A simple method to detect and estimate heterogeneity: application to Huntington disease, diabetes, and HIV seroconversion.

The traditional method for calculating risk in prospective and retrospective studies is based on the assumption that the study population is homogeneous. Risk is therefore estimated as an overall average for the entire population, when in fact some individuals may be at high risk and others at little or no risk. This paper introduces an alternate approach to risk estimation. The calculations are equally simple and utilize the same data. Yet, the new approach allows for heterogeneity and can detect it when it exists. The new method was applied to HIV seroconversion data from a follow-up study, age-at-onset distribution for Huntington disease, and age-specific prevalence of insulin-treated diabetes. These analyses were intended to demonstrate both applicability of the method to different types of data and the accuracy of the estimates when compared with the known parameters. The HIV analysis predicted a high-risk subgroup constituting about 17% of the cohort. This estimate closely approximates the actual 16% who reportedly engaged in high-risk activities and had a 15-fold higher seroconversion rate than the rest of the cohort. There is no evidence from genetic linkage studies for heterogeneity in Huntington disease. The present results, however, suggested that 14%-18% of individuals who are susceptible to the disease have a much lower risk than others. Diabetes data was chosen because the model is clearly too simplistic for this disease, and the analysis did reveal lack of fit of the model.     

Category of exposure to HIV and age in the progression to AIDS: longitudinal study of 1199 people with known dates of seroconversion. HIV Italian Seroconversion Study Group.

OBJECTIVES: To determine whether rate of development of AIDS is affected by category of exposure to HIV and whether the more rapid development found in older subjects persists for each exposure category. DESIGN: Longitudinal study of people with known date of seroconversion to HIV. SETTING: 16 HIV treatment centres throughout Italy. SUBJECTS: 1199 people infected with HIV through use of injected drugs, homosexual sex, or heterosexual sex. MAIN OUTCOME MEASURES: AIDS as defined by 1987 definition of Centers for Disease Control (including and excluding neoplasms) and by 1993 European definition. RESULTS: 225 subjects (18.8%) progressed to AIDS (Centers for Disease Control 1987 definition) during median follow up of 5.8 years. Univariate analyses showed more rapid progression to AIDS for older subjects compared with younger subjects and for homosexual men compared with other exposure categories. The age effect was of similar size in each exposure category and in men and women. In a bivariate model with age and exposure categories simultaneously included as covariates, differences by exposure category disappeared for use of injected drugs and heterosexual sex compared with homosexual sex (relative hazards 1.02 (95% confidence interval 0.71 to 1.45) and 1.07 (0.70 to 1.64) respectively), while the age effect remained (relative hazard 1.55 (1.32 to 1.83) for 10 year increase in age). Analyses using the other definitions for AIDS did not appreciably change these results. CONCLUSIONS: There was no evidence of differences in rate of development of AIDS by exposure category, while there was a strong tendency for more rapid development in older subjects for all three groups. This supports the view that external cofactors do not play major role in AIDS pathogenesis but that age is of fundamental importance.     

Effect of serotesting with counselling on condom use and seroconversion among HIV discordant couples in Africa.

OBJECTIVE--To determine whether HIV testing and counselling increased condom use and decreased heterosexual transmission of HIV in discordant couples. DESIGN--Prospective study. SETTING--Kigali, the capital of Rwanda. SUBJECTS--Cohabiting couples with discordant HIV serology results. MAIN OUTCOME MEASURES--Condom use in the couple and HIV seroconversion in the negative partners. RESULTS--60 HIV discordant couples were identified, of whom 53 were followed for an average of 2.2 years. The proportion of discordant couples using condoms increased from 4% to 57% after one year of follow up. During follow up two of the 23 HIV negative men and six of the 30 HIV negative women seroconverted (seroconversion rates of 4 and 9 per 100 person years). The rate among women was less than half that estimated for similar women in discordant couples whose partners had not been serotested. Condom use was less common among those who seroconverted (100% v 5%, p = 0.01 in men; 67% v 25%, p = 0.14 in women). CONCLUSIONS--Roughly one in seven cohabiting couples in Kigali have discordant HIV serological results. Confidential HIV serotesting with counselling caused a large increase in condom use and was associated with a lower rate of new HIV infections. HIV testing is a promising intervention for preventing the spread of HIV in African cities.     

HIV disease progression in 854 women and men infected through injecting drug use and heterosexual sex and followed for up to nine years from seroconversion. Italian Seroconversion Study.

OBJECTIVE--To compare the progression of HIV-1 infection in men and women followed up for up to nine years after an accurately estimated date of seroconversion. DESIGN--Prospective observational study. SETTING--16 HIV outpatient clinics across Italy. SUBJECTS--321 women and 533 men infected with HIV through injecting drug use or heterosexual sex and with accurately estimated dates of seroconversion. MAIN OUTCOME MEASURES--Progression to severe CD4 lymphocytopenia (CD4 lymphocyte count < 200 x 10(6)/l), development of AIDS defining diseases, and death from AIDS. RESULTS--Thirty two women and 67 men developed AIDS at Kaplan-Meier progression rates of 25% (95% confidence interval 13.8% to 35.5%) and 23% (15.6% to 30.4%), respectively, 7 years after seroconversion. In a Cox proportional hazards model the relative hazard was 0.93 (that is, a slightly lower hazard in women) before and 1.10 (0.70 to 1.72) after adjusting for age, HIV exposure group, and year of seroconversion. When CD4 lymphocytopenia and death from AIDS were used as end points the results were similar, with adjusted relative hazards of 0.95 (0.63 to 1.42) and 0.72 (0.48 to 1.79) respectively. In both women and men the risk of developing AIDS before the CD4 lymphocyte count had declined below 200 x 10(6)/l was small (3% in women, 6% in men). The estimated median count at which AIDS developed in women (34 x 10(6)/l; 10 x 10(6) to 44 x 10(6)) was similar to that for men (44 x 10(6)/l; 22 x 10(6) to 60 x 10(6)). CONCLUSION--There seems to be little evidence for appreciable differences in the natural course of HIV infection between men and women followed up from the time of seroconversion.     

Nitric oxide-induced damage to mtDNA and its subsequent repair.

Mutations in mitochondrial DNA (mtDNA) have recently been associated with a variety of human diseases. One potential DNA-damaging agent to which cells are continually exposed that could be responsible for some of these mutations is nitric oxide (NO). To date, little information has been forthcoming concerning the damage caused by this gas to mtDNA. Therefore, this study was designed to investigate damage to mtDNA induced by NO and to evaluate its subsequent repair. Normal human fibroblasts were exposed to NO produced by the rapid decomposition of 1-propanamine, 3-(2-hydroxy-2-nitroso-1-propylhydrazino) (PAPA NONOate) and the resultant damage to mtDNA was determined by quantitative Southern blot analysis. This gas was found to cause damage to mtDNA that was alkali-sensitive. Treatment of the DNA with uracil-DNA glycosylase or 3-methyladenine DNA glycosylase failed to reveal additional damage, indicating that most of the lesions produced were caused by the deamination of guanine to xanthine. Studies using ligation-mediated PCR supported this finding. When a 200 bp sequence of mtDNA from cells exposed to NO was analyzed, guanine was found to be the predominantly damaged base. However, there also was damage to specific adenines. No lesions were observed at pyrimidine sites. The nucleotide pattern of damage induced by NO was different from that produced by either a reactive oxygen species generator or the methylating chemical, methylnitrosourea. Most of the lesions produced by NO were repaired rapidly. However, there appeared to be a subset of lesions which were repaired either slowly or not at all by the mitochondria.     

Genetic susceptibility to chlamydial salpingitis and subsequent infertility in mice.

Groups of mice from genetically defined inbred strains were infected genitally with a pathogenic human strain of Chlamydia trachomatis and their subsequent fertility was compared. The CBA, C3H (H-2o) and C3H/He-mg (H-2k) mice were less fertile than control mice, at least up to 6 months after infection. In contrast, fertility was not impaired in BALB/c mice or in congenic BALB/K mice, which had the H-2k haplotype. Reduced fertility was paralleled by the extent of histological oviductal inflammation in mice of each strain. No salpingitis was seen 21 days after infection in the BALB strains, but lesions were apparent in CBA and C3H strains up to about 70 days after inoculation and these sometimes developed into hydrosalpinges. These results indicate that susceptibility to chlamydial salpingitis and subsequent infertility is under genetic control. This control was not simply associated with the major H-2 gene complex, as mouse strains of the same haplotype (H-2k) differed in susceptibility. The fertility of BALB/c (H-2d) and BALB/K (H-2k) strains was no different from that of controls, and congenic C3H mice of differing H-2 haplotypes (H-2k and H-2o) showed reduced fertility. Although all the infected F1 (BALB/K x C3H/He-mg) mice produced litters at the same rate as untreated controls, the litters were considerably smaller. This was due to the occurrence of unilateral pregnancies in the mice inoculated under the ovarian bursae and possibly also to early fetal death in mice inoculated directly in the uterus. These findings emphasize the importance of early diagnosis and treatment of infection of the lower genital tract of women.     

IgG opsonization of HIV impedes provirus formation in and infection of dendritic cells and subsequent long-term transfer to T cells

Already at initial phases of infection, HIV is coated with complement fragments. During the chronic phase, when HIV-specific IgGs appear, the virus circulates immune complexed with IgG and complement. Thus, we studied the interaction of dendritic cells (DCs) and DC-T cell cocultures with complement (C)-opsonized and C-IgG-opsonized HIV. HIV infection of monocyte-derived DCs and circulating BDCA-1-positive DCs was significantly reduced upon the presence of virus-specific but non-neutralizing IgGs. DCs exposed to C-Ig-HIV or IgG-opsonized HIV showed an impaired provirus formation and p24 production and a decreased transmission rate to autologous nonstimulated T cells upon migration along a chemokine gradient. This reduced infectivity was also observed in long-term experiments, when T cells were added delayed to DCs exposed to IgG-coated HIV without migration. Similar kinetics were seen when sera from HIV-1-infected individuals before and after seroconversion were used in infection assays. Both C- and C-IgG-opsonized HIV were captured and targeted to a tetraspanin-rich endosome in immature DCs, but differed with respect to MHC class II colocalization. The reduced infection by IgG-opsonized HIV is possibly due to interactions of virus-bound IgG with FcgammaRIIb expressed on DCs. Therefore, the intracellular fate and transmission of immune-complexed HIV seems to differ depending on time and opsonization pattern.     

Approaches to HIV vaccine design.

Contemporary vaccines are relying increasingly on modern biotechnology and a vaccine against the AIDS virus is expected to depend even more on new technological advances. Four basic areas of vaccine development are discussed in this context: (1) selection and preparation of candidate immunogens; (2) presentation of immunogens to the immune system; (3) pre-clinical testing of vaccine candidates; and (4) monitoring of vaccine clinical trials.     

Human immunodeficiency virus (HIV)-positive sera obtained shortly after seroconversion neutralize autologous HIV type 1 isolates on primary macrophages but not on lymphocytes

The aim of this study was to analyze the role of humoral immunity in early human immunodeficiency virus (HIV) infection. As neutralizing activities in HIV-positive sera are rarely detectable earlier than 9 to 12 months after infection using primary lymphocytes as target cells in neutralization assays, humoral immunity is generally thought not to contribute significantly to early virus control in the patients. Besides lymphocytes, cells of the monocyte/macrophage lineage are known to be important target cells for HIV in vivo during the establishment of the infection. Therefore, we studied the neutralization of early primary HIV isolates by autologous serum samples using primary macrophages as target cells in the neutralization assays. We analyzed neutralizing activities against the autologous HIV-1 isolates in 10 patients' sera taken shortly after seroconversion, both on primary macrophages and, for comparison, on lymphocytes. Viruses were isolated and expanded in primary mixed cultures containing macrophages and lymphocytes in order to avoid selection for one particular cell type. All viruses replicated to different degrees in macrophages and lymphocytes; nine had a nonsyncytium-inducing phenotype, and one was syncytium inducing. The detection of neutralizing antibodies in acute primary HIV infection depended on the target cells used. Confirming previous studies, we did not find neutralizing activities on lymphocytes at this early time point. In contrast, neutralizing activities were detectable in the same sera if primary macrophages were used as target cells. Differences in neutralizing activities on macrophages and lymphocytes were not due to different virus variants being present in the different cell systems, as gp120 sequences derived from both cell types were homogeneous. Neutralization activities on macrophages did not correlate with the amount of beta-chemokines in the sera. As affinity-purified immunoglobulin G preparations from an early patient serum also exhibited neutralization of the autologous virus isolate on primary macrophages, but not on lymphocytes, neutralization is very likely due to antibodies against viral epitopes necessary for infection of macrophages but not for infection of lymphocytes. Our data suggest that, along with cell-mediated immunity, humoral immunity may contribute to the reduction of primary viremia in the patient. This was further supported by a certain association between neutralizing antibody titers on macrophages and viral load in the patients.