DNA-VLP prime-boost intra-nasal immunization induces cellular and humoral anti-HIV-1 systemic and mucosal immunity with cross-clade neutralizing activity.

The immune response to HIV-1 virus-like particles (VLPs), presenting a clade A Ugandan gp120, has been evaluated in a mouse model by intra-nasal (i.n.) administration by a VLP+VLP homologous or a DNA+VLP heterologous prime-boost immunization protocol, including a HIV-1 DNA gp160/rev plasmid. Furthermore, the effect of the Eurocine lipid-based mucosal L3 adjuvant on the VLP immunogenicity has been assessed as well. The designed heterologous protocol is able to increase the env-specific humoral and cellular immune response, compared to the homologous protocol, which is to some extent increased by the administration of L3-adjuvanted VLP boosting dose. The anti-gag response is statistically increased in both homologous and heterologous protocols, particularly when the VLP boosting dose is adjuvanted. Immune sera from immunized animals exhibit >50% ex vivo neutralizing activity against heterologous A and B-clade viral isolates. An envelope B-cell epitope mapping shows an enhanced response against V3 epitopes all across the C2-V5 region in the heterologous prime-boost immunization strategy. The induction of humoral immunity at mucosal sites, which represents the main port of entry for the HIV-1 infection, is extremely relevant. In this framework, the DNA-VLP heterologous prime-boost protocol appears a promising preventive vaccine approach which can significantly benefit from specific mucosal adjuvants, as the Eurocine L3.

A new method of preemptive analgesia in laparoscopic cholecystectomy.

BACKGROUND: Although laparoscopic cholecystectomy (LC) results in less pain then open cholecystectomy, it is not a pain-free procedure. The aim of this study was to test a new method of preemptive analgesia. METHODS: By simple randomization 60 patients were assigned to two groups (30 in each group). Group A, the placebo group, received 200 ml of 0.9% saline, and group B received 5 mg/kg of a local anesthetic solution (ropivacaine) in 200 ml of 0.9% saline. Local anesthetic or placebo solution were administer before creation of the pneumoperitoneum. RESULTS: Pain intensity, as rated by visual analog and verbal rating scales, and stress response data were significantly less in the group receiving ropivacaine than in the placebo group. No patients in treatment group received an additional dose of analgesic, whereas two patients in placebo group needed an additional analgesic. CONCLUSIONS: Our results support the clinical validity of preemptive analgesia, but the timing of intraperitoneal administration of local anesthetic is very important. Only application before creation of the pneumoperitoneum may preempt every neuronal central sensitization.

Functional HIV-1 specific IgA antibodies in HIV-1 exposed, persistently IgG seronegative female sex workers.

Although HIV-specific cellular immune responses are found in a number of HIV highly-exposed, persistently seronegative (HEPS) cohorts, late seroconversion can occur despite pre-existing cytotoxic T lymphocytes (CTL), suggesting that a protective HIV vaccine may need to induce a broader range of HIV-specific immune responses. Low levels of HIV-specific IgA have been found in the genital tract and plasma of the majority of Nairobi HEPS sex workers and appeared to be independent of HIV-specific cellular responses. IgA purified from genital tract, saliva and plasma of most HEPS sex workers were able to neutralize infection of PBMC by a primary (NSI) clade B HIV isolate, as well as viral isolates from clades A and D, which predominate in Kenya. In addition, these IgA were able to inhibit transcytosis of infective HIV virions across a transwell model of the human mucosal epithelium in an HIV-specific manner. Preliminary work in other HEPS cohorts has suggested the recognition of different gp41 epitopes in HEPS and HIV-infected subjects. Although present at low levels, these IgA demonstrated cross-clade neutralizing activity and were able to inhibit HIV mucosal transcytosis, suggesting an important functional role in protection against HIV infection.

Attachment, parenting, and marital dissatisfaction as predictors of disruptive behavior in preschoolers.

The aim of this study was to examine if an insecure coercive attachment pattern is associated with disruptive behavior in preschoolers, as well as to examine the concurrent and joint effects of attachment pattern, marital dissatisfaction, and ineffective parenting practices on disruptive behavior. Participants included 60 preschoolers and their mothers, recruited from three sites to ensure an adequate range of disruptive behavior. The Preschool Assessment of Attachment (Crittenden, 1992) was used to measure attachment pattern. Results of an analysis of variance revealed that children in the coercively attached dyads scored significantly higher on the measure of disruptive behavior than either the defended or secure children. Results of a hierarchical regression analysis indicated that the combination of a coercive pattern of attachment, marital dissatisfaction, and permissive parenting practices accounted for a significant proportion of the variance in disruptive behavior in preschoolers. These data suggest that a specific type of insecure attachment, a coercive pattern, is associated with disruptive behavior in preschoolers. Also, the data are consistent with previous findings of associations among marital dissatisfaction, ineffective parenting practices, and disruptive behavior.

Oppositional defiant disorder with onset in preschool years: longitudinal stability and pathways to other disorders.

OBJECTIVE: To examine the stability and change in oppositional defiant disorder (ODD) with onset among preschool children in a pediatric sample. METHOD: A total of 510 children aged 2-5 years were enrolled initially in 1989-1990 (mean age 3.42 years); 280 participated in five waves of data collection over a period of 48 to 72 months (mean wave 5 age, 8.35 years). Test batteries varied by age, but they included the Child Behavior Checklist, developmental evaluation, Rochester Adaptive Behavior Inventory, and a play session (before age 7 years) and a structured interview (Diagnostic Interview for Children and Adolescents, parent and child versions) at ages 7+ years. Consensus diagnoses were assigned by using best-estimate procedures. RESULTS: Wave 1 single-diagnosis ODD showed a significant relationship with both single-diagnosis ODD and single-diagnosis attention-deficit hyperactivity disorder (ADHD) at subsequent waves, but not with single-diagnosis anxiety or mood disorders. Single-diagnosis ODD at wave 1 was associated with later comorbidity of ODD/ADHD, ODD/anxiety, and ODD/mood disorders. Stability across waves 2 through 5 was moderate to high for comorbid ODD/anxiety and ODD/ADHD; low to moderate stability for single-diagnosis ODD and single-diagnosis mood disorder; and low for mood disorder, single-diagnosis ADHD, and single-diagnosis anxiety disorder. CONCLUSIONS: Preschool children with ODD are likely to continue to exhibit disorder, with increasing comorbidity with ADHD, anxiety, or mood disorders.

Mucosal and plasma IgA from HIV-1-exposed uninfected individuals inhibit HIV-1 transcytosis across human epithelial cells.

HIV-1-specific IgA has been described in the genital tract and plasma of HIV-1 highly exposed, persistently seronegative (HEPS) individuals, and IgA from these sites has been shown to neutralize HIV-1. This study examines the ability of IgA isolated from HEPS individuals to inhibit transcytosis across a tight epithelial cell layer. A Transwell system was established to model HIV-1 infection across the human mucosal epithelium. The apical-basolateral transcytosis of primary HIV-1 isolates across this mucosal model was examined in the presence and the absence of IgA isolated from the genital tract, saliva, and plasma of HEPS individuals enrolled in both a sex worker cohort in Nairobi, Kenya, and a discordant couple cohort in Italy. In the absence of IgA, HIV-1 primary isolates were actively transported across the epithelial membrane and were released on the opposite side of the barrier. These transcytosed HIV-1 particles retained their ability to infect human mononuclear cells. However, IgA purified from the mucosa and plasma of HEPS individuals was able to inhibit HIV-1 transcytosis. Inhibition was seen in three of six cervicovaginal fluid samples, five of 10 saliva samples, and three of six plasma samples against at least one of the two primary HIV-1 isolates tested. IgA from low risk, healthy control subjects had no inhibitory effect on HIV-1 transcytosis. The ability of mucosal and plasma IgA to inhibit HIV-1 transcytosis across the mucosal epithelium may represent an important mechanism for protection against the sexual acquisition of HIV-1 infection in HEPS individuals.

Mucosal and plasma IgA from HIV-exposed seronegative individuals neutralize a primary HIV-1 isolate.

OBJECTIVE: To characterize functional properties of HIV-specific IgA in samples representing both systemic and mucosal compartments of HIV-1 highly exposed persistently seronegative (HEPS) individuals. METHODS: IgA was purified from plasma and mucosal samples from HEPS individuals and tested for the ability to neutralize infection of peripheral blood mononuclear cells (PBMC) by a non-syncytium inducing HIV-1 (clade B) primary isolate. None of these individuals had measurable HIV-1-specific IgG. RESULTS: HIV-1-specific neutralizing activity of the purified IgA from plasma (n = 15), saliva (n = 15) and cervicovaginal fluid (CVF) (n = 14) were found in the majority of samples (73, 73 and 79%, respectively). In contrast, plasma, saliva and CVF samples of low-risk, uninfected HIV-seronegative individuals lacked neutralizing IgA, with the exception of two out of 34 (6%) saliva samples. CONCLUSION: Mucosal and plasma IgA from HEPS individuals can neutralize HIV-1 infection.

Human immunodeficiency virus type 1 Nef epitopes recognized in HLA-A2 transgenic mice in response to DNA and peptide immunization.

We investigated the immune response against a human immunodeficiency virus type 1 (HIV-1) nef DNA sequence administered epidermally in mice transgenic for the human major histocompatibility complex (MHC) class I molecule HLA-A201. Ten potential HLA-A2 binding 9-mer Nef peptides were identified by a computer-based search algorithm. By a cell surface MHC class I stabilization assay, four peptides were scored as good binders, whereas two peptides bound weakly to HLA-A2. After DNA immunization, cytotoxic T lymphocyte (CTL) responses were predominantly directed against the Nef 44-52, 81-89, and 85-93 peptides. Interestingly, the 44-52 epitope resides outside the regions of Nef where previously described CTL epitopes are clustered. Dominance among Nef-derived peptides did not strictly correlate with HLA-A2 binding, in that only one of the high-affinity binding peptides was targeted in the CTL response. The 44-52, 85-93, and 139-147 peptides also generated specific CTLs in response to peptide immunization. T helper cell proliferation was detected after stimulation with 20-mer peptides in vitro. Three Nef regions (16-35, 106-125, and 166-185) dominated the T helper cell proliferation. The implications of these results for the development of DNA-based vaccines against HIV is discussed.

Mapping of B-cell epitopes in rabbits immunised with various gag antigens for the production of HIV-1 gag capture ELISA reagents.

An HIV-1 p24 capture enzyme linked immunosorbent assay (ELISA) was developed and used in a study of B-cell epitopes in rabbits immunised with different gag p24 antigens. Rabbits were immunised with virion HIV-1/Lai, baculovirus recombinant p24, Escherichia coli recombinant p24-15 and a mixture of synthetic peptides representing sequences of HIV-1 gag p24 protein, respectively. Five out of nine rabbits developed antibodies that could be used for an antigen capture ELISA. No significant differences in IgG titers to the whole gag protein were seen when comparing rabbits immunised with four different antigens. Three major common linear epitope regions were mapped in the rabbits immunised with virion HIV-1/Lai and baculovirus recombinant p24. The rabbit immunised with HIV-1 gag peptides had the broadest linear epitope reactive responses whereas animals immunised with E. coli recombinant antigen had the most restricted linear epitope response. The capture ELISA method thus developed using the different rabbit anti-p24 IgG preparations was shown to capture isolates from HIV-1 subtypes or clades A to G. Only rabbits immunised with virion HIV-1/Lai and baculovirus recombinant p24 developed IgG that was capable of efficiently capturing HIV-1 p24 in ELISA, indicating the importance of preparing antibodies able to recognise native or discontinuous and linear antigen configurations.

Medicare HMO disenrollment and selective use of medical care: osteoarthritis-related joint replacement.

OBJECTIVE: Recent Medicare health maintenance organization (HMO) disenrollees use a high level of medical services. This study examined admissions for total hip arthroplasty (THA) and osteoarthritis-related knee replacements (OKR) among Medicare HMO disenrollees and continuously enrolled fee-for-service (FFS) beneficiaries to determine whether Medicare beneficiaries are returning to the FFS system to receive quality-of-life enhancing elective care. STUDY DESIGN: Retrospective analysis of Medicare inpatient claims for elderly Medicare beneficiaries residing in South Florida between 1990 and 1993. METHODS: Inpatient admission rates for THA, OKR, and for 2 acute conditions--total hip replacements related to fracture of the hip (HRF) and acute myocardial infarction (AMI)--were estimated for Medicare HMO disenrollees over the 3-month period immediately following their disenrollment. These rates were compared with standardized rates for Medicare FFS enrollees. RESULTS: The annualized adjusted rates of both THA and OKR were 3.5 to 4 times higher among Medicare HMO disenrollees than among FFS beneficiaries (P < or = .0001 for both procedures); substantially smaller differences were noted for HRF (P < or = .05), and no difference was present for AMI. HMO disenrollees and FFS enrollees did not differ in their levels of comorbidity at the time of admission. CONCLUSIONS: These data provide indirect evidence that Medicare HMOs in South Florida are rationing THA and OKR and that beneficiaries respond by returning to the FFS system to seek care. This apparent rationing has important implications regarding for the management of serious, but nonemergent, medical conditions within the evolving Medicare system.

Do Medicare HMOs and Medicare FFS differ in their use of the Medicare hospice benefit?

This study compares use of the hospice benefit in Medicare fee-for-service (FFS) and Medicare risk-health maintenance organization (HMO) options in South Florida in 1992. A higher percentage of deaths occurred in hospice in the HMO option than in the FFS option. Compared to individuals in the FFS option, HMO-enrolled hospice users had longer lengths of hospice stay, lower 7-day mortality and higher 180-day (6 month) survival. These differences are consistent with the physician's financial incentives associated with the two programs.

Racial and income differences in use of the hospice benefit between the medicare managed care and medicare fee-for-service.

OBJECTIVE: To examine whether use of the Medicare Hospice Benefit between health maintenance organization (HMO) and Fee-For-Service (FFS)-enrolled beneficiaries varies by income or race. DATA SOURCE: Medicare enrollment and claims data for South Florida. RESULTS: In the FFS system, rate of death in hospice varied by income. In the HMO system, it did not. Time spent in hospice varied by income in the HMO system and not in the FFS system. There was little evidence that racial differences in hospice use differed between FFS and HMO options. CONCLUSIONS: These differences raise questions about whether some hospice use may be in response to system-level incentives.

[Evaluation of indirect immunofluorescence as a supplementary test for the diagnosis of HIV-1 infection]. / Evaluación de la inmunofluorescencia indirecta como prueba suplementaria para el diagnóstico de la infección por HIV-1.

In order to be used as an alternative or complementary test to confirm HIV-1 infection, the efficiency of indirect immunofluorescence assay (IFA) was compared with Western blot (WB) in 362 samples from persons with high and low risk behaviour. A panel of sera with 220 WB positive, 122 WB negative and 20 WB indeterminate sera were tested by an "in house" IFA. The sensitivity of IFA was found to be 98.63% and the specificity 98.36%. Therefore, IFA appeared to be an efficient alternative method to WB, since the cost of testing by IFA is less than 10% of WB testing. We observed a direct relationship between WB protein reactivity and IFA results. In 15 samples with coincident indeterminate results for WB and IFA, antibody reactivity to p24 and gp160 presented the highest frequency. On the other hand, antibodies to viral glycoproteins were always present in IFA weak positive samples, showing their high predictive value.

Home environmental hazards and the risk of fall injury events among community-dwelling older persons. Study to Assess Falls Among the Elderly (SAFE) Group.

OBJECTIVE: To determine if home environmental hazards increase the risk of fall injury events among community-dwelling older persons. DESIGN: Population-based case-control study. SETTING: South Miami Beach, Florida. PARTICIPANTS: 270 persons aged 65 years and older who sought treatment at six area hospitals for injuries resulting from falls within the dwelling unit and 691 controls, frequency matched for sex and age, selected randomly from Health Care Financing Administration (Medicare) files. MAIN INDEPENDENT VARIABLES: The home environment of each person, assessed directly by interviewers using a standardized instrument. RESULTS: Environmental hazards were present in nearly all dwelling units. After adjusting for important confounding factors, most of these hazards were not associated with an increased risk of fall injury events among most older persons. Increasing numbers of tripping hazards, or total hazards in the dwelling unit, did not increase the risk of fall injury events, nor was there an increasing trend in risk. CONCLUSIONS: Current fall-prevention strategies of finding and changing all environmental hazards in all community-dwelling older persons' homes may have less potential effect than previously thought. The usefulness of grab bars, however, appears to warrant further evaluation.

Evaluación de la inmunofluorescencia indirecta como prueba suplementaria para el diagnóstico de la infección por HIV-1 / Evaluation of indirect inmunofluorescence as a confirmatory test for the diagnosis of HIV-1 infection

Se comparó la eficiencia diagnóstica de la inmunofluorescencia indirecta (IFI) como método confirmatorio de la infección por HIV-1 en muestras de suero de 362 personas con conductas de alto y bajo riesgo. El panel compuesto por 220 positivos, 122 negativos y 20 indeterminados por Western blot (WB) fue ensayado por una técnica de IFI desarrollada en nuestro laboratorio. La sensibilidad calculada fue 98,63 por ciento y la espicificidad 98,36 por ciento, indicando que la IFI es un método alternativo para la confirmación de la presencia de anticuerpos contra el HIV-1. Dado que su costo es menor que el 10 por ciento comparado con el del WB, se justifica su introducción en el algoritmo de diagnóstico serológico de HIV-1. Se observó también una relación directa entre la reactividad de las proteínas del WB y los resultados de IFI. En 15 muestras con resultado indeterminado por WB e inespecífico por IFI, las bandas más observadas fueron la p24 seguida de la gp160; por otro lado los anticuerpos contra las glicoproteínas virales son los que presentan mayor frecuencia en las muestras positivas débiles, demostrando su alto valor predictivo (AU)

Evaluación de la inmunofluorescencia indirecta como prueba suplementaria para el diagnóstico de la infección por HIV-1 / Evaluation of indirect inmunofluorescence as a confirmatory test for the diagnosis of HIV-1 infection

Se comparó la eficiencia diagnóstica de la inmunofluorescencia indirecta (IFI) como método confirmatorio de la infección por HIV-1 en muestras de suero de 362 personas con conductas de alto y bajo riesgo. El panel compuesto por 220 positivos, 122 negativos y 20 indeterminados por Western blot (WB) fue ensayado por una técnica de IFI desarrollada en nuestro laboratorio. La sensibilidad calculada fue 98,63 por ciento y la espicificidad 98,36 por ciento, indicando que la IFI es un método alternativo para la confirmación de la presencia de anticuerpos contra el HIV-1. Dado que su costo es menor que el 10 por ciento comparado con el del WB, se justifica su introducción en el algoritmo de diagnóstico serológico de HIV-1. Se observó también una relación directa entre la reactividad de las proteínas del WB y los resultados de IFI. En 15 muestras con resultado indeterminado por WB e inespecífico por IFI, las bandas más observadas fueron la p24 seguida de la gp160; por otro lado los anticuerpos contra las glicoproteínas virales son los que presentan mayor frecuencia en las muestras positivas débiles, demostrando su alto valor predictivo

Medicare HMOs: who joins and who leaves?

Medicare risk health maintenance organizations (HMOs) are an increasingly common alternative to fee-for-service Medicare. To date, there has been no examination of whether the HMO program is preferentially used by blacks or by persons living in lower-income areas or whether race and income are associated with reversing Medicare HMO selection. This question is important because evidence suggests that these beneficiaries receive poorer care under the fee-for-service-system than do whites and persons from wealthier areas. Medicare enrollment data from South Florida were examined for 1990 to 1993. Four overlapping groups of enrollees were examined: all age-eligible (age 65 and over) beneficiaries in 1990; all age-eligible beneficiaries in 1993; all age-eligible beneficiaries residing in South Florida during the period 1990 to 1993; and all beneficiaries who became age-eligible for Medicare benefits between 1990 and 1993. The associations between race or income and choice of Medicare option were examined by logistic regression. The association between the demographic characteristics and time staying with a particular option was examined with Kaplan-Meier methods and Cox Proportional Hazards modeling. Enrollment in Medicare risk HMOs steadily increased over the 4-year study period. In the overall Medicare population, the following statistically significant patterns of enrollment in Medicare HMOs were seen: enrollment of blacks was two times higher than that of non-blacks; enrollment decreased with age; and enrollment decreased as income level increased. For the newly eligible population, initial selection of Medicare option was strongly linked to income; race effects were weak but statistically significant. The data for disenrollment from an HMO revealed a similar demographic pattern. At 6 months, higher percentages of blacks, older beneficiaries (older than 85), and individuals from the lowest income area (less than $15,000 per year) had disenrolled. A small percentage of beneficiaries moved between HMOs and FFS plans multiple times. These data on Medicare HMO populations in South Florida, an area with a high concentration of elderly individuals and with one of the highest HMO enrollment rates in the country, indicate that enrollment into and disenrollment from Medicare risk HMOs are associated with certain demographic characteristics, specifically, black race or residence in a low-income area.

Seroreactivity to HIV-1 V3 subtypes A to H peptides of Argentinian HIV-positive sera.

Serologic assays could be useful for determining circulating subtypes in different geographic regions. A total of 175 serum samples from the same number of Argentinian HIV-infected patients from Buenos Aires and Rosario were tested against a panel of peptides representing V3 consensus subtypes A through H. A V3 peptide enzyme immunoassay was used for screening the sera. Most sera were reactive with peptides representing subtypes B (58.28%), F (13.14%), and A (8.57%). Cross-reactivity between the remainder of the peptides was observed. Genotypes of eight patients from Rosario were determined and compared with serotyping. Results showed that seven of eight genotyped patients reacted with their respective consensus B peptide and one reacted with consensus B and F. V3 peptide serology proved to be useful for determining HIV-1 clades circulating in Argentina.

PIP: 175 serum samples were collected from 175 HIV-infected Argentineans in Buenos Aires and Rosario during 1987-95, for testing against a panel of peptides representing V3 consensus HIV-1 subtypes A through H. A V3 peptide enzyme immunoassay was used to screen the sera. 58.28% of the sera were infected with HIV-1 subtype B, 13.14% with subtype F, 8.57% with subtype A, 4% with subtype H, 2.85% with subtype D, 2.28% with subtype G, and 1.71% with subtype C. Some cross-reactivity between peptides was observed. Peripheral blood mononuclear cells (PBMCs) were obtained from 8 HIV-infected subjects from Rosario for use in determining genotypes. 7 of the 8 genotyped patients reacted with their respective consensus B peptide and 1 reacted with consensus B and F. V3 peptide serology proved useful in determining which HIV-1 clades are circulating in Argentina.

The Medicare-HMO revolving door--the healthy go in and the sick go out.

BACKGROUND: Enrollment in Medicare health maintenance organizations (HMOs) is encouraged because of the expectation that HMOs can help slow the growth of Medicare costs. However, Medicare HMOs, which are paid 95 percent of average yearly fee-for-service Medicare expenditures, are increasingly believed to benefit from the selective enrollment of healthier Medicare recipients. Furthermore, whether sicker patients are more likely to disenroll from Medicare HMOs, thus raising average fee-for-service costs, is not clear. METHODS: We used Medicare enrollment and inpatient billing records for southern Florida from 1990 through 1993 to examine differences in the use of inpatient medical services by 375,406 beneficiaries in the Medicare fee-for-service system, 48,380 HMO enrollees before enrollment, and 23,870 HMO enrollees after disenrollment. We also determined whether these differences were related to demographic characteristics and whether the pattern of use after disenrollment persisted over time. RESULTS: The rate of use of inpatient services in the HMO-enrollment group during the year before enrollment was 66 percent of the rate in the fee-for-service group, whereas the rate in the HMO-disenrollment group after disenrollment was 180 percent of that in the fee-for-service group. Beneficiaries who disenrolled from HMOs re-enrolled at about the time that their level of use dropped to that in the fee-for-service group. CONCLUSIONS: These data show marked selection biases with respect to HMO enrollment and disenrollment. These biases undermine the effectiveness of the Medicare managed-care system and highlight the need for longitudinal and population-based studies.