A microELISA system for the detection of both anti-HIV-1 and anti-HIV-2 antibodies.

Current anti-HIV-1 screening tests combine an excellent anti-HIV-1 sensitivity with a sensitivity of only 28-93% for anti-HIV-2 positive plasma or serum samples. The reactivity of anti-HIV-2 sera in anti-HIV-1 screening tests is based mainly on the immunological cross-reactivity of the GAG and POL proteins of HIV-1 and HIV-2. We describe here a sandwich immunoassay, in which HIV-1 viral lysate is combined with an HIV-2 ENV synthetic peptide, corresponding to the immunodominant envelope epitope, as the coating antigens on microELISA plates. This immunoassay has a sensitivity of 100% for anti-HIV-1 (128 sera tested) and 100% for anti-HIV-2 (109 sera tested). Assay specificity with fresh human donor sera was 99.9% (2256 sera tested).

Potent antiretroviral therapy initiates normalization of hypergammaglobulinemia and a decline in HIV type 1-specific antibody responses.

Next to a profound T cell immunodeficiency, HIV-1 infection induces activation and dysfunction of B cells, resulting in hypergammaglobulinemia. Whereas T cell immune reconstitution with potent antiretroviral therapy has been extensively documented, limited data are available on B cell immune reconstitution. We studied the effect of potent antiretroviral therapy on antibody titers to the viral proteins gp120 and p24 and on total IgG concentrations. Three retrospectively chosen groups were studied: a successfully treated group, untreated controls, and subjects with virological failure after several months of successful therapy. In the successfully treated group, the median total IgG concentrations normalized, whereas they remained elevated in the untreated group and rebounded after an initial decline in the therapy failure group. The HIV-1-specific antibody titers declined in the successfully treated group and followed the rebound of the HIV RNA levels in the therapy failure group. With potent antiretroviral therapy the hypergammaglobulinemia normalized whereas HIV-1-specific immune responses were weakened. The weakening of antiviral immunity with therapy may be relevant for current attempts to gain immunological control over the virus through structured treatment interruptions or therapeutic vaccinations.

Sertraline response in adults with mental retardation and autistic disorder.

BACKGROUND: Self-injury and aggression are common reasons for urgent psychiatric referral of persons with mental retardation and autistic spectrum disorders. Although the treatment prescribed for these problems has traditionally been neuroleptic medication, serotonin reuptake inhibitors such as sertraline may result in significant clinical improvement as well as fewer side effects. METHOD: The authors administered sertraline in an open trial to nine consecutively admitted adult mentally retarded outpatients presenting with target behaviors of self-injury and/or aggression. Most patients (N = 6) were mildly or moderately mentally retarded by DSM-III-R criteria; five had comorbid autistic disorder. Prescribed dosages ranged from 25 mg to 150 mg daily, based on observed clinical responses. Clinical Global Impressions (CGI) ratings were made at baseline and again after sertraline treatment for at least 28 days. RESULTS: Sertraline led to improvement in CGI ratings of overall clinical severity in eight of nine subjects; mean +/- SD improvement in CGI ratings was 2.44 points +/- 1.67. Discontinuation of the treatment was necessary in only one patient, after 18 weeks of sertraline treatment, because of agitation and worsening of self-picking. Side effects were otherwise minimal. CONCLUSION: These findings from a clinical sample suggest that sertraline is promising in the treatment of self-injury and aggression. Double-blind controlled studies of sertraline and other serotonin reuptake inhibitors in the treatment of self-injury and aggression in patients with mental retardation and with autistic disorder are warranted.

Characterization of DS-antigen, an antigen related to non-A, non-B hepatitis. I. Physico-chemical properties.

An enzyme-linked immunosorbent assay for an antigen (termed DS-Ag) related to non-A, non-B hepatitis has recently been developed in our laboratory. The DS-Ag was derived from a proven infectious serum obtained from a patient with haemophilia and it was also detected in the acute phase serum of an experimentally infected chimpanzee. The DS-Ag has now been shown to have a buoyant density in CsCl of 1.32 g/cm2 and a sedimentation coefficient of 20 S. The mean molecular weight, determined by gel chromatography on 4% agarose columns, was found to be 0.9 x 10(6). The DS-Ag recovered from acute-phase chimpanzee serum has identical characteristics. The DS-Ag is stable for 1 h at 56 degrees C, at pH 3, in high concentration of CsCl or Nal and in 20% ether. Complete inactivation occurred after 1 min at 98 degrees C, at pH 2, in chloroform (1:1 mixture), in 1 ppm chlorine and in formalin (1:4000, 72 h 37 degrees C). Our studies showed that DS-Ag bears no resemblance to particles related to well-documented forms of viral hepatitis types A and B or other known pathogens. The DS-Ag is a complex substance which forms an antigenic entity related to non-A, non-B hepatitis.

New developments in ELISA verification of anti-HIV screening of blood donors.

First generation ELISA screening assays for antibodies to HTLV-III (HIV) generated between 0.1 and 1.0% false positive results. Western blot analysis in specialized reference centers is almost uniformly used as a method to confirm the specificity of the ELISA results. Yet, the high cost, time delay and lack of standardization in these systems cause a growing demand for tests that can be performed on site and that can at least reduce the number of sera that have to be sent to reference centers. Such tests thus should primarily be aimed at the detection of false positive results. Ancillary to the Vironostika anti-HTLV-III screening test, we developed a set of reagents (VERIFY) which can be used for the verification of initially or repeatedly positive screening results. The test employs a reagent specifically blocking true HTLV-III-anti HTLV-III reactions, a reagent blocking HLA-anti HLA reactions and a control reagent. Use of this test may reduce the number of sera found false positive by reference methods by more than 90%. The introduction of improved versions and second generation screening assays obviously will reduce the number of false positive results. Yet the significant results of this verification assay and the ease with which it can be integrated in the screening procedures will make it a valuable tool in the blood bank screening program.

Preliminary results of transmission experiments of non-A, non-B hepatitis by mononuclear leucocytes from a chronic patient.

Mononuclear leucocytes (mainly lymphocytes), isolated by Ficoll-Paque gradient centrifugation of blood freshly drawn from a haemophilia A patient with chronic non-A, non-B hepatitis (NANB), caused NANB when infused in a susceptible chimpanzee. Plasma from the same patient also transmitted the disease, as witnessed by elevated levels of liver enzymes in serum, histopathological signs of viral hepatitis and submicroscopic cytoplasmic alterations in the hepatocytes, considered to be specific for NANB in chimpanzees. In contrast, neither lymphocytes nor plasma from a chimpanzee apparently fully recovered from two episodes of NANB had the same effect.

Weight gain in a controlled study of risperidone in children, adolescents and adults with mental retardation and autism.

As part of an ongoing, prospective, ABA design, double-blind crossover study of risperidone versus placebo for the treatment of aggressive, destructive and self-injurious behavior in persons aged 6-65 years with mental retardation (MR) and autism, we measured the weight of 19 subjects at each study visit. We compared mean weight gain during the 16-week acute phase and 24-week open maintenance phase with that during the initial and middle placebo phases statistically, using a linear mixed model procedure. Results of the linear mixed model analysis showed that relative weight gain observed during the acute and maintenance drug phases was significantly greater than that observed during the initial and middle placebo phases respectively (p = .0001 and p = .0001). Over approximately a year, children aged 8-12 (n = 5) gained a mean of 8.2 kg (range = 2.7-17.7 kg); adolescents (n = 6) aged 13-16 gained a mean of 8.4 kg (range 3.6-15.5 kg); adults aged 21-51 (n = 8) gained a mean of 5.4 kg (range 0-9.5 kg). Weight gain observed in this controlled study of risperidone treatment in children, adolescents, and adults with MR and autism was significant. It may be greater in this population than in others reported and in this study was not limited to an acute effect only. Rate of weight gain diminished rapidly on tapering and stopping the drug. Further studies are urgently needed, including those incorporating diet and exercise programming.

Effects of risperidone on aberrant behavior of persons with developmental disabilities: I. A double-blind crossover study using multiple measures.

The efficacy of the atypical antipsychotic risperidone was evaluated in the treatment of aberrant behavior (e.g., aggression, self-injury) in 20 individuals with developmental disabilities. A double-blind, crossover design was used to compare risperidone with placebo in a 22-week trial with a 6-month follow-up phase. Based on a 50% reduction in mean Aberrant Behavior Checklist--Community total scores, 50% of the participants were identified as responders. Naturalistic observations of a subset of five individuals showed that for 4 out of 5 participants, risperidone was effective in reducing aberrant behavior. Side effects included weight gain (84% of participants) and sedation (40% of participants). The advantages of conducting a comprehensive analysis of the effects of medication on aberrant behavior are discussed.

SPECT brain imaging abnormalities in attention deficit hyperactivity disorder.

SPECT using N-Isopropyl I-123 IMP was performed, as part of a neuropsychiatric evaluation, on 10 patients with the DSM III-R diagnosis of Attention Deficit Hyperactivity Disorder (ADHD) and 6 patients from a non-ADHD mixed psychiatric group used as controls for comparison. Mean regional I-123 IMP SPECT region of interest (ROI) count ratios (left to right) demonstrated that the ADHD patients had greater overall hemispheric I-123 IMP uptake asymmetry with less activity in the left frontal and left parietal regions in comparison to control patients. Both groups demonstrated similar I-123 IMP uptake asymmetry in the temporal regions. These findings are consistent with previous studies of brain physiology in ADHD implicating regional cortical perfusion and metabolism abnormalities in areas which are involved in the control of attentional processes.

A new HBsAg screening assay designed for sensitive detection of HBsAg subtypes and variants.

The design of a new HBsAg screening assay, the Hepanostika HBsAg Ultra is based on the use of monoclonal antibodies raised against native wild-type HBsAg and reactive with HBsAg in which the common 'a'-determinant is modified by site-directed mutagenesis of four of the cysteine moieties. The design was checked using the same cysteine variants and samples from patients known to be infected with HBsAg variants. The results found were compared with other state-of-the-art commercial screening assays. The design of the Hepanostika HBsAg Ultra enabled detection of all variant HBsAg-positive samples in contrast to the other commercial assays. An additional 980 samples were tested to assess the specificity and sensitivity of the Hepanostika HBsAg Ultra. Screening of presumed negative serum and plasma samples resulted in a specificity of 100%. This makes the Hepanostika HBsAg Ultra the first screening assay with a design able to detect HBsAg variants with high sensitivity and specificity.

Non-A, non-B hepatitis: an update.

The definition of non-A, non-B hepatitis (NANB) is improved by further characterization of what it is not (like the delta agent or non-A epidemic hepatitis) rather than by providing convincing evidence of isolation of the agent responsible for blood transfusion- or blood product-related NANB or specific markers thereof. Yet, NANB research is in disquieting movement. Modern biotechnology yielded its blessings to the field. However, monoclonal antibodies and molecular probes will have to be evaluated with the same scrutiny that unmasked so many test systems and viral agents thus far. Recent victims appear to be published reports on NANB being identified as a retroviral agent and NANB virus being propagated in primary cultures of chimpanzee hepatocytes. Yet the application of these powerful new tools, together with the availability of cultured human and chimpanzee hepatocytes for propagation of the agent may improve the chances for substantial progress. Our finding of involvement of lymphocytes in transmission of the disease may add another approach to reach the ultimate goal of characterization of the causative agent and development of diagnostic methods to detect it in patients and biological materials derived from carriers of the disease.

Self-injurious behavior and serotonin in Prader-Willi syndrome.

Low central nervous system (CNS) serotonin levels have been associated with impulsive, aggressive and self-injurious behavior (SIB). Persons with Prader-Willi Syndrome (PWS) often engage in self-injury by severe compulsive skin picking and gouging and often manifest compulsive eating, hoarding, and explosive outbursts. Some of the compulsive behaviors seen in patients with obsessive-compulsive disorder (OCD) bear similarity to behaviors associated with PWS: Skin picking, trichotillomania, and onychophagia (nail biting). There is abundant evidence that selective serotonin reuptake inhibitors (SSRIs) are effective in treating OCD. Three cases are described in which persons with PWS responded favorably to SSRI treatment. Two persons showed a significant decrease in skin picking. The third case showed a significant decrease in hoarding and explosive outbursts. Strategies are discussed for investigating the possibility of a shared neurochemical basis for the self-injurious, aggressive, and compulsive behaviors in persons with PWS. PWS may provide a relatively homogenous model for the study of skin picking and explosive outbursts among other populations.

Nucleosomal fragments in serum may directly reflect cell-mediated cytotoxic activity in vivo.

In in vitro experiments the activity of cytotoxic T-cells and natural killer cells has been shown to cause nuclear DNA fragmentation leading to the release of nucleosomal multimers from the target cells. These multimers form a ladder-like pattern with a periodicity of approximately 200 bp during gel electrophoresis. The objective of the present study was to show the relevance of the presence of these nucleosomal multimers in vivo during diseases that show cell-mediated cytotoxicity. Nucleosomal multimers (n greater than 5) could be detected using nick translation followed by electrophoresis in a series of sera of a chimpanzee infected with hepatitis A virus and in sera drawn from several hepatitis B patients. The multimers were present during periods expected to show an increased activity of cell-mediated cytotoxicity in the liver. During these periods the injury of the liver cells was also mirrored by the classical parameter, the release of a specific liver enzyme into the serum. The liver enzyme activity in the serum and the detection of the nucleosomal multimers did not completely overlap, however. It is postulated that the proposed nick translation assay is useful as a simple diagnostic test for cell-mediated cytotoxicity since it reflects this activity under different in vivo situations.

An enzyme-linked immunosorbent assay for the detection of hepatitis non-A, non-B antigen and antibody.

An enzyme-linked immunosorbent assay (ELISA) was developed, based on sera from patients convalescent from non-A, non-B hepatitis and haemophilia A patients. Using this ELISA an antigen (DS-Ag) was detected which appears to be related to non-A, non-B hepatitis. The serum of another patient with haemophilia A, which was strongly positive for the DS-Ag, caused non-A, non-B hepatitis in a chimpanzee. DS-Ag could be detected in the serum of the chimpanzee during the acute phase of the infection. The ELISA for DS-Ag did not react with either hepatitis A or B virus antigens, or with Epstein-Barr virus or Cytomegalovirus. The assay was provisionally evaluated using sera from different groups of patients. From 17 patients with post-transfusion hepatitis non-A, non-B, 11 were found positive in the ELISA for DS-Ag (65%). A relatively high prevalence of DS-Ag (17%) and -antibodies (20%) was found in patients with haemophilia, who are regularly treated with blood clotting factor concentrates. The DS-Ag was found in 8 of 1400 (0,6%) volunteer blood donors, and antibody to DS-Ag in 3% of the sera. Remarkably, a high incidence (41%) of antibodies to DS-Ag was found in prostitutes, suggesting that this antigen may be transmitted by a sexual route as well. The DS-Ag was pelleted by ultracentrifugation for 4 h at 100.000 xg and was found to have a buoyant density of 1.32 g/cm3 in a CsCl gradient.

An enzyme-linked immunosorbent assay for an antigen related to non-A, non-B hepatitis and its antibody: partial characterization of the antigen and chimpanzee transmission.

An enzyme-linked immunosorbent assay (ELISA) was developed based on sera from patients convalescent from non-A, non-B hepatitis and haemophilia A patients who had been frequently treated with commercial blood products. Using this ELISA, an antigen was detected which appears to be related to non-A, non-B hepatitis. The antigen is provisionally designated as DS-antigen (DS-Ag). The serum of another patient with haemophilia A, which was strongly positive for the DS-Ag, caused a typical case of non-A, non-B hepatitis in a chimpanzee. DS-Ag could be detected in the serum of the chimpanzee during the acute phase of the infection. The ELISA for DS-Ag reacted with neither hepatitis A or B virus antigens, nor Epstein-Barr virus or cytomegalovirus. The assay was provisionally evaluated using sera from different groups of patients. Out of 17 patients with posttransfusion hepatitis non-A, non-B, 11 were found positive in the ELISA for DS-Ag (65%). As expected, a relatively high prevalence of DS-Ag (9%) was found in patients with haemophilia, who are regularly treated with blood-clotting factor-concentrates. Antibodies to DS-Ag were found in 48% of these patients. The DS-Ag was found in 8 of 1400 (0.6%) volunteer blood donors, and antibody to DS-Ag in 3% of the sera. Remarkably, a high incidence (41%) of antibodies to DS-Ag was found in prostitutes, suggesting that this antigen may be transmitted by a sexual route. The DS-Ag was pelleted by ultracentrifugation for four hours at 100,000g and was found to have a buoyant density of 1.32 g/cm3 in a CsCl gradient.

The effect of storage of whole blood on the association of factor VIII-related antigen and factor VIII-coagulant antigen.

To evaluate the extent of denaturation of factor VIII-coagulant activity (VIII: C) during production of factor VIII concentrates, the factor VIII-coagulant antigen (VIII: CAg)/VIII: C ratio was measured in plasma, cryoprecipitate and cryosupernatant from fresh and stored blood. This ratio was close to unity for both cryoprecipitate and other concentrates, suggesting that VIII: CAg is lost concurrently with VIII: C during cryoprecipitation and further fractionation. Storage of blood (18 h, 22 degrees C) before processing resulted in a 30% loss of VIII: C from the separated plasma; however, VIII: CAg was not affected. In cryoprecipitate prepared from this plasma, VIII: C and VIII: CAg both were 30% lower than when prepared from fresh plasma. In the corresponding cryosupernatant, however, more VIII: CAg but less VIII: C was present compared with fresh material. Gel chromatography revealed that the rise of VIII: CAg in cryosupernatant prepared from stored blood, was due to an increased amount of VIII: CAg of low molecular weight, not being associated with factor VIII-related antigen. Such an increase in dissociated VIII: CAg was not detected in the plasma prior to cryoprecipitation. It is concluded that during storage of blood, molecular changes are induced in the factor VIII-VWF complex, possibly by limited proteolysis, which make the complex more liable to dissociation during subsequent cryoprecipitation.

Using the Diagnostic Assessment of the Severely Handicapped-II (DASH-II) to measure the therapeutic effects of risperidone.

BACKGROUND: Within the scope of a double-blind, placebo-controlled, crossover medication study, the Diagnostic Assessment of the Severely Handicapped-II (DASH-II) was evaluated as a measurement for determining the effectiveness of the medication risperidone in treating the problem behaviour of 21 people with intellectual disabilities (ID). METHOD: Participants' caregivers completed the DASH-II during the placebo/baseline phase of the study and the maintenance phase of the study, and completed the Aberrant Behavior Checklist - Community (ABC-C) weekly throughout the entire study. The results obtained using the DASH-II were compared to those obtained using the ABC-C, an instrument shown to be well correlated with the DASH-II. RESULTS: Results suggest that while the DASH-II and the ABC-C were well correlated during the placebo/baseline phase of the current study, they were not well correlated at completion of the 6-month maintenance phase of the medication trial. CONCLUSION: The DASH-II, while appropriate for assisting in the diagnosis of psychopathology in people with ID, does not appear to monitor changes in problem behaviour as a result of risperidone use as well as the ABC-C. Differences in the frequency of problem behaviour that each measure evaluated and the applicability of using the DASH-II to measure medication effects on problem behaviour are discussed.

Inhibition of human hemopoiesis by non-A, non-B hepatitis virus.

All etiologies of acute viral hepatitis are associated with a transient suppression of hemopoiesis and, rarely, with the development of aplastic anemia. Both hepatitis A and hepatitis B viruses directly inhibit the growth and differentiation of human bone marrow progenitor cells in vitro. We now report a similar effect of a non-A, non-B (NANB) hepatitis agent on human bone marrow progenitor cells. Three chimpanzees were inoculated with a putative NANB agent. Coded sera were blindly evaluated for their ability to affect human bone marrow colony formation in vitro. Sera obtained during the acute phase of NANB hepatitis inhibited the in vitro growth of human erythroid (CFU-E, BFU-E) and granulocyte-macrophage (CFU-GM) progenitor cells, compared with sera obtained before inoculation. Sera obtained after remission of both the biochemical and histological hepatitis and sera obtained from a chimpanzee who underwent biochemical but not histological remission did not inhibit the stem cell assays as much as the acute phase sera. These results suggest an approach to identifying the viremic phase of NANB hepatitis. Inhibition of human bone marrow proliferation appears to be a common property of all known hepatitis viruses.

Contributions to the optimal use of human blood. VIII. Stability of blood coagulation factor VII during collection and storage of whole blood and plasma.

Investigations were performed concerning the influence of the pH on the stability of factor VIII during the collection of blood and during the storage of blood and plasma for varying periods and at varying temperatures. It was found that the low pH of ACD anticoagulant solution (pH 4.9) caused a loss of factor VIII procoagulant activity of 10-15% during the collection of blood. However, when less acidic anticoagulant solutions were used, substantial losses of factor VIII occurred during the storage of blood. We concluded that the optimal pH of both the anticoagulant solution and the stored blood, should be between 6.7 and 7.0. However, no anticoagulant solution is known that meets these requirements. In practice ACD ensures the highest recovery of factor VIII in cryoprecipitate, at least in those cases where the blood donations are stored for several hours before separation and freezing of the plasma.