Oral DNA vaccination promotes mucosal and systemic immune responses to HIV envelope glycoprotein.

In this report, we described induction of HIV envelope (env)-specific systemic and mucosal immune responses by oral vaccination of BALB/c mice with env-encoded plasmid DNA encapsulated in poly(dl-lactide-co-glycolide) (PLG) microparticles. We demonstrated that intragastric administration of the encapsulated plasmid DNA resulted in transduced expression of the env glycoprotein in the intestinal epithelium. Mice immunized orally exhibited env-specific type 1 and cytotoxic T lymphocyte (CTL) responses in spleen and the inductive (Peyer's patches) and effector (lamina propria) mucosal tissues of gut. Oral administration of PLG-encapsulated plasmid DNA encoding gp160 also induced env-specific serum antibodies, and an increased level of IgA directed to gp160 was detected in fecal washes of the immunized mice. In contrast, intramuscular (i.m.) administration of naked or PLG-encapsulated DNA vaccine induced only systemic cellular and humoral responses to the env glycoprotein. Using an HIV env-expressing recombinant vaccinia viral intrarectal murine challenge system, we observed higher resistance to mucosal viral transmission in mice immunized orally than in animals injected i.m. with PLG-encapsulated plasmid DNA encoding gp160. Results of these studies demonstrate the feasibility of using orally delivered PLG microparticles containing plasmid DNA-encoded HIV gp160 for induction of env-specific systemic and mucosal immune responses and protection against recombinant HIV env vaccinia virus challenge.

Association between HIV-specific T helper responses and CTL activities in pediatric AIDS.

We examined the relationship between the profile of HIV-specific T helper (Th) cell responses, cytotoxic T lymphocyte (CTL) activity, HIV viral load, and CD4(+) T cell counts during longitudinal studies in children with perinatal HIV infection. Patients with AIDS demonstrated undetectable or low levels of HIV-specific Th and CTL activities, and exhibited almost exclusively Th0 type of responses with low IFN-gamma and IL-4 production. The levels of IL-2 expression in the envelope (env) peptide-stimulated peripheral blood mononuclear cells were increased in children with a slowly progressive disease, concomitant with higher numbers of CD45RO(+) memory T cells and increased proportions of Th1 clones. In these patients, high levels of env peptide-specific IL-2 expression correlated with increases in HIV-specific CTL responses, whereas a delay in the generation of HIV-specific CTL activity was associated with lower IL-2 production and elevated Th2 responses. Patients with slow disease progression produced higher levels of beta-chemokines than those detected in children with AIDS. These results suggest that an impaired development of HIV-specific cellular responses and inhibition of T cell differentiation during infancy are associated with fast disease progression. They also point to a protective role of noncytotoxic antiviral activity that might complement HIV-specific CTL responses in children with a slowly progressive disease.

New generation dopaminergic agents. 6. Structure-activity relationship studies of a series of 4-(aminoethoxy)indole and 4-(aminoethoxy)indolone derivatives based on the newly discovered 3-hydroxyphenoxyethylamine D2 template.

A series of 4-(aminoethoxy)indoles 7 and a related series of 4-(aminoethoxy)indolones 8 were synthesized and evaluated for their affinity for both the high- and low-affinity agonist states (D2High and D2Low, respectively) of the dopamine (DA) D2 receptor. The 4-aminoethoxy derivatives (i.e., 7 and 8) were designed as bioisosteric analogues based on the phenol prototype 4. The indolones 8 were observed to have high affinity for the D2High receptor. Comparison of their previously reported chroman analogues with the more flexible 4-(aminoethoxy)indoles revealed the chroman analogues to be more potent, whereas little loss in D2High affinity was observed when comparing the 4-(aminoethoxy)indolones with their respective chroman analogues. Several regions of the phenoxyethylamine framework were modified and recognized as potential sites to modulate the level of intrinsic activity. A conformational analysis was performed and a putative bioactive conformation was proposed which fulfilled the D2 agonist pharmacophore criteria based on the McDermed model. Structure-activity relationships gained from these studies have aided in the synthesis of D2 partial agonists of varying intrinsic activity levels. These agents should be of therapeutic value in treating disorders resulting from hypo- and hyperdopaminergic activity, without the side effects associated with complete D2 agonism or antagonism.

Protective role of beta-chemokines associated with HIV-specific Th responses against perinatal HIV transmission.

To examine the protective role of cellular immunity in the vertical transmission of HIV, we analyzed HIV-specific IL-2 and CTL responses, as well as beta-chemokine expression in HIV-infected and uninfected infants of HIV+ mothers. Our results showed that HIV envelope (env) peptide-specific IL-2 responses associated with beta-chemokine production were detectable at birth in the majority of uninfected infants of HIV+ mothers. The responses falling to background before the infants were 1 yr old were rarely associated with HIV-specific CTL activity. Conversely, HIV-specific Th and CTL cellular responses were absent at birth in HIV-infected infants. Infants with AIDS-related symptoms exhibited undetectable or very low levels of HIV-specific cellular immunity during the first year of life, whereas those with a slowly progressive disease showed evidence of such immunity between their second and ninth month. The latter group of infected infants tested negative for plasma HIV RNA levels shortly after birth, suggesting lack of intrauterine exposure to HIV. The presence of HIV-specific Th responses at birth in uninfected newborns of HIV+ mothers, but absence of such activities in HIV-infected infants without evidence of intrauterine HIV infection, suggests that in utero development of HIV-specific Th responses associated with beta-chemokines could mediate nonlytic inhibition of infection during vertical transmission of HIV.

The effect of deletion of the V3 loop of gp120 on cytotoxic T cell responses and HIV gp120-mediated pathogenesis.

New strategies for improving the efficacy of HIV vaccines are of significant importance. In this study, we analyzed the effect of deletion of the hypervariable V3 loop of gp120 on envelope (env)-specific CTL responses in PBMC of HIV-infected individuals. We showed increased CTL activities against conserved epitopes of the env glycoprotein in cultures induced with the AV3 mutant compared with those stimulated with the full-length env gene products. In contrast to the wild-type env, the AV3 mutant-expressing cells were resistant to Ab-dependent cell-mediated cytotoxicity, formed no syncytia, and neither underwent nor induced apoptosis in CD4+ cells. Thus, the AV3 mutant may redirect immune responses toward conserved epitopes of gp160, has longer expression time due to increased resistance to Ab-dependent cell-mediated cytotoxicity, and does not trigger cytopathic effects associated with apoptosis and syncytium formation. This approach may apply to other Ags of HIV, where deletions of highly variable or immunosuppressive epitopes may improve the efficacy of HIV vaccines.

The effect of epitope variation on the profile of cytotoxic T lymphocyte responses to the HIV envelope glycoprotein.

To address the relationship between viral and host factors during HIV infection, we analyzed the effect of viral mutations on T cell responses in seropositive, asymptomatic HLA-A2+ individuals using four envelope (env)-specific peptides with the HLA-A*0201 binding motif. We showed that the natural sequence variation was frequent within epitopes located in the C-terminal region of the env glycoprotein and was largely responsible for a lower env-specific cytotoxic T lymphocyte (CTL) activity in the peptide-stimulated cultures. The highest CTL responses in vitro were induced with conserved epitopes D1 and 4.3 that mapped to the N-terminal region of the env glycoprotein. These peptides exhibited high binding affinity for HLA-A*0201 molecules and stimulated CD8+ T cells of relatively limited TCR Vbeta chain repertoire. Decreased CTL activities to the D1 epitope were observed in the absence of any detectable viral mutation, and were associated with lower proliferative responses and expression of the CD28 antigen. Results of this study demonstrate that the degree of sequence variation within a stimulatory epitope of the viral quasispecies, as well as proliferative potential of the effector cells, are among the factors underlying decreased CTL activity in HIV-infected patients. These experiments also provide evidence that the D1 peptide might be useful for the development of vaccines and immune-based therapy.

Diminished HIV-specific CTL activity is associated with lower type 1 and enhanced type 2 responses to HIV-specific peptides during perinatal HIV infection.

The early development of symptoms and the rapid progression of disease in some vertically infected infants are thought to reflect in part the immaturity of their immune systems. We examined the relationship between HIV-specific CTL activity and the profile of cytokine production induced by mAb to CD3 and HIV envelope (env) peptides P18 and T1 in PBMC derived from 0.6- to 3.6-yr-old children with perinatal HIV infection. Cellular immunity against HIV was demonstrated only during early stages of disease, whereas the responses were either undetectable or at background levels in HIV-infected children with rapidly progressing disease and in uninfected children of HIV+ and HIV- mothers. Levels of IL-2 mRNA in anti-CD3 mAb- and env peptide-induced PBMC varied and were increased in the infected children with high frequencies of HIV-specific CTL precursors. Analysis of IFN-gamma and IL-4 production by CD4+ T cell clones obtained from cultures stimulated with anti-CD3 mAb or the env peptides showed an increased proportion of Th2 and Th0 clones in HIV-infected children with lower HIV-specific CTL activity, whereas children with high CTL activity had increased numbers of Th1 clones. The results of these studies suggest that decreases in CTL activity to the virus might be associated with the induction of a type 2 cytokine response. These findings underline the role of cytokines in the generation of HIV-specific CTL responses and may be important for the development of immunomodulatory and vaccine strategies to interrupt vertical transmission of HIV.

Evidence for B cell-mediated activation of V delta 1+ T lymphocytes during progression of HIV infection.

Progression of HIV-induced immunodeficiency is associated with both B cell activation and an increased proportion of Vdelta1+ T cells in PBL. To examine whether the peripheral expansion of Vdelta1+ cells is driven by activated B cells, we isolated CD19+ PBL from HIV+ individuals at different stages of infection and used them to stimulate Vdelta1+ T cell clones. The Vdelta1+ T cell clones were isolated from HIV+ individuals and selected on the basis of cytotoxic activity and IFN-gamma expression in response to lymphoblastoid cell lines (LCLs) established from patients with AIDS (AIDS-related LCLs) but not LCLs of HIV- donors. Peripheral blood B cells from HIV+ patients induced IFN-gamma expression in these Vdelta1+ clones, and their stimulatory ability was associated with up-regulated expression of the CD38 activation Ag and with a 6- to 10-fold increased spontaneous Ig production. Stimulation of CD19+ PBL from HIV+ individuals with cross-linked anti-CD40 mAb or rgpl20 further augmented induction of IFN-gamma expression in the Vdelta1+ cells. The isolated Vdelta1+ T cell clones expressed the Jdelta1 gene segment, but differed in Vgamma gene segment usage and in the junctional region of TCR-delta chains, indicating Vdelta gene-determined recognition. These results provide evidence that the peripheral expansion of Vdelta1+ cells in HIV infection is associated with phenotypic and functional alterations of B cells, due to chronic activation during progression to AIDS.

New generation dopaminergic agents. 1. Discovery of a novel scaffold which embraces the D2 agonist pharmacophore. Structure-activity relationships of a series of 2-(aminomethyl)chromans.

A series of 2-(aminomethyl)chromans (2-AMCs) was synthesized and evaluated for their affinity and selectivity for both the high- and low-affinity agonist states (D2High and D2Low, respectively) of the dopamine (DA) D2 receptor. The 7-hydroxy-2-(aminomethyl)chroman moiety was observed to be the primary D2 agonist pharmacophore. The 2-methylchroman moiety was discovered to be an entirely novel scaffold which could be used to access the D2 agonist pharmacophore. Attaching various simple alkyl and arylalkyl side chains to the 7-hydroxy 2-AMC nucleus had significant effects on selectivity for the D2High receptor vs the 5HT1A and alpha 1 receptors. A novel DA partial agonist, (R)-(-)-2-(benzylamino)methyl)chroman-7-ol [R-(-)-35c], was identified as having the highest affinity and best selectivity for the D2High receptor vs the alpha 1 and 5HT1A receptors. Several regions of the 2-AMC nucleus were modified and recognized as potential sites to modulate the level of intrinsic activity. The global minimum conformer of the 7-hydroxy-2-AMC moiety was identified as fulfilling the McDermed model D2 agonist pharmacophoric criteria and was proposed as the D2 receptor-bound conformation. Structure-activity relationships gained from these studies have aided in the synthesis of D2 partial agonists of varying intrinsic activity levels. These agents should be of therapeutic value in treating disorders resulting from hypo- and hyperdopaminergic activity, without the side effects associated with complete D2 agonism or antagonism.

Role of the V2, V3, and CD4-binding domains of GP120 in curdlan sulfate neutralization sensitivity of HIV-1 during infection of T lymphocytes.

A sulfated polysaccharide, curdlan sulfate (CRDS) with 1,3-beta-D-glucan as a main chain, inhibits HIV-1 infection of human peripheral blood lymphocytes (PBLs) by binding to the V3 region of gp 120. We previously showed that T cell (T)-tropic HIV-1 isolates are over 10-fold more sensitive to neutralization by CRDS than macrophage (MT)-tropic viruses, which possesses a relatively less charged amino acid composition in the V3 sequence. To analyze the interaction of CRDS with V3 and its association with neutralization sensitivity of HIV-1 isolates, we examined the effect of CRDS on the binding of neutralizing antibodies to monomeric and oligomeric gp 120 mutants of T- and MT-tropic HIV-1 clones in which the V3 loop was either deleted or substituted by V3 of another isolate. Our results showed that the presence and the amino acid composition of the V3 loop appears to determine the extent of interaction of CRDS with the V2 and CD4-binding regions on native gp 120 monomers; however, the positive charge of V3 has less effect on this interaction on oligomeric gp 120. Furthermore, our results established that only the CRDS-induced masking of V3 on oligomeric gp120 appears to be associated with the anti-HIV-1 activity of CRDS in vitro. Our findings underline the usefulness of CRDS for understanding the structural constraints on gp 120 that drive the transition from MT- to T-tropic isolates in vivo and enable the virus to use multiple fusion cofactors.

Effect of hemodialysis on lipid peroxidation and antioxidant system in patients with chronic renal failure.

Plasma lipid peroxidation, activity of erythrocyte superoxide dismutase (SOD) and catalase, and serum antioxidant activity (AOA) in uremic patients were examined before and after hemodialysis. An increased level of lipid peroxidation, a decreased serum AOA level, and elevated SOD and normal catalase activity before hemodialysis were observed in uremic patients compared with controls. Hemodialysis was found to produce increased lipid peroxidation, a simultaneous decrease of SOD activity, and lack of any changes in serum AOA and erythrocyte catalase. It is suggested that intensification of lipid peroxidation during hemodialysis could account for accelerated progress of atherosclerosis in patients with renal insufficiency.

[The fate of young individuals with a history of myocardial infarction]. / Los mlodych ludzi po przebytym zawale serca.

The fate of young individuals (to 45 years) with a history of myocardial infarction during 12 years was analysed with the aid of a questionnaire containing questions of both social and medical character. Sudden cardiac death or the second infarction were the most frequent causes of death during the first two years following myocardial infarction. Change in the physical activity mainly involved the return to work. Only 47% of young men and 29.8% of women started full-time jobs. They mainly belonged to so-called white collars. The lack of patients' physicians permission was a main cause of the abstinence of young men from the occupation. A position within the family and social activity usually remained unchanged in the majority of patients, but every third patient greatly reduced sexual activity. Every third patients continued smoking, and did not observe recommended diet despite the systematical medical check-ups. The course of the disease is unclear in the majority of young patients. It is often deformed and requires further, detailed information on young patients' style of life.