Cytokine profiles in seronegative volunteers immunized with a recombinant canarypox and gp120 prime-boost HIV-1 vaccine. NIAID AIDS Vaccine Evaluation Group.

OBJECTIVES: To study memory T cell proliferative responses and cytokine profiles induced in HIV-1 seronegative volunteers immunized with a live recombinant canarypox vector expressing HIV-1 antigens (ALVAC-HIV) and boosted with a recombinant gp120 subunit vaccine. DESIGN: HIV-specific T cell proliferative responses and cytokines were measured 2 weeks after vaccination. Cytokines secreted by T helper 1 cells (Th1) [interleukin (IL)-2 and interferon-gamma (IFN-gamma)] and T helper 2 (Th2) cells (IL-4, IL-5, IL-6, and IL-10) were assessed both at the mRNA and the protein level. METHODS: Peripheral blood mononuclear cells (PBMC) were stimulated in vitro with HIV antigens. Subsequently, T cell proliferation was measured in a standard lymphoproliferation assay; secreted cytokines were measured using an enzyme-linked immunosorbent assay and upregulation of cytokine mRNA was measured using reverse transcriptase polymerase chain reaction. RESULTS: All individuals who had received ALVAC-HIV followed by the protein vaccine exhibited HIV-1-specific T cell proliferative responses. Moreover, the PBMC of all prime-boost vaccinated individuals produced detectable IFN-gamma and IL-10 in response to stimulation with HIV-1 envelope glycoprotein antigens; 83% also had detectable levels of IL-2 and IL-6, 71% had detectable levels of IL-4, and 86% had detectable levels of IL-5. CONCLUSIONS: These data indicate that this vaccination regimen was inducing both Th1- and Th2-type responses to HIV-1 envelope antigens. This prime-boost vaccination approach elicited T cell help for the generation of cytotoxic T lymphocyte responses as well as help for antibody production and so promises to generate a broad HIV-1-specific immune response.

HIV-1MN recombinant glycoprotein 160 vaccine-induced cellular and humoral immunity boosted by HIV-1MN recombinant glycoprotein 120 vaccine. National Institute of Allergy and Infectious Diseases AIDS Vaccine Evaluation Group.

We evaluated prime-boost immunization with two recombinant envelope glycoprotein subunit vaccines (HIV-1MN recombinant gp160 vaccine in alum adjuvant [MN rgp160] and HIV-1MN recombinant gp120 vaccine in alum adjuvant [MN rgp120]) for safety and immunogenicity in healthy, HIV-1-uninfected adults. The rationale was to combine the helper T cell memory and binding antibody responses typically induced by rgp160 vaccines with the superior neutralizing antibody responses induced by rgp120 vaccines. In a double-blinded, controlled trial, volunteers were randomly assigned to receive MN rgp160 or adjuvant placebo, and a subset later received MN rgp120. The two vaccines were safe, but reactions to MN rgp160 and its adjuvant placebo exceeded those to MN rgp120. MN rgp160 induced IgG binding antibodies, including all IgG subclasses, to MN rgp160 in all vaccine recipients. HIV-1MN-neutralizing and anti-V3 MN peptide-binding antibodies were observed in a majority of volunteers after the fourth MN rgp160 immunization, but at lower levels compared with immunization with MN rgp120 in historical controls. HIV-1-binding, neutralizing, and fusion inhibition antibodies were boosted to the highest levels among MN rgp160 recipients after MN rgp120 booster injections. MN rgp120 boosting appeared to alter the distribution of MN rgp160 vaccine-induced, anti-MN rgp160 IgG subclass antibodies. MN rgp160 induced helper T cell memory, measured by lymphocyte proliferation, Thl and Th2 cytokine production, and skin testing. Strategies including both subunit vaccines may help maximize antibody and helper T cell memory responses to HIV-1 envelope glycoprotein.

Safety and immunogenicity of Env 2-3, a human immunodeficiency virus type 1 candidate vaccine, in combination with a novel adjuvant, MTP-PE/MF59. NIAID AIDS Vaccine Evaluation Group.

We investigated the safety and immunogenicity of a candidate HIV-1 vaccine, Env 2-3 (Chiron Biocine Co.), in combination with an adjuvant emulsion, MF59, with or without an additional immune modulator, MTP-PE 78 healthy HIV-1-seronegative adults. Sixteen subjects participated in a dose escalation study of MTP-PE in MF59 without Env 2-3, given at 0 and 1 months; 48 subjects participated in a study of a fixed dose of 30 micrograms of Env 2-3 in MF59 with increasing doses of MTP-PE (0, 5, 10, 25, 50, and 100 micrograms), and 14 subjects participated in a study of 100 micrograms of Env 2-3 in MF59 without MTP-PE. Subjects were assigned to study groups under a randomized, double-blind allocation. Subjects received immunization at 0, 1, and 6 months, and had the option of receiving a fourth dose at 12-18 months. Env 2-3 in MTP-PE/MF59 was associated with significant reactogenicity, in that severe, although self-limited systemic and/or local reactions occurred in 15 of 30 vaccinees. In contrast, Env 2-3 in MF59 without MTP-PE was relatively well tolerated, and severe local and/or systemic reactions occurred in only 2 of 18 subjects. Env 2-3 stimulated serum antibodies to HIV-1 envelope protein (gp120) as detected by Western blot in 39 of 43 subjects and to HIV-1 virus lysate by EIA in 28 of 43 subjects after three injections. The majority of subjects also developed EIA antibodies to recombinant gp120 (SF-2), gp120 (LAI), and V3 peptide (SF-2). Neutralizing antibodies to the homologous SF-2 strain developed in 30 of 43 and 27 of 34 subjects, and fusion inhibition antibodies in 25 of 43 and 15 of 36 subjects after three and four injections, respectively. Lymphoproliferative responses to the immunogen, Env 2-3 were observed in over 80% of the vaccinees examined, and CD4+ cytotoxic T cell activity directed against HIV-1 was noted transiently in 2 of 20 vaccinees. Addition of MTP-PE to Env 2-3 or increasing the dose of Env 2-3 from 30 to 100 micrograms did not augment immunogenicity. Env 2-3 in MF59 was well tolerated and immunogenic in HIV-1-seronegative individuals. The addition of MTP-PE significantly increased reactogenicity, but had little, if any, effect on immunogenicity.

Oral toxicity evaluation of kefir-isolated Lactobacillus kefiranofaciens M1 in Sprague-Dawley rats.

Lactobacilli kefiranofaciens M1 has shown novel immunomodulation and anti-allergy probiotic attributes in cell and animal models. An acute oral toxicity assessment of L. kefiranofaciens M1 was evaluated in Sprague-Dawley rats. The rats were randomly assigned to four groups (12 rats/sex/group): the low dose group was orally gavaged with L. kefiranofaciens M1 at 3.0×10(8)cfu/kg bw while the medium dose and high dose groups received 9.0×10(9)cfu/kg bw and 1.8×10(10)cfu/kg bw, respectively, for 28days. The control group received phosphate buffer saline. The body weights were measured weekly while blood samples were collected for haematology and serum biochemistry tests. Histopathology of the organs (heart, liver, kidney, adrenal glands, spleen, ovary, testis), and urinalysis were conducted on study termination. The body weight gain of the L. kefiranofaciens M1 and control groups were comparable during the administration period. Overall, L. kefiranofaciens M1 did not induce adverse effects on haematology, serum biochemistry, and urinalysis parameters. Gross and microscopic histopathology of the organs revealed no toxicity effect of L. kefiranofaciens M1. In conclusion, 1.8×10(10)cfu/kg bw of L. kefiranofaciens M1 was considered as the no-observed-adverse-effect-level (NOAEL), which was the highest dose tested in the present study.

Aerodynamic characteristics and design guidelines of push-pull ventilation systems.

Aerodynamic characteristics such as the flow patterns, velocity field, streamline evolutions, characteristic flow modes and characteristic flow regimes of the push-pull ventilation system are cross-examined by using the laser-light sheet smoked-flow visualization method and laser Doppler velocimetry. Four characteristic flow modes, which are denoted as dispersion, transition, encapsulation and strong suction, are identified in the domain of the push-jet and pull-flow velocities at various open-surface tank widths and rising gas velocities. It is argued phenomenologically, from the aerodynamic point of view, that operating the system in the strong suction regime would be a better strategy than operating it in other characteristic regimes for the consideration of capture efficiency. Design guidelines are developed and summarized based on the results obtained from this study. The regression formulas for calculating the critical values of the push-jet and the pull-flow velocities are provided for easy access. The sulfur hexafluoride tracer gas validation technique is performed to measure the capture efficiency. The results of tracer gas validations are consistent with those obtained from the aerodynamic visualization and measurements. The operation points obtained by employing the American Conference of Governmental Industrial Hygienists design criteria are compared with the results obtained in this study for both the aerodynamics and the capture efficiency. Methods for improving the capture efficiency and energy consumptions are suggested.

Age-dependent respiratory function decline and DNA deletions in human muscle mitochondria.

The electron transport activities of various respiratory enzyme complexes in the muscle mitochondria of subjects of different ages without known mitochondriopathies were investigated. The results showed that the activity of cytochrome c oxidase declined with age more drastically than the activities of other respiratory enzyme complexes. NADH-cytochrome c reductase activity decreased mildly with age, whereas succinate cytochrome c reductase activity did not show significant age-dependent changes. Deletions in the muscle mitochondrial DNA (mtDNA) by use of PCR techniques were investigated. Three different age-dependent deletions in the muscle mtDNA of old individuals were found. These were identified to have sizes of 4,977 bp (8470 to 13446), 6,063 bp (7,842 to 13,904) and 7,436 bp (8,649 to 16,084). The 4,977 bp deleted mtDNA (dmtDNA) started to appear in the muscle mtDNA of subjects over 36 years of age and was found to exist in the muscle of more than 70% of the study subjects over 60 years of age. The 6,063 bp dmtDNA was detected in the muscle of the subjects above 25 years of age and was present in the muscle of about 91% of the individuals above 60 years old. However, the 7,436 bp deletion was less prevalent and was only seen in the muscle mtDNA of about 47.2% of the study subjects that were above 60 years. Using a quantitative PCR method, we found that the proportion of the 7,436 bp-deleted mtDNA increased with age, although with notable individual differences. Some of the subjects were found to have multiple deletions in their muscle mtDNAs, but some others carried only a specific type of deletion. The frequency of occurrence of multiple deletions in the muscle mtDNA was significantly increased with the age of the study subjects. The age-dependent increase in the proportions of various deleted mtDNA molecules may cause deleterious effects on the expression of mitochondrial genes in the muscle of old humans. This may account, at least in part, for the age-dependent decline of respiratory functions in the mitochondria of a broad spectrum of human tissues. We suggest that deletions of mtDNA are early molecular events that are associated with and contribute to the ageing processes of the human.

A novel mutation in the mitochondrial 16S rRNA gene in a patient with MELAS syndrome, diabetes mellitus, hyperthyroidism and cardiomyopathy.

Using RNase protection analysis, we found a novel C to G mutation at nucleotide position 3093 of mitochondrial DNA (mtDNA) in a previously reported 35-year-old woman exhibiting clinical features of mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS) syndrome together with diabetes mellitus, hyperthyroidism and cardiomyopathy. The patient also had an A3243G mutation in the tRNA(Leu(UUR)) gene and a 260-base pair duplication in the D-loop of mtDNA. The fibroblasts of the patient were cultured and used for the construction of cybrids using cytoplasmic transfer of the patient's mtDNA to the mtDNA-less rho(0) cells. RNA isolated from the cybrids was subjected to RNase protection analysis, and a C3093G transversion at the 16S rRNA gene and a MELAS-associated A3243G mutation of mtDNA were detected. The novel C3093G mutation together with the A3243G transition were found in muscle biopsies, hair follicles and blood cells of this patient and also in her skin fibroblasts and cybrids. The proportion of the C3093G mutant mtDNA in muscle biopsies of the patient was 51%. In contrast, the mutation was not detected in three sons of the proband. To characterize the impact of the mtDNA mutation-associated defects on mitochondrial function, we determined the respiratory enzyme activities of the primary culture of fibroblasts established from the proband, her mother and her three sons. The proportions of mtDNA with the C3093G transversion and the A3243G transition in the fibroblasts of the proband were 45 and 58%, respectively. However, the fibroblasts of the proband's mother and children harbored lower levels of mtDNA with the A3243G mutation but did not contain the C3093G mutation. The complex I activity in the proband's fibroblasts was decreased to 47% of the control but those of the fibroblasts of the mother and three sons of the proband were not significantly changed. These findings suggest that the C3093G transversion together with the A3243G transition of mtDNA impaired the respiratory function of mitochondria and caused the atypical MELAS syndrome associated with diabetes mellitus, hyperthyroidism and cardiomyopathy in this patient.

Age-dependent 6kb deletion in human liver mitochondrial DNA.

Using PCR technique, restriction mapping and DNA sequencing, we analyzed liver mitochondrial DNA (mtDNA) of 2 stillborn babies and 62 Chinese subjects with non-liver disease from 27 to 86 years old. The results showed an age-dependent 6,063 bp deletion in the liver mtDNA of older subjects. We found a TAACAGAC sequence flanking the 5'-end breakpoint at 7,842 nucleotide position and an imperfect repeat sequence CAACATAC flanking the 3'-end breakpoint at 13,905 nucleotide position. The incidence of the deleted mtDNA was found to increase with age. The deleted mtDNA was not detected in the liver of the stillbirth or blood cells of all the subjects. This is the first account that an age-related 6,063 bp deletion occurs in the liver mtDNA of old humans. The occurrence of this and previously reported 4,977 bp deletions is consistent with our recent finding that liver mitochondrial respiratory functions decline with age and support the hypothesis that continuous accumulation of mtDNA mutations is an important contributor to ageing process in the human.

Age-related 4,977 bp deletion in human lung mitochondrial DNA.

The accumulation of mitochondrial DNA (mtDNA) mutations has been suggested to be an important contributor to human aging and degenerative diseases. The lung is exposed to ambient air and makes direct contact with the external environment. Numerous potentially noxious agents may damage lung tissues directly or indirectly through free-radical-mediated reactions. In previous studies, we demonstrated an age-dependent increase of mtDNA mutations in various human tissues. We hypothesize that the accumulation of the 4,977 bp (base pairs) deleted mtDNA in human lung tissues is also age-dependent. Using the polymerase chain reaction technique, we determined the incidence of the 4,977 bp-deleted mtDNA in 127 human lung specimens from 34-wk gestation to 79 yr of age. The results showed that 77 lung biopsies (60.6%) contained the 4,977 bp-deleted mtDNA, which started to appear in lung tissues after the fourth decade of life. The incidence apparently increased from 14.3% (one of seven) of the subjects in the 30- to 39-yr age group to 77.8% (two of 27) of the subjects in the 70- to 79-yr age group (p < 0.0001). The mean (+/- SEM) proportion of the 4,977 bp-deleted mtDNA in lung tissues significantly increased from 0.007 +/- 0.007% of the subjects in the 30- to 39-yr age group to 0.833 +/- 0.330% of those in the 70- to 79-yr age group (p < 0.005). Other factors such as sex, pulmonary function indices, and smoking status did not have statistically significant impact on the amount of the deleted mtDNA. These findings suggest that the accumulation of the 4,977 bp-deleted mtDNA is associated with aging human lung.

Modulation of immunologic responses to HIV-1MN recombinant gp160 vaccine by dose and schedule of administration. National Institute of Allergy and Infectious Diseases AIDS Vaccine Evaluation Group.

The safety and immunogenicity of HIV-1MN recombinant gp160 (MN rgp160) vaccine in healthy, uninfected volunteers was tested in a double-blind study with a factorial design. By random assignment, 20 volunteers received three 200 micrograms doses of MN rgp160 and four volunteers received placebo at days 0, 28, and 168 or 0, 56, and 224. Of the 24 volunteers, 16 received 200 micrograms or 800 micrograms of MN rgp160 and two received placebo at day 532 (month 18). The vaccine was safe. It induced T cell memory measured by Th1 cytokine production and lymphocyte proliferation, and serum anti-MN rgp160 IgG (all subclasses) and IgA antibodies. Fifteen of 20 vaccinees developed neutralizing antibody. The regimen including immunizations on days 0, 28, and 168 followed by the 800 micrograms fourth dose was most immunogenic.

A dose-ranging study of a prototype synthetic HIV-1MN V3 branched peptide vaccine. The National Institute of Allergy and Infectious Diseases AIDS Vaccine Evaluation Group.

A phase I double-blind trial was done to examine the safety and immunogenicity of a prototype synthetic human immunodeficiency virus type 1 MN strain (HIV-1MN) third variable region domain (V3) branched peptide vaccine in HIV-1-uninfected healthy adult volunteers. Subjects were randomly assigned to receive 20, 100, or 500 micrograms of vaccine or alum adjuvant control on days 0, 28, and 168. The vaccine was well-tolerated and appeared safe. Induction of binding antibody to V3 MN branched peptide was vaccine dose-related and was detectable in 9 of 10 subjects in the highest-vaccine-dose group. HIV-1MN-neutralizing antibody was detected after the third 500-micrograms dose in 8 of 10 subjects at the 90% neutralization end point. V3 MN peptide stimulated lymphocyte proliferation in 15 (75%) of 20 subjects after vaccination. In conclusion, this prototype vaccine was safe and it induced humoral and cell-mediated immune responses.

Immunization with envelope MN rgp120 vaccine in human immunodeficiency virus-infected pregnant women.

Twenty-six human immunodeficiency virus (HIV)-infected pregnant women participated in a placebo-controlled study of immunogenicity and safety of multiple doses of MN rgp120 vaccine over the last half of pregnancy. The women had CD4 lymphocyte counts>400/mm3, no AIDS-defining illness and normal pregnancies. Vaccination was well tolerated, with no significant local or systemic reactions in the women and no adverse outcomes in the infants attributable to the vaccine. Vaccination did not alter plasma RNA reverse transcriptase-polymerase chain reaction copy number; moreover, immunization was not associated with changes in CD4 counts or HIV binding and neutralization antibody titers. Infants were followed up until 18 months of age. Five of 26 infants (19%) were HIV infected, with infection occurring in children of both vaccinated and placebo women. Analysis of factors that influence transmission did not disclose associations with immunization status, viral load, CD4 count, or maternal viral neutralization titers.

Immune responses to human immunodeficiency virus (HIV) type 1 induced by canarypox expressing HIV-1MN gp120, HIV-1SF2 recombinant gp120, or both vaccines in seronegative adults. NIAID AIDS Vaccine Evaluation Group.

A safety and immunogenicity trial was conducted in vaccinia-immune and vaccinia-naive human immunodeficiency virus (HIV)-uninfected adults who were randomized to receive 10(6) or 10(7) TCID50 of canarypox (ALVAC) vector expressing HIV-1MN gp160 or 10(5.5) TCID50 of ALVAC-rabies virus glycoprotein control at 0 and 1 or 2 months and ALVAC-gp160 or 50 microg of HIV-1SF2 recombinant (r) gp120 in microfluidized emulsion at 9 and 12 months; others received rgp120 at 0, 1, 6, and 12 months. All vaccines were well-tolerated. Neither vaccinia-immune status before vaccination nor ALVAC dose affected HIV immune responses. HIV-1MN and HIV-1SF2 neutralizing antibodies were detected more often (100%) in ALVAC-gp160/rgp120 recipients than in recipients of ALVAC-gp160 (<65%) or rgp120 (89%) alone. ALVAC-gp160/rgp120 also elicited more frequent HIV V3-specific and fusion-inhibition antibodies, antibody-dependent cellular cytotoxicity, lymphoproliferation, and cytotoxic CD8+ T cell activity than did either vaccine alone. Trials with ALVAC expressing additional HIV components and rgp120 are underway.