TL1A/TNFSF15 directly induces proinflammatory cytokines, including TNFα, from CD3+CD161+ T cells to exacerbate gut inflammation.

Tumor necrosis factor (TNF)-like cytokine 1A (TL1A)/TNF superfamily member 15 (TNFSF15) is a proinflammatory cytokine and TNFα superfamily member that is linked preclinically and clinically to inflammatory bowel disease (IBD). By homology and function, TNFα is its closest family member. In this study, we investigated the mechanism of TL1A-induced inflammation in CD4+ T cells and compared it with the TNFα pathway. We found that TL1A induces proinflammatory cytokines, including TNFα, from isolated human CD4+CD161+ T cells, whereas these cells were resistant to TNFα treatment. Anti-TNFα failed to block TL1A-induced cytokine production, indicating that the effects of TL1A are direct. Lastly, CD161 and TL1A expression were significantly and selectively increased in gut tissue biopsies, but not in the peripheral blood, from IBD patients. Thus, TLIA not only functions upstream of TNFα, driving its expression from CD161+ T cells, but is also independent of TNFα. These findings may have therapeutic IBD implications.

Mucosal correlates of isolated HIV semen shedding during effective antiretroviral therapy.

Effective antiretroviral therapy (ART) suppresses the blood HIV RNA viral load (VL) below the level of detection. However, some individuals intermittently shed HIV RNA in semen despite suppression of viremia, a phenomenon termed "isolated HIV semen shedding (IHS)". In a previously reported clinical study, we collected blood and semen samples from HIV-infected men for 6 months after ART initiation, and documented IHS at ≥1 visit in almost half of the participants, independent of ART regimen or semen drug levels. We now report the mucosal immune associations of IHS in these men. Blood and semen plasma cytokine levels were assayed by multiplex enzyme-linked immunosorbent assay, T-cell populations were evaluated by flow cytometry in freshly isolated blood and semen mononuclear cells, and semen cytomegalovirus (CMV) DNA levels were measured by PCR. Although IHS was not associated with altered blood or semen cytokine levels, the phenomenon was associated with a transient, dramatic increase in CD4+ and CD8+ T-cell activation that was restricted to the semen compartment. All participants were CMV infected, and although semen CMV reactivation was common despite ART, this was not associated with T-cell activation or IHS. Further elucidation of the causes of compartmentalized mucosal T-cell activation and IHS may have important public health implications.

Lipidic implants for controlled release of bioactive insulin: effects on cartilage engineered in vitro.

Controlled release systems for growth factors and morphogens are potentially powerful tools for the engineering or the treatment of living tissues. However, due to possible instabilities of the protein during manufacture, storage, and release, in the development of new release systems it is paramount to investigate into the maintenance of bioactivity of the protein. Within this study, recently developed protein releasing lipid matrix cylinders of 2 mm diameter and 2 mm height made from glycerol tripalmitate were manufactured in a compression process without further additives. Insulin in different concentrations (0.2%, 1%, and 2%) served as model protein. The bioactivity of the protein released from the matrices was investigated in a long-term cartilage engineering culture for up to four weeks; additionally, the release profiles were determined using ELISA. Insulin released from the matrices increased the wet weights of the cartilaginous cell-polymer constructs (up to 3.2-fold), the amount of GAG and collagen in the constructs (up to 2.4-fold and 3.2-fold, respectively) and the GAG and collagen content per cell (1.8-fold and 2.5-fold, respectively), compared to the control. The dose-dependent effects on tissue development correlated well with release profiles from the matrices with different insulin loading. In conclusion, the lipid matrices, preserving the bioactivity of incorporated and released protein, are suggested as a suitable carrier system for use in tissue engineering or for the localized treatment of tissues with highly potent protein drugs such as used in the therapy of brain cancer or neurodegenerative CNS diseases.

nef gene sequence variation among HIV-1-infected African children.

BACKGROUND: There are few data on African children infected with nonclade B HIV-1 in endemic settings, which limits generalizations about pathogenesis and progression. Genotypic and phenotypic variations in host immunogenetics and HIV-1 negative factor (nef) accessory protein may influence disease progression and have frequently been characterized in subjects infected with clade B HIV-1. METHODS: In this descriptive study, we report nef gene sequence variation and host genetic polymorphisms in 32 Kenyan children, including 12 slow progressors. RESULTS: Phylogenetic analysis identified HIV-1 clades A, C and D and a recombinant A/D subtype. Grossly defective nef genes or significant changes from relevant clade reference sequences were not identified in children with delayed disease progression. CONCLUSIONS: nef sequence variations may not be common in perinatally infected African children. Further studies are warranted in HIV-1-infected subjects in settings where infection is endemic.

Analysis of transition from long-term nonprogressive to progressive infection identifies sequences that may attenuate HIV type 1.

Long-term nonprogressive human immunodeficiency virus type 1 (HIV-1) infection and its transition to progressive infection presents an opportunity to identify the molecular determinants of HIV-1 attenuation and pathogenesis. We studied an individual who underwent a transition from long-term nonprogressive to rapidly progressive infection. Because HIV-1 RNA genomes in plasma represent replicating virus, we developed a technique to clone full-length HIV-1 RNA genomes from plasma and used this technique to obtain clones from this individual before and during the transition. Most clones assayed were infectious, demonstrating that the RNA genomes encoded viable virus. Analysis of 20 complete HIV-1 RNA genomic sequences revealed one major difference between sequences found during the two phases of infection. During the nonprogressive phase, the predominant sequences had a large deletion in an Sp1-binding site and adjacent promoter in the U3 part of the long terminal repeat (LTR); when the infection became progressive, all viruses had intact Sp1 and promoter sequences and were derived from a minor species present earlier. Analysis of 184 clones of the LTR region obtained at five time points spanning a 7-year period confirmed this switch. In an in vitro assay, the deletion downregulated LTR-driven transcription of a reporter gene. In addition, analysis of cytotoxic T lymphocyte (CTL) epitopes predicted from the complete viral RNA genomes revealed multiple potential escape mutants that accumulated by the time of progression. These studies suggest that during the nonprogressive phase, the Sp1 enhancer-promoter deletion is likely to have played a role in decreasing replication, thereby attenuating HIV-1. The accumulation of CTL escape mutants suggests that a breakdown in immunologic surveillance may have allowed proliferation of intact virus, thus leading to rapid disease progression. These data reveal the viral and immune interactions characterizing a transition from long-term nonprogressive to rapidly progressive infection.

The nuclear factor kappa B (NF-kappa B): a potential therapeutic target for estrogen receptor negative breast cancers.

The effect of a kinase inhibitor Go6796 on growth of epidermal growth factor (EGF)-stimulated estrogen receptor negative (ER-) breast cancer cells in vivo and role of nuclear factor kappa B (NF-kappaB) on tumorogenesis have been investigated. This was studied in an animal model by implanting ER- mouse mammary epithelial tumor cells (CSMLO) in syngeneic A-J mice. (i) Local administration of Go6976 an inhibitor of protein kinases C alpha and beta inhibited growth of tumors and caused extensive necrotic degeneration and regression of the tumors without causing any microscopically detectable damage to the vital organs liver and lung. (ii) Stable expression of dominant-negative mutants of the beta subunit (dnIkkbeta) of the inhibitory kappa B (IkappaB) kinase (dnIkk) that selectively blocked activation of NF-kappaB caused loss of tumorigenic potential of CSMLO cells. Stable expression of dnIkkbeta also blocked phorbol 12-myristate 13-acetate (PMA)-induced activation of NF-kappaB and overexpression of cyclin D1, concomitantly with the loss or reduced tumorigenic potential of these cells. Thus, results from in vivo and in vitro experiments strongly suggest the involvement of NF-kappaB in ER- mammary epithelial cell-mediated tumorigenesis. We propose that blocking NF-kappaB activation not only inhibits cell proliferation, but also antagonizes the antiapoptotic role of this transcription factor in ER- breast cancer cells. Thus, NF-kappaB is a potential target for therapy of EGFR family receptor-overexpressing ER- breast cancers.

Biology of HIV-1 in women and men.

The HIV epidemic continues to spread worldwide, particularly among women and nonwhites. Development of vaccines and improved treatments depend on understanding pathogenesis. In the past few years, studies have begun to focus on HIV-1 pathogenesis in women, in whom some differences have been found in comparison with men. Attention is now focused on HIV-1 infection in reservoirs other than blood, particularly the genital tract. In addition, study of the genetic determinants of susceptibility to HIV-1 infection, including the chemokine receptors, have provided knowledge useful for the design of new treatments.

Preferential suppression of CXCR4-specific strains of HIV-1 by antiviral therapy.

To initiate infection, HIV-1 requires a primary receptor, CD4, and a secondary receptor, principally the chemokine receptor CCR5 or CXCR4. Coreceptor usage plays a critical role in HIV-1 disease progression. HIV-1 transmitted in vivo generally uses CCR5 (R5), but later CXCR4 (X4) strains may emerge; this shift heralds CD4+ cell depletion and clinical deterioration. We asked whether antiretroviral therapy can shift HIV-1 populations back to R5 viruses after X4 strains have emerged, in part because treatment has been successful in slowing disease progression without uniformly suppressing plasma viremia. We analyzed the coreceptor usage of serial primary isolates from 15 women with advanced disease who demonstrated X4 viruses. Coreceptor usage was determined by using a HOS-CD4+ cell system, biological and molecular cloning, and sequencing the envelope gene V3 region. By constructing a mathematical model to measure the proportion of virus in a specimen using each coreceptor, we demonstrated that the predominant viral population shifted from X4 at baseline to R5 strains after treatment. Multivariate analyses showed that the shift was independent of changes in plasma HIV-1 RNA level and CD4+ cell count. Hence, combination therapy may lead to a change in phenotypic character as well as in the quantity of HIV-1. Shifts in coreceptor usage may thereby contribute to the clinical efficacy of anti-HIV drugs.

Association of race and gender with HIV-1 RNA levels and immunologic progression.

CONTEXT: HIV-1 RNA and lymphocyte subset levels are the principal indications for antiretroviral treatment. Past reports have differed with regard to the effect of gender and race on these measures and in measures of disease progression. OBJECTIVE: To assess racial and gender differences in HIV-1 RNA levels and CD4+ lymphocyte decline. DESIGN: A longitudinal study based in the two largest HIV natural history cohort studies conducted in 7 metropolitan areas of the United States. RESULTS: In all, 1256 adult women and 1603 adult men for whom multiple data points were available prior to initiation of antiretroviral therapy were included. Women were more likely to be nonwhite, to have a history of injection drug use, and to have HIV-associated symptoms. After adjustment for differences in measurement method, baseline CD4+ cell count, age, and clinical symptoms, HIV-1 RNA levels were 32% to 50% lower in women than in men at CD4+ counts >200 cells/mm3 (p <.001) but not at CD4+ cell counts <200 cells/mm3. HIV-1 RNA levels were also 41% lower in nonwhites than in whites (p <.001) and 21% lower in persons reporting a prior history of injection drug use (p <.001). Women had more rapid declines in CD4+ cell counts over time than men (difference in slope of 46 cells/year) and nonwhite individuals had slower decline in CD4 cell counts than whites (difference of 39 cells/year). CONCLUSIONS: Both race and gender influence the values of HIV-1 RNA and the rate of HIV-1 disease progression as indicated by decline in CD4 cell counts over time. These effects could provide clues regarding the factors that influence HIV-disease progression and may indicate that guidelines for therapy should be adjusted for demographic characteristics.

The relative value of CD4 cell count and quantitative HIV-1 RNA in predicting survival in HIV-1-infected women: results of the women's interagency HIV study.

OBJECTIVES: To determine factors associated with survival and to assess the relative strength of CD4 cell count and HIV-1 RNA in predicting survival in a cohort of HIV-1-infected women. DESIGN: Prospective cohort, enrolled during 1994-1995, with median follow-up of 29 months RESULTS: Of 1769 HIV-infected women 252 died. In multivariate analyses, lower CD4 cell count, higher quantitative plasma HIV-1 RNA, and the presence of a self-reported AIDS-defining (Class C) condition were significantly associated with shorter survival: the relative hazard (RH) of dying was 1.17, 3.27, and 8.46, respectively for women with baseline CD4 cell count of 200-349, 50-199, and < 50 x 10(6) cells/l, compared with women with CD4 cell count of > or = 350 x 10(6) cells/l. Compared with women with HIV-1 RNA levels of < 4000 copies/ml plasma, the RH of dying for women with baseline quantitative HIV-1 RNA measurements of 4000-20,000, 20,000-100,000, 100,000-500,000 and > 500,000 copies/ml, was 2.19, 2.17, 3.16, and 7.25, respectively. CD4 cell count had as strong a prognostic value as HIV-1 RNA level, particularly among participants with more advanced immunodeficiency. When the analysis was adjusted to eliminate the distortion created by having disproportionately sized strata of the categorized variables, the relative hazard of death associated with CD4 cell count became even larger in comparison with that for HIV-1 RNA. Eliminating from the analysis all follow-up time during which participants could have received highly active antiretroviral therapy did not change these findings. Age was not a predictor of survival after adjustment for covariates. CONCLUSIONS: CD4 cell count and HIV-1 RNA had similar prognostic value in this cohort of HIV-1-infected women. Even in the presence of a low viral burden, a substantially decreased CD4 cell count remained a strong predictor of mortality.

CCR5 genotype and resistance to vertical transmission of HIV-1.

A human gene has been identified that affects susceptibility to HIV-1 infection. The gene codes for CCR5, the coreceptor for macrophage-tropic strains of HIV-1. Individuals who are homozygous for a deleted, mutant form of the gene, delta32, display a high degree of natural resistance to sexual and parenteral transmission of HIV-1. To investigate whether delta32 plays a role in vertical transmission, we determined the CCR5 genotype of 552 children born to infected mothers in the United States and correlated the genotypes with HIV-1 infection status. Of these children, 13% were white, 30% Latino, and 56% African American, reflecting the ethnic makeup of infected women in the United States. The delta32 gene frequency varied among these groups, ranging from 0.08 in whites to 0.02 in both Latinos and African Americans. Approximately 27% of the children in each ethnic group were infected. Four children were identified as delta32 homozygotes, two uninfected whites (3.77%) and two uninfected Latinos (1.68%). None of the infected children displayed the delta32 homozygous genotype. Among Latinos and whites, the number of uninfected children who carried the homozygous delta32 mutation was significantly greater than that predicted by the Hardy-Weinberg equilibrium (p < .001 for Latinos, p = .044 for whites). This association was noted in Latino and white children whose mothers were either treated or untreated with zidovudine. These data document the occurrence of the homozygous delta32 genotype among children of HIV-1-infected mothers and suggest that this mutant genotype may confer protection from mother-to-child transmission of HIV-1. They also suggest that sexual, parenteral, and vertical transmission all involve processes that use CCR5 as a coreceptor for primary HIV-1 infection. Therefore, blocking the CCR5 receptor may provide an additional strategy to prevent HIV-1 vertical transmission.

PCR-mediated recombination: a general method applied to construct chimeric infectious molecular clones of plasma-derived HIV-1 RNA.

A PCR-based approach was developed that provides a powerful tool for engineering recombinant molecules without reliance on restriction sites. DNA sequences were first amplified by high-fidelity PCR using Pfu polymerase; they were then used both as 'megaprimers' and templates in subsequent asymmetric long PCR amplifications to form chimeric clones. To demonstrate the technique, we constructed chimeric full-length HIV-1 clones derived from reverse-transcribed plasma viral RNA and proviral LTRs. Biologic characterization of these clones showed that most were infectious in tissue culture and sequence analysis demonstrated an error rate of only one base change in 20 kb of DNA sequence. For PCR-mediated recombination, it is necessary to know the sequence of the 3' and 5' overlapping regions of the desired PCR products. This method may be extended to include construction of chimeras between any DNA fragments lacking sequence homology. Such chimeras may be constructed by introducing overlapping sequences to one of the fragments. To ensure that unwanted mutations have not been introduced into the clones constructed by this method, each clone should be sequenced. Our results demonstrate that by using a high-fidelity polymerase and highly controlled PCR conditions, the PCR-introduced error rate can be greatly minimized. This new procedure may be used to construct infectious chimeras of HIV or SIV for studies of vaccines and pathogenesis. Moreover, the method is designed to exchange viral genes at precise boundaries to study individual gene products from different HIV genomes. It can also be used to construct expression vectors for production of specific proteins or delivery vectors for gene transfer and gene therapy. Finally, the technique described here provides a versatile tool to transfer genes or gene fragments from different sources for genetic investigation and engineering.

Minimizing DNA recombination during long RT-PCR.

Recent developments have made it possible to reverse transcribe RNA and amplify cDNA molecules of > 10 kb in length, including the HIV-1 genome. To use long reverse transcription combined with polymerase chain reaction (RT-PCR) to best advantage, it is necessary to determine the frequency of recombination during the combined procedure and then take steps to reduce it. We investigated the requirements for minimizing DNA recombination during long RT-PCR of HIV-1 by experimenting with three different aspects of the procedure: conditions for RT, conditions for PCR, and the molar ratios of different templates. We used two distinct HIV-1 strains as templates and strain-specific probes to detect recombination. The data showed that strategies aimed at completing DNA strand synthesis and the addition of proofreading function to the PCR were most effective in reducing recombination during the combined procedure. This study demonstrated that by adjusting reaction conditions, the recombination frequency during RT-PCR can be controlled and greatly reduced.

Mapping the inside of the ribosome with an RNA helical ruler.

The structure of ribosomal RNA (rRNA) in the ribosome was probed with hydroxyl radicals generated locally from iron(II) tethered to the 5' ends of anticodon stem-loop analogs (ASLs) of transfer RNA. The ASLs, ranging in length from 4 to 33 base pairs, bound to the ribosome in a messenger RNA-dependent manner and directed cleavage to specific regions of the 16S, 23S, and 5S rRNA chains. The positions and intensities of cleavage depended on whether the ASLs were bound to the ribosomal A or P site, and on the lengths of their stems. These data predict the three-dimensional locations of the rRNA targets relative to the positions of A- and P- site transfer RNAs inside the ribosome.

Maternal serum vitamin A levels are not associated with mother-to-child transmission of HIV-1 in the United States.

HIV-1 transmission from mother to child has been associated with maternal vitamin A status in studies of women living in Africa. This finding has raised the question of whether vitamin A supplementation might help reduce transmission in the United States as well as worldwide. In industrialized nations, however, both the vitamin A nutritional status of HIV-1-infected pregnant women and the association of vitamin A levels with vertical transmission were unknown. Furthermore, vitamin A is teratogenic, and supplements during pregnancy have caused birth defects. To investigate whether maternal serum levels of vitamin A (retinol) and three other micronutrients correlate with vertical transmission of HIV-1 in the United State, we studied 95 HIV-1-infected pregnant women and followed their infants to determine whether transmission occurred. Sera were obtained during the third trimester of pregnancy from 95 HIV-1-infected women living in the New York and Los Angeles metropolitan areas. The two cohorts were established to study vertical transmission of HIV-1 and to reflect the racial, ethnic, and socioeconomic status of HIV-1-infected in women in the United States. We measured serum levels of vitamin A (retinol) and three other micronutrients, vitamin E (alpha-tocopherol), beta-carotene, and lycopene, in the mothers using reverse-phase high-performance liquid chromatography and determined the HIV-1 infection status of their infants using virus cultivation and polymerase chain reaction. Sixteen of the 95 women transmitted HIV-1 to their infants. Statistical analysis of the data indicated that low maternal serum retinol levels during the third trimester of pregnancy were not associated with mother-to-child transmission of HIV-1. None of the women had retinol levels so low as to have clinical symptoms of vitamin A deficiency. The serum levels of alpha-tocopherol, beta-carotene, and lycopene, three micronutrients that act as antioxidants and enhance immune function, were also measured. Statistical analysis of the data revealed no association of the levels of these three micronutrients with vertical transmission of HIV-1. Analysis of the data obtained from 95 women in the United States indicates that vitamin A deficiency is rare, and serum retinol levels are not associated with risk of vertical HIV-1 transmission. In view of the teratogenic effects of vitamin A when taken as a supplement during pregnancy, pregnant HIV-1-infected women living in nations where vitamin A deficiency is not a public health problem should not be advised to take extra vitamin A supplements.

Biology of HIV-1 in women and men.

The 1990s have been marked by tremendous progress in understanding HIV-1 infection and disease progression in infected individuals. The new discoveries have direct applications in predicting clinical outcomes and monitoring antiviral therapies. With the identification of secondary receptors for HIV-1 cell entry, the CCR-5 receptor was found to be a single genetically determined factor influencing both HIV-1 transmission and disease progression. Quantitation of HIV-1 RNA led to the discoveries that detectable or even high levels of HIV-1 replication occur during all phases of infection, and that plasma HIV-1 RNA levels are powerful predictors of clinical outcome. These findings have increased the ability to predict disease progression and to monitor-antiviral therapy in infected individuals.

HIV type 1 RNA expression in bone marrows of patients with a spectrum of disease.

HIV-1-infected individuals at various stages of disease harbor virus in their lymphoid organs, which serve as reservoirs of viral replication throughout the course of infection. Hematologic abnormalities are extremely common in HIV-1-infected individuals and occur at all stages of disease. To determine if the bone marrow is a reservoir of HIV-1 in vivo and if active HIV-1 RNA expression in that site is related to hematologic disease in infected individuals, we examined HIV-1 RNA expression in bone marrow biopsies from 37 patients with a broad spectrum of hematologic and HIV-1-related disease. To detect HIV-1 RNA expression, we performed in situ hybridization. Double-label in situ hybridization-immunohistochemistry was used for precise identification of the type of cell expressing viral RNA. Six of 37 (16%) patients demonstrated HIV-1 RNA expression in the bone marrow. Double-label analysis performed on two marrows localized HIV-1 RNA to cells of the macrophage lineage. Active HIV-1 expression correlated with advanced HIV-1-related disease and CD4 cell depletion rather than a specific hematologic or clinical diagnosis. These data suggest that although the bone marrow does not serve as a reservoir of viral expression throughout the course of infection as do the lymphoid organs, HIV-1-expressing cells are present in the bone marrow during late stages of disease. These data also suggest that hematologic abnormalities in the majority of infected individuals may result from indirect effects of HIV-1 such as cytokine dysregulation rather than HIV-1 expression in the bone marrow itself.

Molecular cloning of full-length HIV-1 genomes directly from plasma viral RNA.

Human immunodeficiency virus type 1 (HIV-1) in plasma reflects the replicating virus population at any point in time in vivo. Studies of the relationship of the complete HIV-1 genome to pathogenesis therefore need to focus on plasma virions. Since dual infections and recombination can occur in vivo, cloning an intact plasma virus genome as a single full-length molecule is desirable. For these reasons, we developed an efficient method to clone full-length HIV-1 genomes directly from plasma viral RNA. This method used reverse transcription and long polymerase chain reaction (PCR) amplification. Virion-associated RNA was isolated from plasma samples and then reverse-transcribed to make cDNA for PCR amplification. Two different strategies were employed to amplify the full-length genome: one amplified a 9-kb fragment, and the other amplified two overlapping 5-kb fragments. Although both strategies were successful, the second was preferable for amplifying HIV-1 genomes from samples with low viral titers. By directly ligating the PCR-derived fragments into a phagemid vector, we constructed clones that comprised full-length HIV-1 RNA genomes. Using this technique, we have constructed hundreds of clones containing full-length HIV-1 genomes derived from the plasma of HIV-1-infected individuals, some of whom had low HIV-1 titers. Different HIV-1 molecular species were cloned from a single clinical sample, as demonstrated by restriction site polymorphism. This method provides a tool for studying complete HIV-1 genomes in relation to pathogenic processes.