourable in the context of HIV-1 infection, being associated with accelerated acquisition of HIV-1 infection in two African cohorts. To begin elucidating the functional mechanism, we studied NK-mediated killing of CD4(+) T cells and monocyte-derived macrophages infected with two laboratory-adapted HIV-1 strains and two transmitted/founder (T/F) viruses. In the presence of target cells derived from individuals with the three HLA-B P2 genotypes (M/M, M/T and T/T), NK-mediated cytolysis was elevated consistently for P2-T in a dose-dependent manner for all cell and virus combinations tested (P = 0·008-0·03). Treatment of target cells with an anti-HLA-E monoclonal antibody restored NK-mediated cytolysis of cells expressing P2-M. Observations on cell lysis were also substantiated by measurements of HIV-1 p24 antigen in the culture supernatants. Overall, our experiments indicate that the anti-HIV-1 function mediated by NK cells is compromised by P2-M, corroborating the association of HLA-B genotype encoding P2-M with accelerated HIV-1 acquisition.

The foamy virus envelope glycoproteins.

The main functions of retroviral glycoproteins are recognition and binding to the cellular virus receptor as well as fusion of viral and cellular lipid membranes to release the viral particle into the cytoplasm of the host cell. Foamy viruses (FVs) are a special group of retroviruses with a very broad host range that use a currently unknown cellular receptor for entry. Nevertheless, many functions of the FV envelope glycoproteins in the viral replication cycle have been characterized in detail over the last years. Several unique features not found for any other retrovirus were identified. These include the presence of two types of FV Env proteins, gp170(Env-Bet) and gp130Env, and the strict requirement of gp130Env coexpression for the FV budding and particle release process, a function that cannot be compensated for by any other viral glycoprotein tested so far. Furthermore, domains in gp130Env could be characterized that influence its intracellular distribution, cell surface transport, and its specific interaction with the viral capsid during particle egress. In addition, it has recently been shown that gp130Env expression alone induces release of subviral particles from cells. This review summarizes the current knowledge about the nature of the FV Env proteins and their function in the viral replication cycle.

CD8 CTL responses in vaccines: emerging patterns of HLA restriction and epitope recognition.

We evaluated MHC-class I-restricted CTL responses induced by HIV-1 clade B-based vaccines in nine HIV-1 seronegative vaccine recipients with regard to their patterns of HLA restriction and epitope recognition. We found that seven of nine volunteers developed detectable CTL reactivities against novel epitopes within the HIV-1 Env and Gag proteins. Although four of nine subjects were HLA-A*0201, none of the cellular responses was restricted in the context of this allele. The type of responses observed in this sampling of vaccines appeared similar to those reported during primary infection and among long term non-progressors, with three out of nine subjects recognizing HLA-B27 or HLA-B17(57)-restricted epitopes. Although the majority of CTL responses were directed against novel epitopes, these effectors were still able to mediate cross-clade reactivities.

Polymorphisms in HLA class I genes associated with both favorable prognosis of human immunodeficiency virus (HIV) type 1 infection and positive cytotoxic T-lymphocyte responses to ALVAC-HIV recombinant canarypox vaccines.

Carriers of certain human leukocyte antigen class I alleles show favorable prognosis of human immunodeficiency virus type 1 (HIV-1) infection, presumably due to effective CD8(+) cytotoxic T-lymphocyte responses, but close relationships between class I variants mediating such responses to natural and to vaccine HIV-1 antigen have not been established. During 6 to 30 months of administration and follow-up in trials of ALVAC-HIV recombinant canarypox vaccines, cells from 42% of 291 HIV-1-negative vaccinated subjects typed at class I loci responded to an HIV-1 protein in a lytic bulk CD8(+) cytotoxic T-lymphocyte assay. By 2 weeks after the second dose, higher proportions of vaccinees carrying one of two alleles consistently associated with slower progression of natural HIV-1 infection reacted at least once: B*27 carriers reacted to Gag (64%; odds ratio [OR] = 10.3, P = 0.001) and Env (36%; OR = 4.6, P = 0.04), and B*57 carriers reacted to Env (44%; OR = 6.6, P < 0.05). By 2 weeks after the third or fourth dose, B*27 carriers had responded (two or more reactions) to Gag (33%; OR = 4.4, P < 0.05) and B*57 carriers had responded to both Gag (39%; OR = 5.3, P = 0.013) and Env (39%; OR = 9.5, P = 0.002). Homozygosity at class I loci, although conferring an unfavorable prognosis following natural infection, showed no such disadvantage for vaccine response. Individual class I alleles have not previously demonstrated such clear and consistent relationship with both the clinical course of an infection and cellular immunity to a vaccine against the infectious agent. This proof of principle that class I an alleles modulate both processes has implications for development of HIV-1 and presumably other vaccines.

[Prognosis and risk evaluation of postoperative coronary patients (PERISCOP). Methodology and study population characteristics]. / Pronostic et évaluation du risque chez le coronarien opéré (PERISCOP). Méthodologie et caractéristiques de la population incluse.

The aim of the PERISCOP study was to evaluate the predictive value of cardiological investigations performed after recent coronary bypass surgery with regards to cardiac event and mortality at one year. The treatment of lipid abnormalities was also analysed. This first article describes the methodology and patient characteristics at inclusion. This prospective national multicenter trial included 2065 patients (86% men) with an average age of 63.1 +/- 9.9 years. The number of diseased vessels was 2.6 +/- 0.6. Preoperative left ventricular function was normal (ejection fraction 60 +/- 13%). Revascularisation was complete in 73% of cases (22% of arterial grafts). The cardiological investigations were performed at Day 20 +/- 10 after surgery. The duration of exercise on stress testing was 429 +/- 170 seconds. It was positive or doubtful in 9% of cases. Ventricular arrhythmias were observed in 6.5% of cases. The blood pressure response was abnormal in 6% of cases. Holter monitoring showed a median number of ventricular extrasystoles over 24 hours of 44. Three per cent of patients had one episode of ventricular tachycardia and 7% had ischaemic episodes. The echocardiographic index of segmental contractility was on average 1.75 (ejection fraction: 52.6%). The lipid analysis performed at one month, under lipid therapy in 34% of cases, showed a total cholesterol level at 1.91 +/- 0.10 g/l, an LDL-cholesterol of 1.27 +/- 0.08 g/l. The therapeutic target (LDL-cholesterol < 1 g/l) was attained in 46% of cases with treatment and in 18% of cases without treatment.

QS-21 promotes an adjuvant effect allowing for reduced antigen dose during HIV-1 envelope subunit immunization in humans.

Three separate studies were undertaken in HIV-1 uninfected persons to determine if the adjuvant QS-21 improves the magnitude or kinetics of immune responses induced by recombinant soluble gp120 HIV-1(MN) protein (rsgp120) immunization. The QS-21 was administered at two doses (50 and 100 microg), either alone or in combination with aluminum hydroxide (600 microg). At the highest doses of rsgp120 (100, 300, and 600 microg), QS-21 exerted no significant effect on either binding or neutralizing antibody titers. Antibody binding and neutralizing responses fell dramatically when rsgp120, formulated with alum alone, was given at low doses (3 and 30 microg). In contrast, antibody responses similar in titer to those in the high dose antigen groups were induced with the low dose rsgp120 formulated with QS-21. In addition, the lymphocyte proliferation and delayed type hypersensitivity skin testing were superior in the QS-21 recipients compared with the alum recipients at the low antigen doses. Moderate to severe pain was observed in majority of the volunteers receiving QS-21 formulations, and vasovagal episodes and hypertension were not infrequent. Thus, the use of QS-21 may provide a means to reduce the dose of a soluble protein immunogen.

A significant number of human immunodeficiency virus epitope-specific cytotoxic T lymphocytes detected by tetramer binding do not produce gamma interferon.

Despite the seemingly important role of cytotoxic T-lymphocyte (CTL) responses in human immunodeficiency virus (HIV) disease pathogenesis, their measurement has relied on a variety of different techniques. We utilized three separate methodologies for the detection of CTLs in a cohort of HIV-infected individuals who were also human leukocyte antigen A2 (HLA-A2) positive. Among the different CTL assays, a correlation was seen only when the Gag epitope-specific HLA A*0201-restricted tetramer assay was compared with the ELISPOT assay performed after stimulation with the Gag epitope; however, this correlation was of borderline statistical significance. On average, the tetramer reagent detected a 10-fold-higher number of cells than were seen to produce gamma interferon by the ELISPOT assay. The implications of this CTL assay comparison and the possibility of phenotypic differences in HIV-specific CD8(+) T lymphocytes are discussed.

Recurrence of the acute HIV syndrome after interruption of antiretroviral therapy in a patient with chronic HIV infection: A case report.

BACKGROUND: Clinical and virologic consequences of temporary interruption of HIV therapy are incompletely understood. OBJECTIVE: To describe a febrile illness that was consistent with the acute HIV syndrome and occurred after interruption of antiretroviral therapy. DESIGN: Case report. SETTING: University clinic. PATIENT: HIV-infected man. MEASUREMENTS: Plasma viral load, lymphocyte subsets, diagnostic evaluation (including cultures and serologic tests), and analysis of lymph node tissue. RESULTS: The patient began antiretroviral therapy 3 months after initial HIV exposure and had sustained viral suppression, except during a brief scheduled treatment interruption. One hundred sixty-nine days after resuming therapy, the patient discontinued it again immediately following an influenza vaccination. Eleven days later, he presented with a febrile mononucleosis-like syndrome associated with dramatic shifts in plasma HIV RNA level (<50 to >1 000 000 copies/mL) and CD4 cell count (0.743 x 10(9) cells/L to 0.086 x 10(9) cells/L). Evaluation for alternative causes of fever was unrevealing. Symptoms resolved rapidly with resumption of HIV therapy. CONCLUSION: Therapeutic interruption may be associated with profound viral rebound and recurrence of the acute HIV syndrome.

Characterization of the R572T point mutant of a putative cleavage site in human foamy virus Env.

A putative cleavage site of the human foamy virus (HFV) envelope glycoprotein (Env) was altered. Transient env expression revealed that the R572T mutant Env was normally expressed and modified by asparagine-linked oligosaccharide chains. However, this single-amino-acid substitution was sufficient to abolish all detectable cleavage of the gp130 precursor polyprotein. Cell surface biotinylation demonstrated that the uncleaved mutant gp130 was transported to the plasma membrane. The uncleaved mutant protein was incapable of syncytium formation. Glycoprotein-driven virion budding, a unique aspect of HFV assembly, occurred despite the absence of Env cleavage. We then substituted the R572T mutant env into a replication-competent HFV molecular clone. Transfection of the mutant viral DNA into BHK-21 cells followed by viral titration with the FAB (foamy virus-activated beta-galactosidase expression) assay revealed that proteolysis of the HFV Env was essential for viral infectivity. Wild-type HFV Env partially complemented the defective virus phenotype. Taken together, these experimental results established the location of the HFV Env proteolytic site; the effects of cleavage on Env transport, processing, and function; and the importance of Env proteolysis for virus maturation and infectivity.

An endoplasmic reticulum retrieval signal partitions human foamy virus maturation to intracytoplasmic membranes.

Among all retroviruses, foamy viruses (FVs) are unique in that they regularly mature at intracytoplasmic membranes. The envelope glycoprotein of FV encodes an endoplasmic reticulum (ER) retrieval signal, the dilysine motif (KKXX), that functions to localize the human FV (HFV) glycoprotein to the ER. This study analyzed the function of the dilysine motif in the context of infectious molecular clones of HFV that encoded mutations in the dilysine motif. Electron microscopy (EM) demonstrated virion budding both intracytoplasmically and at the plasma membrane for the wild-type and mutant viruses. Additionally, mutant viruses retained their infectivity, but viruses lacking the dilysine signal budded at the plasma membrane to a greater extent than did wild-type viruses. Interestingly, this relative increase in budding across the plasma membrane did not increase the overall release of viral particles into cell culture media as measured by protein levels in viral pellets or infectious virus titers. We conclude that the dilysine motif of HFV imposes a partial restriction on the site of viral maturation but is not necessary for viral infectivity.

A sorting motif localizes the foamy virus glycoprotein to the endoplasmic reticulum.

We recently identified an endoplasmic reticulum (ER) retrieval signal-the dilysine motif-in the glycoproteins of all five foamy viruses (FVs) for which sequences were available (P. A. Goepfert, G. Wang, and M. J. Mulligan, Cell 82:543-544, 1995). In the present study, expression of recombinant human FV (HFV) glycoprotein and analyses of oligosaccharide modifications and precursor cleavage indicated that the protein was localized to the ER. HFV glycoproteins encoding seven different dilysine motif mutations were then expressed. The results indicated that disruptions of the dilysine motif resulted in higher levels of forward transport of the HFV glycoprotein from the ER through the Golgi apparatus to the plasma membrane. We conclude that the dilysine motif is responsible for ER sorting of the FV glycoprotein. Signal-mediated ER localization has not previously been described for a retroviral glycoprotein.

Analysis of west African hunters for foamy virus infections.

Foamy viruses are a genus of complex retroviruses that infect a wide variety of mammals. However, a clear association with any disease process has yet to be proven for these viruses. A higher human seroprevalence was reported in African populations, perhaps due to exposure to simian foamy viruses (SFV) endemic in primates. However, the earlier serologic surveys were not confirmed by studies employing nucleic acid amplification. Foamy virus infections of humans clearly do occur as rare zoonoses among primate center or laboratory workers exposed to captive primates or their blood. We sought to detect foamy virus infections in a cohort of humans also presumed to be exposed to SFV, i.e., West African hunters. We constructed recombinant vaccinia viruses that expressed human foamy virus (HFV) Gag or Env polyproteins in mammalian cells. The sera from 17 monkey hunters or several controls were tested in radioimmunoprecipitation assays (RIPAs) against the recombinant HFV proteins. Chimpanzee sera or HFV-positive human sera immunoprecipitated gp130, the HFV Env precursor, as well as p74, the HFV Gag polyprotein. None of the hunters' sera recognized both of these recombinant proteins. We then employed a nested polymerase chain reaction (PCR) analysis of the hunters' DNA but also failed to detect foamy virus infections. Therefore, by utilizing a recombinant RIPA and a nested PCR assay, we have not identified foamy virus infections occurring naturally in hunters exposed to wild monkeys in West Africa.

An outbreak of Burkholderia (formerly Pseudomonas) cepacia respiratory tract colonization and infection associated with nebulized albuterol therapy.

OBJECTIVE: To investigate an outbreak of Burkholderia (formerly Pseudomonas) cepacia respiratory tract colonization and infection in mechanically ventilated patients. DESIGN: A retrospective case-control and bacteriologic study. SETTING: Veterans Affairs medical center. PATIENTS: 42 mechanically ventilated patients who developed respiratory tract colonization or infection with B. cepacia and 135 ventilator-dependent controls who were not colonized and did not develop infections. MEASUREMENTS: Clinical and demographic data; benzalkonium chloride concentrations and pH levels in albuterol sulfate solutions; repetitive-element polymerase chain reaction (PCR)-mediated molecular fingerprinting on eight patient isolates and three environmental B. cepacia isolates that were available for study. RESULTS: 42 patients had B. cepacia respiratory tract colonization or infection. Observation of intensive care unit and respiratory care personnel showed faulty infection control procedures (for example, the same multiple-dose bottle of albuterol was used for many mechanically ventilated patients). More case patients (39 [92.9%]) than controls (95 [70.4%]; P = 0.006) received nebulized albuterol, and case patients (67.5 treatments) received more treatments than controls (18 treatments; P < 0.001). In-use albuterol solutions had pH values that were unstable, and benzalkonium chloride concentrations declined over time to levels capable of supporting bacterial growth. Medication nebulizers and in-use bottles of albuterol harbored B. cepacia. Molecular fingerprints of patient isolates and environmental B. cepacia isolates were identical using repetitive-element PCR. No further isolates of B. cepacia were identified after institution of appropriate infection control procedures. CONCLUSIONS: Multiple-dose medications and reliance on benzalkonium chloride as a medication preservative provide a mechanism for nosocomial spread of microorganisms, particularly if infection control procedures are not carefully followed. Repetitive-element PCR is a useful fingerprinting technique for molecular epidemiologic studies of B. cepacia.

[Rehabilitation centers: for whom? Why and how long?]. / Le centre de réadaptation: pour qui? pourquoi? combien de temps?

The clinical profile of coronary patients admitted to cardiac rehabilitation centres after myocardial infarction has changed considerably in the last 15 years. Complementary investigations (coronary angiography, studies of left ventricular function) provide accurate information which improves the process of rehabilitation. Global management of the coronary patient requires and justifies, especially in young adults, taking into consideration the physical, psychological and socio-professional consequences of a myocardial infarction. The indications of rehabilitation are much more comprehensive nowadays. Even patients with significant haemodynamic impairment can benefit from a stay in a specialised centre. Contraindications are usually only temporary. Finally, an enquiry performed in 33 French cardiac rehabilitation centres shows large variations in methods, personnel and organisation. However, as a general rule, a 3 week stay seems to be adequate but it is logical to continue rehabilitation when the patients goes home to pursue and maintain at long term the results obtained on discharge from a specialised centre.

Exercise training, cardiac rehabilitation and return to work in patients with coronary artery disease.

Few studies have analysed the influence of exercise training or cardiac rehabilitation on return to work in patients with coronary artery disease. In a consecutive series of 41 men less than 60 years of age admitted for acute myocardial infarction, 19 participated in a comprehensive rehabilitation programme focused on physical training, and 22 received standard care. The baseline clinical and social variables of the two groups were comparable except for left ventricular ejection fraction which was significantly lower in the intervention group. Work resumption was not significantly different between the two groups (9/19, 47% in the intervention group vs 17/22, 77% in the control group); after three years, 42% and 73%, respectively, were still employed. These results are in keeping with that of most controlled studies of cardiac rehabilitation after myocardial infarction or coronary artery surgery, which fail to demonstrate a significant improvement in work resumption. Psychosocial factors are probably more important determinants of work resumption than is exercise training.

Studies on the citryl-CoA-dependent inhibition of citrate-synthase with source variants from baker's yeast, Escherichia coli and Sulfolobus solfataricus.

1) Citrate synthase from pig heart has previously been shown to display complex kinetic characteristics in the reactions with citryl-CoA, resulting in inhibition. The synthase from another eukaryotic source, baker's yeast, yields the same complex kinetics. 2) Synthases from a Gram-negative prokaryote, E. coli, and from an archaebacterium, S. solfataricus, catalyse the reactions of citryl-CoA in kinetics of the Michaelis-Menten type. A comparison of the rates of citryl-CoA hydrolysis (V') and physiological reaction (V), determined with these enzymes, corresponds to ratios of V'/V approximately 1 and approximately 2, respectively. Thus, and for the first time, there is no reason left to doubt the intermediate formation of citryl-CoA in the physiological reaction. 3) The complex kinetics indicated under 1) are related to efficient formation of citrate from citryl-CoA-derived acetyl-CoA and oxaloacetate in the presence of NADH and malate dehydrogenase. These conditions are not met by the enzymes from E. coli, S. solfataricus and by proteolytically nicked synthase species from pig heart. All these enzyme variants have low affinities to either one or both of the physiological substrates. Consistent with earlier ideas, the results indicate that the inhibition mechanism is related to high affinities of the enzyme for both acetyl-CoA and oxaloacetate.

Purification and properties of an archaebacterial enzyme: citrate synthase from Sulfolobus solfataricus.

Citrate synthase from the thermoacidophilic archaebacterium Sulfolobus solfataricus was purified to homogeneity. The synthase is a dimer composed of subunits of Mr approximately equal to 40,000. The Km values of acetyl-CoA and oxalacetate are 7 microM and 20 microM, respectively. NADH (Ki = 3.5mM) and ATP (Ki = 0.36mM) are competitive inhibitors vs acetyl-CoA. The dimeric structure and the inhibition by nucleotides (ATP greater than NADH) correlate the archaebacterial enzyme to synthases from eukaryotes and Gram-positive eubacteria.

Hysteretic behaviour of citrate synthase. Isotopically chiral citryl-CoA reveals increased number of reversals of the synthase condensation step under steady-state conditions.

Citrate specimens derived from chiral acetates were converted to the CoA derivatives. These were reconverted with citrate synthase to citrates under conditions of either predominating hydrolytic burst or predominating steady-state period. The stereochemical purity of substrates and products was determined. Reversal of the synthase condensation step occurs under both conditions but is markedly increased during the steady-state period. The results indicate that citryl-CoA-derived acetyl-CoA and oxaloacetate create the steady-state conditions. The hydrolase state and the ligase/lyase state of the synthase predominate under burst and steady-state conditions, respectively. This result indicates a conversion of the hydrolase state into the ligase/lyase state during the transition.

[Importance of the exercise test in the follow-up of surgically treated congenital aortic stenoses]. / Intérêt de l'épreuve d'effort dans la surveillance des sténoses aortiques congénitales opérées.

Sixty four children with isolated congenital aortic stenosis (39 valvular, 16 fixed subvalvular, 4 supravalvular and 5 multiple) were operated at a mean age of 11,5 years. Valve repair was possible in all but three patients who had to undergo valvular replacement. Myotomy was associated in 18 cases (28 p. 100). The mean systolic pressure gradient was 79,9 mmHg (+/- 17,8); there was associated aortic regurgitation in 21 patients but this was minimal except in one case. Twenty children (31 p. 100) had symptoms on effort and the basal ECG showed ST-T wave changes in the left precordial leads in 30 cases (47 p. 100). Several preoperative exercise ECGs were performed in 29 patients without ST-T changes on the resting ECG. The exercise ECG was positive in 15 patients, providing one of the arguments for surgery; a poor blood pressure response to exercise was observed in 12 patients with a negative test. Out of the 28 patients with a positive preoperative exercise ECG, 7 (25 p. 100) went on having a positive result after surgery (p less than 0,05). The maximal heart rate was not significantly higher after surgery but the total work was significantly greater (p less than 0,01) and the increase in systolic blood pressure was even more significant (p less than 0,001). Out of 14 patients undergoing repeat catheterisation for a continuing positive exercise ECG or for ST-T wave changes on the resting ECG, there were 6 residual severe stenoses, 3 severe aortic regurgitations, 3 hypertrophic cardiomyopathies which were obstructive in 2 cases. The exercise ECG is a means of appreciating the consequences of the stenosis which are the cause of the complications (myocardial ischemia and poor blood pressure adaptation). This justifies its use in assessing the surgical indications and for the follow-up of the surgical result. A persistantly positive exercise ECG and continuing ST-T wave changes on the resting ECG are signs of a poor surgical result and hemodynamic revaluation should be considered; besides severe postoperative aortic regurgitation, residual or recurrent stenosis and, above all, asymmetric septal hypertrophy, obstructive or not, are the main causes of poor postoperative results.