Potentially exposed but uninfected individuals produce cytotoxic and polyfunctional human immunodeficiency virus type 1-specific CD8(+) T-cell responses which can be defined to the epitope level.

We measured CD8(+) T-cell responses in 12 potentially exposed but uninfected men who have sex with men by using cytokine flow cytometry. Four of the individuals screened exhibited polyfunctional immune responses to human immunodeficiency virus type 1 Gag or Vif. The minimum cytotoxic T lymphocyte epitope was mapped in one Gag responder.

The outcome of hepatitis C virus infection is predicted by escape mutations in epitopes targeted by cytotoxic T lymphocytes.

CD8(+) cytotoxic T lymphocytes (CTL) are thought to control hepatitis C virus (HCV) replication and so we investigated why this response fails in persistently infected individuals. The HCV quasispecies in three persistently infected chimpanzees acquired mutations in multiple epitopes that impaired class I MHC binding and/or CTL recognition. Most escape mutations appeared during acute infection and remained fixed in the quasispecies for years without further diversification. A statistically significant increase in the amino acid replacement rate was observed in epitopes versus adjacent regions of HCV proteins. In contrast, most epitopes were intact when hepatitis C resolved spontaneously. We conclude that CTL exert positive selection pressure against the HCV quasispecies and the outcome of infection is predicted by mutations in class I MHC restricted epitopes.

Identification of five different Patr class I molecules that bind HLA supertype peptides and definition of their peptide binding motifs.

We have sequenced the Pan troglodytes class I (Patr) molecules from three common chimpanzees and expressed them as single molecules in a class I-deficient cell line. These lines were utilized to obtain purified class I molecules to define the peptide binding motifs associated with five different Patr molecules. Based on these experiments, as well as analysis of the predicted structure of the B and F polymorphic MHC pockets, we classified five Patr molecules (Patr-A*0101, Patr-B*0901, Patr-B*0701, Patr-A*0602, and Patr-B*1301) within previously defined supertype specificities associated with HLA class I molecules (HLA-A3, -B7, -A1, and -A24 supertypes). The overlap in the binding repertoire between specific HLA and Patr class I molecules was in the range of 33 to 92%, depending on the particular Patr molecule as assessed by the binding of HIV-, hepatitis B virus-, and hepatitis C virus-derived epitopes. Finally, live cell binding assays of nine chimpanzee-derived B cell lines demonstrated that HLA supertype peptides bound to Patr class I molecules with frequencies in the 20-50% range.

Development and validation of the mini-osteoporosis quality of life questionnaire (OQLQ) in osteoporotic women with back pain due to vertebral fractures. Osteoporosis Quality of Life Study Group.

The objective of the study was to evaluate a shortened osteoporosis quality of life questionnaire (OQLQ) in osteoporotic women with back pain due to vertebral fractures. From the longer 30-item OQLQ (four to nine items per domain) we created the mini-OQLQ by choosing the two items with the highest impact in each of five domains (symptoms, physical function, activities of daily living, emotional function, leisure). We administered the OQLQ, the Sickness Impact Profile, the SF-36 and the Brief Pain Index to patients at baseline, after 2 weeks and after 6 months. The intraclass correlations between baseline and the 2-week follow-up for the five mini-OQLQ domains ranged from 0.72 to 0.86. Cross-sectional correlations between the domains of the mini-OQLQ and other health instruments were moderate to large (0.35-0.80) and greater than predicted. The mini-OQLQ items showed moderate to large correlations with items omitted from the shortened questionnaire (0. 44-0.88). Correlations between the OQLQ domains and the other three instruments were greater than those of the mini-OQLQ, and partial correlations between OQLQ items omitted from the mini-OQLQ and the other three instruments after considering mini-OQLQ items were substantial (0.19-0.71) and statistically significant. Sample sizes of less than 200 per group should be required to detect minimally important differences in parallel-group clinical trials. Longitudinal correlations between the mini-OQLQ and the other measures were often significant but generally lower than predicted (0.10-0.49). The partial correlations revealed that the omitted items explained a significant portion of the longitudinal variance in each domain. We conclude that in a selected group of patients with back pain caused by vertebral fractures, the mini-OQLQ demonstrated good discriminative and adequate evaluative properties. The mini-questionnaire should be useful in clinical settings.

The simian immunodeficiency virus envelope glycoprotein contains two epitopes presented by the Mamu-A*01 class I molecule.

Cytotoxic T lymphocyte (CTL) responses against the simian immunodeficiency virus (SIV) envelope and Gag proteins were monitored in a Mamu-A*01-positive rhesus macaque infected with SIVsmE660. Peripheral blood mononuclear cells (PBMC) cultured with synthetic peptides spanning the entire gp160 and Gag coding region recognized a total of three epitopes. One located in Gag was identified as the previously described Mamu-A*01-restricted p11cC-->M epitope (CTPYDINQM). The other two epitopes, designated p15m and p54m, were located in the gp160 envelope protein. Both were nine amino acids in length and were predicted to bind Mamu-A*01 because they contained proline and leucine residues at positions 3 and 9, respectively. Indeed, expression of this class I major histocompatibility complex molecule was required for target cell recognition by envelope-specific CD8(+) T cells directed against both epitopes. These Mamu-A*01-restricted epitopes in the SIV envelope will be useful for monitoring immune responses in vaccinated or infected animals.

Analysis of a successful immune response against hepatitis C virus.

To investigate the type of immunity responsible for resolution of hepatitis C virus (HCV) infection, we monitored antibody and intrahepatic cytotoxic T lymphocyte (CTL) responses during acute (<20 weeks) infection in chimpanzees. Two animals who terminated infection made strong CTL but poor antibody responses. In both resolvers, CTL targeted at least six viral regions. In contrast, animals developing chronic hepatitis generated weaker acute CTL responses. Extensive analysis of the fine specificity of the CTL in one resolver revealed nine peptide epitopes and restriction by all six MHC class I allotypes. Every specificity shown during acute hepatitis persisted in normal liver tissue more than 1 yr after resolution. These results suggest that CD8+CTL are better correlated with protection against HCV infection than antibodies.

Patr-A and B, the orthologues of HLA-A and B, present hepatitis C virus epitopes to CD8+ cytotoxic T cells from two chronically infected chimpanzees.

Common chimpanzees (Pan troglodytes) infected with hepatitis C virus (HCV) show a disease progression similar to that observed for human patients. Although most infected animals develop a chronic hepatitis, virus persistence is associated with an ongoing immune response, for which the beneficial or detrimental effects are uncertain. Lines of virus-specific cytotoxic CD8+ T lymphocytes (CTL) have been previously established from liver biopsies of two common chimpanzees chronically infected with HCV-1. The viral epitopes recognized by six lines of CTL have been defined using synthetic peptides and shown to consist of 8 to 9-residue peptides derived from various viral proteins. Five of the epitopes derive from sequences that vary among strains of HCV. The majority of the corresponding variant epitopes from different HCV strains were either recognized less efficiently or not at all by the CTL, suggesting their response may have limited potential for controlling replication of HCV variants. Complementary DNAs encoding class I alleles of the two common chimpanzees, Patr-A, -B, and -C were cloned, sequenced, and transfected individually into a class I-deficient human cell line. Analysis of peptide presentation by the class I transfectants to CTL identified the Patr class I allotypes that present the six epitopes defined here and an additional epitope defined previously. The assignment of epitopes to class I allotypes based upon analysis of the transfected cells correlates precisely with the segregation of antigen-presenting function within a panel of common chimpanzee cell lines and the expression of class I heavy chains as defined by isoelectric focusing. Five of the HCV-1 epitopes are presented by Patr-B allotypes, two epitopes are presented by a Patr-A allotype, and none is presented by Patr-C allotypes.

The presentation of a hepatitis C viral peptide by distinct major histocompatibility complex class I allotypes from two chimpanzee species.

A cytotoxic T lymphocyte (CTL) line, derived from the liver of a common chimpanzee (Pan troglodytes) with hepatitis C, specifically recognized a hepatitis C viral 9-mer peptide (KHP-DATYSR in single-letter amino acid code) bound by the major histocompatibility complex (MHC) class I molecule, Patr-A04. This same CTL line also recognized the identical peptide bound by a structurally different class I molecule, Papa-A06, derived from the separate chimpanzee species, Pan paniscus or pygmy chimpanzee. These class I allotypes differ by six amino acids but, in spite of the structural differences, share the same antigen-presenting function. This is the first observation of antigen presentation to a given T cell receptor by different MHC class I allotypes from separate species.

Delivery of purified, functional CFTR to epithelial cells in vitro using influenza hemagglutinin.

To assess the feasibility of protein replacement as a potential therapy for cystic fibrosis, we have evaluated the ability of influenza hemagglutinin (HA) to mediate the delivery of purified cystic fibrosis transmembrane conductance regulator (CFTR) to recipient cells in vitro. CFTR was purified from both CHO cells and Sf9 cells and reconstituted into two different types of vesicular delivery vehicles. In one, CFTR and HA were co-reconstituted into the same lipid vesicle. After binding to the cell surface, delivery of CFTR to the recipient cell was achieved by a transient, low-pH activation of the fusion activity of HA. A second delivery strategy used HA virosomes together with purified CFTR that had been reconstituted into vesicles containing gangliosides, a receptor for HA. After binding of the HA virosomes and CFTR-containing vesicles to the recipient cells, delivery to the plasma membrane again was achieved by a transient pH drop. Delivery of functional CFTR was assessed using the SPQ fluorescence assay. Functional CFTR was detected in a fraction (> 20%) of the recipient cells using this assay. Quantitative binding and fusion assays using radiolabeled virosomes and lipid vesicles showed that on the order of 1,000 of the added CFTR-containing vesicles bound to each C127 cell under the conditions of our delivery protocols. However, only a fraction of these vesicles fused and delivered CFTR to the cell plasma membrane. The two delivery strategies were found to be approximately equivalent in their ability to deliver active CFTR, and there were no significant differences between deliveries using purified CFTR from either cell source. These feasibility studies suggest that purified CFTR can be delivered to a recipient cell in a functional form and therefore represent a significant step in establishing the concept of protein replacement as a therapy for cystic fibrosis.

Persistent hepatitis C virus infection in a chimpanzee is associated with emergence of a cytotoxic T lymphocyte escape variant.

Hepatitis C virus (HCV) establishes a persistent infection in humans and chimpanzees despite the presence of virus-specific, class I major histocompatibility complex-restricted CD8+ cytotoxic T lymphocytes (CTLs) in the liver. The data presented here demonstrate that CTLs directed against a conserved epitope in the HCV nonstructural 3 protein persist in the liver of a chronically infected chimpanzee for at least 2 years after infection. However, these CTLs did not recognize the HCV quasi-species present in the plasma of this animal at week 16 postinfection or at later time points. Escape from the CTL response was facilitated by an aspartic acid to glutamic acid (D-->E) substitution at amino acid position 1449 in all HCV genomes that were sequenced. The results of this study strongly support the concept that CTL responses can select for variant viruses with an enhanced ability to persist in a host and have important implications for the design of vaccines against HCV.

Association of cytotoxic T lymphocyte (CTL) escape mutations with persistent hepatitis C virus (HCV) infection.

Mechanisms by which HCV evades the cellular immune response in persistently infected humans and chimpanzees are poorly defined, but could involve mutations in epitopes recognized by class I MHC restricted CTLs. To investigate this possibility, we identified an epitope in the NS3 protein of HCV that was recognized by intrahepatic CTLs from a chimpanzee that developed persistent HCV infection after experimental challenge with the virus. Fine mapping studies with truncated synthetic peptides revealed that the epitope was 9 amino acids in length, encompassing residues 1445 to 1454 (GDFDSVIDC) of NS3. This sequence was completely conserved in all full-length NS3 genomes described to date. In view of the fact that the major genotypes of HCV may differ by up to -30% in overall amino acid homology, it appears in contrast that this epitope is highly conserved. The role of CTL escape mutations in HCV persistence was assessed in the virus inoculum used to infect this chimpanzee and in post-inoculation plasma samples. Sequencing of 6-10 M13 clones containing a 232-nucleotide fragment amplified with NS3-specific primers revealed that the epitope in the challenge inoculum and a post-inoculum plasma sample obtained at week 4 were identical to the published sequence of HCV-I. In contrast, all molecular clones sequenced from week 16, 25 and 28 plasma samples contained a single Asp--> Glu (D-->E) amino acid substitution at residue 1449. Significantly, four independently derived CTL clones established from the liver of this chimpanzee at various times up to two years after infection recognized target cells pulsed with a nonameric peptide representing the wild-type HCV-I sequence, but not those pulsed with a peptide containing the D-->E mutation. These data suggest that CTL escape mutations may play a role in viral persistence.

An epitope in the V1 domain of the simian immunodeficiency virus (SIV) gp120 protein is recognized by CD8+ cytotoxic T lymphocytes from an SIV-infected rhesus macaque.

Cytotoxic T-lymphocyte (CTL) responses against the external envelope glycoprotein (gp120) of the simian immunodeficiency virus (SIV) were studied in a rhesus macaque infected with SIVmac/239. CD8+ T cells enriched from concanavalin A-stimulated peripheral blood mononuclear cells lysed autologous target cells infected with recombinant vaccinia virus vectors expressing the SIVmac/239 or SIVsm/H4 envelope protein, which share approximately 80% identity in amino acid sequence. A CD8+ CTL line derived by limiting dilution culture of the concanavalin A-stimulated lymphocytes was also specific for the envelope proteins of both SIV isolates. Mapping studies revealed that this cell line recognized an epitope between amino acids 113 and 121 (CNKSETDRW) in the V1 domain of gp120. Amino acid substitutions are observed at positions 116 and 120 among viruses of the SIVsm/mac/human immunodeficiency virus type 2 group, and thus synthetic peptides representing these variants were tested for the ability to sensitize target cells for lysis by the CTL line. Autologous target cells sensitized with a synthetic peptide representing the SIVmac/239 sequence were efficiently killed. In contrast, recognition of target cells was reduced or abolished when peptides representing the amino acid substitutions at position 116 or 120 of other SIVmac, SIVsm, SIVmne, or SIVstm strains were tested. Further studies of CTL responses against this epitope could provide insights into mechanisms of variability within the gp120 V1 domain and its importance in evasion of immunity in infected or vaccinated monkeys.

Hepatitis C virus-specific CTL responses in the liver of chimpanzees with acute and chronic hepatitis C.

Hepatitis C virus (HCV)-specific CTL responses were evaluated in two chimpanzees (Pan troglodytes) during the acute and chronic phases of HCV infection. CD8+ T lymphocytes were isolated from liver tissue homogenates using anti-CD8 antibody-coated magnetic beads and then stimulated with anti-CD3 antibodies, IL-2, and irradiated human PBMC using limiting dilution culture conditions. HCV-specific cytotoxic activity of expanding CD8+ cell lines was assessed against autologous lymphoblastoid cell lines infected with recombinant vaccinia virus vectors encoding HCV Ag. CD8+ T cell lines specific for structural and nonstructural proteins of HCV were established from both animals. Cytolytic activity was blocked with anti-CD8 or anti-class I MHC antibodies, indicating that class I MHC molecules were involved in presentation of viral Ag to the CTL. Overlapping synthetic peptides were used to define a 12 amino acid segment of the nonstructural 3 (NS3) protein recognized by CTL lines from both chimpanzees. Studies with truncated peptides revealed that these CD8+ cell lines were directed against overlapping epitopes presented by distinct class I restriction elements of the chimpanzee MHC complex. CD8+ cell lines with identical specificities for an NS3 epitope were generated from one chronically infected animal at 16 and 28 wk postinfection. These results indicate that virus-specific CTL populations persist in the liver for months, but are unable to resolve chronic HCV infection.

Class I major histocompatibility complex-restricted cytotoxic T cell responses to vaccinia virus in humans.

Cytotoxic T lymphocyte (CTL) responses to vaccinia virus (VV) were studied in human subjects receiving smallpox vaccine by dermal scarification. After in vitro restimulation with VV, peripheral blood mononuclear cells (PBMCs) from three of six vaccinated subjects killed virus-infected target cells. VV-specific, HLA-restricted CTL activity was mediated primarily by CD8+ cells, although low levels of lytic activity by CD4+ cells were observed in some experiments. Two of the three responders had no history of exposure to VV prior to vaccination, whereas all three non-responders were vaccinated against smallpox during childhood. PBMCs from an additional three subjects who had received smallpox vaccine at least 20 years previously were negative for CTL activity. These data suggest that the duration of memory CTL populations against VV in the peripheral blood is limited, and that pre-existing immunity to VV may interfere with boosting of the CTL response upon revaccination.

Inhibition of human immunodeficiency virus replication in acutely infected CD4+ cells by CD8+ cells involves a noncytotoxic mechanism.

The mechanism by which CD8+ T cells from human immunodeficiency virus (HIV)-infected individuals suppress HIV replication in acutely infected CD4+ T cells was investigated. Cytotoxicity was not involved, as the antiviral activity of the CD8+ cells did not correlate with the ability to lyse HIV-infected or uninfected CD4+ T cells. In addition, the frequency of HIV-infected CD4+ cells increased during coculture with CD8+ T cells even in the absence of detectable levels of virus replication. Moreover, separation of the CD4+ and CD8+ cells by a 0.4-micron-pore-size filter delayed HIV replication, indicating a role, at least in part, for a soluble factor. However, cell contact was required for optimal antiviral activity. These results extend further the observation on the mechanism of antiviral HIV activity by CD8+ cells from infected individuals. They support the conclusion that CD8+ cells can play a major role in preventing development of disease in HIV-infected individuals.

CD8+ T cells from HIV-1-infected individuals inhibit acute infection by human and primate immunodeficiency viruses.

T lymphocytes expressing the CD8 surface antigen block HIV replication in CD4+ peripheral blood cells from HIV-infected individuals. We report here that CD4+ cells from HIV seronegative donors, when infected in vitro with HIV, also do not replicate virus when cocultured with CD8+ T cells from HIV-infected individuals. CD8+ cells from HIV-uninfected donors did not show this effect on virus replication. HLA-restriction of the antiviral response was not observed, and virus-containing cells were not eliminated from culture. The antiviral activity was broadly cross-reactive, as CD8+ cells from individuals infected only with HIV-1 suppressed the replication of diverse strains of HIV-1 and HIV-2, as well as the simian immunodeficiency virus. This ability of CD8+ cells to control HIV replication could play an important role in the maintenance of an asymptomatic state in HIV-infected individuals.

Effects of angiotensin II on plasma antidiuretic hormone and renal water excretion.

The effects of intravenous (i.v.) and intracarotid (IC) angiotensin II (AII) infusion on systemic and renal hemodynamics, renal water excretion, and plasma antidiuretic hormone (ADH) levels were examined in six conscious dogs under water loaded and hydropenic conditions. In the first group of seven studies, AII in a mean dose of 12.7 ng/kg/min was administered i.v. to water loaded dogs. The infusion induced a significant increase in mean arterial pressure (MAP, 99 to 118 mm Hg, P less than 0.001), and significant reductions in both glomerular filtration rate (GFR, 67 to 57 ml/min, P less than 0.05) and para-aminohippurate clearance (CPAH, 280 to 212 ml/min, P less than 0.005) occurred. Despite this decrement in renal hemodynamics, urine remained maximally dilute (Uosm, 58 to 61 mOsm/kg H2O, NS). Furthermore, plasma ADH was suppressed maximally after water load and did not increase after i.v. AII infusion. The IC infusion of AII (mean dose 5.8 ng/kg/min) produced similar changes in hemodynamics; plasma ADH remained undetectable. When AII was administered i.v. to hydropenic animals (mean dose 8.3 ng/kg/min), MAP again increased (86 to 111 mm Hg, P less than 0.001) as GFR (81.3 to 68.6 ml/min, NS) and CPAH (291 to 223 ml/min, P less than 0.05) declined modestly. In these animals, Uosm decreased significantly (1429 to 1114 mOsm/kg H2O, P less than 0.005) and plasma ADH did not change significantly (1.66 to 1.88 pg/ml, NS). When IC AII (4 ng/kg/min) was repeated in hydropenic dogs pretreated with indomethacin, neither Usom (1787 to 1664 mOsm/kg H2O, NS) nor plasma ADH were altered.(ABSTRACT TRUNCATED AT 250 WORDS)

Reduced osmotic and nonosmotic release of vasopressin after meclofenamate in the conscious dog.

Both in vivo as well as in vitro experiments suggest that prostaglandins (PG) may influence arginine vasopressin (AVP) release. Recent studies on conscious dogs have shown that cyclooxygenase inhibition with meclofenamate reduces basal AVP release as well as AVP release in response to hypoxia. The current experiments were performed in order to test whether PG synthesis inhibition affects osmotic- and nonosmotic-stimulated AVP release in a similar manner. Osmotic AVP release was tested by slowly infusing hypertonic saline intravenously in water-diuresing dogs and serially sampling plasma for AVP concentration. Experiments were performed both with and without meclofenamate (2 mg/kg and 2 mg X kg-1 X h-1 iv) pretreatment. AVP release to a comparable osmotic stimulus was greatly reduced after meclofenamate administration. Nonosmotic AVP release was tested by inducing systemic hypotension with an intravenous infusion of nitroprusside. Hypotension was associated with an increase in AVP concentration, which was partially blunted after meclofenamate administration. Experiments performed with only a saline vehicle administered showed no decrease in AVP release in response to comparable hypotension. The findings of these studies suggest that endogenous PG may be involved in both osmotic and nonosmotic AVP release in the conscious dog.

Cardiovascular and renal effects of the beta-blocker SE2395 in conscious normotensive dogs.

The cardiac and renal hemodynamic effects of SE2395, a beta-blocking agent, were examined after intravenous administration (50 micrograms/kg) in normotensive conscious dogs. Beta-blocking action of SE2395 was evidenced by a reduction in heart rate and cardiac output without change in the mean arterial pressure. An increase in glomerular filtration rate (inulin clearance) and a slight enhancement in effective renal plasma flow (para-aminohippurate clearance) were observed without attendant decreases in plasma renin activity. An increase in urinary sodium excretion was also observed; urinary prostaglandin E2 excretion was, however, unchanged. We conclude that at effective beta-blocking dosage SE2395 does not induce any detrimental effect on renal function in conscious dogs.

Effect of indapamide on volume-dependent hypertension, renal haemodynamics, solute excretion and proximal nephron fractional reabsorption in the dog.

Indapamide, a newly developed antihypertensive agent with modest diuretic properties, reduced mean arterial pressure toward normal in dogs made hypertensive with salt and DOCA, while low-dose furosemide (0.1 mg per day) and hydrochlorothiazide (1 and 50 mg per day) did not result in similar degrees of blood pressure control. Indapamide, low-dose furosemide and hydrochlorothiazide treatment all resulted in similar decreases in body weight suggesting that the antihypertensive effect of indapamide occurs through a mechanism independent of contraction of extracellular fluid volume. Intravenous indapamide at doses of 0.3, 1.0 and 3.0 mg/kg caused progressive increases in sodium, potassium and chloride excretion when urine losses were replaced by isotonic saline to prevent extracellular fluid volume contraction. Only at 3.0 mg/kg did plasma potassium decrease significantly (2.92 +/- 0.03 to 2.69 +/- 0.05 mmol/l; p less than 0.05). Neither glomerular filtration rate (GFR) nor renal blood flow (flowmeter) decreased in a dose-related manner; however, effective renal plasma flow assessed by para-aminohippurate clearance did decrease about 15% at the highest dose (p less than 0.05). Proximal re-collection micropuncture studies demonstrated decreased proximal reabsorption. Cortical diluting segment reabsorption was decreased, but CNa + CH2O/GFR increased from 7% to 11% (p less than 0.05). These results indicate that, at doses up to 3.0 mg/kg, indapamide causes a natriuresis which is modest and similar to that seen with thiazides. No decrease in GFR or renal blood flow was observed. This drug apparently exerts a natriuretic effect through an inhibition of solute reabsorption in both the proximal nephron and the cortical diluting segment.