Qualitative and quantitative detection of cytomegalovirus DNA in sera by PCR as a clinical marker

The amount of human cytomegalovirus (CMV) DNA in sera is considered to be a direct marker for CMV infection. We established conditions for nested PCR that detected one copy of CMV DNA, and for competitive PCR, which detected five or more copies of CMV DNA quantitatively. We tested 50 microl each of 16 freeze-stored and 5 fresh sera from patients, for CMV DNA. In sera obtained from the same patient at different time points, small amounts of CMV DNA were detected before the onset of CMV pneumonia. In sera from certain CMV-infected patients who were treated with the anti-CMV agent, ganciclovir, CMV DNA was not detected. Quantitative PCR detection of CMV DNA seems to be suitable for predicting early recurrent CMV infection and monitoring the efficacy of antiviral therapy. The qualitative nested PCR examination of CMV DNA in 40 cord blood plasma samples was carried out for the purpose of preventing CMV infection by cord blood stem cell transplantation, and they were all negative for CMV DNA.     

Cross-clade p17 peptide recognition by antisera to HGP-30, a 30-amino acid synthetic peptide antigen from HIV type 1 p17

HGP-30, a 30-amino acid synthetic peptide analog of HIV-1SF2 p17 (aa 86-115), was used to immunize both mice and humans. Since the amino acid sequence of HGP-30 is relatively conserved among different HIV-1 strains and clades, experiments were carried out to determine if antisera obtained by immunizing animals and humans can recognize HGP-30-related peptide consensus sequences belonging to different clades. Results show that antisera from mice immunized with HGP-30 can recognize clade B and C and to a lesser degree clade A and E consensus sequences of HIV-1, in addition to recognizing HGP-30 sequence. The cross-clade recognition was higher in mouse sera obtained on day 42 than on day 14 or 28. MPL/SE and Novasomes were better adjuvants than alum in inducing antibodies that showed cross-clade recognition and IgG2a and IgG2b antibody isotypes. Similar cross-clade recognition was observed in several sera from humans immunized with an HGP-30/KLH/alum formulation. The human sera from HGP-30-immunized subjects evaluated for cross-clade recognition of HGP-30 peptides were from subjects whose cells showed significant protection from HIV infection on virus challenge in the hu-PBL-SCID mouse model. These studies suggest that HGP-30 may be useful as a candidate vaccine antigen for populations in countries with prevalence of different HIV clades.     

Absence of nonpercutaneous transmission of hepatitis C virus in a colony of chimpanzees

Transmission of hepatitis C virus (HCV) was studied in a colony of 85 chimpanzees using assays for anti-HCV and HCV-RNA. Thirteen of the 85 sera were positive for anti-HCV, and 12 of the 13 were also positive for HCV-RNA. All of the anti-HCV positive sera except one were obtained from chimpanzees which had been inoculated with non-A, non-B hepatitis virus. On the other hand, only one of 63 sera of chimpanzees without history of experimental infection of the virus was positive for anti-HCV. Transmission to this chimpanzee was thought to be a needle contaminated with HCV. All 39 samples of chimpanzees born in the center were negative for both anti-HCV and HCV-RNA. Sixteen of their mothers had undergone experimental infection, and 6 of them were positive for both anti-HCV and HCV-RNA. These results suggest that nonpercutaneous transmission, including sexual and mother-to-infant transmissions, is not an important mode of transmission. If these findings apply to humans, definition of inapparent sources of the infection is needed.     

Epidural spinal infection. Variability of clinical and magnetic resonance imaging findings

STUDY DESIGN: This study evaluates the magnetic resonance characteristics of spinal epidural abscesses and their associated disc space infections. OBJECTIVES: The results were correlated with history, clinical, and laboratory findings to provide guidelines for early and appropriate diagnosis of epidural spinal infections. SUMMARY OF BACKGROUND DATA: Imaging signs of spinal infections have been reported before, but not with special attention to early clinical and imaging findings. METHODS: Thirteen patients (10 men, 3 women; age range, 32-64 years) with progressive sensorimotor deficit were studied. All patients had a neurologic examination after admission and a magnetic resonance imaging scan done within the first 48 hours. In all cases, T1-weighted images before and after administration of gadolinium were obtained. T2-weighted images were acquired in eight cases as well. Ten patients subsequently underwent open surgery; in three cases, a percutaneous biopsy and drainage was performed. RESULTS: Cervical discitis was found in five patients, and thoracic discitis was seen in another five cases. Three patients had an epidural infection without a concomitant discitis. Neurologic and clinical findings varied considerably. Despite clinical signs of spinal cord involvement, a spinal cord lesion was demonstrated only once. Signal change in T2-weighted images may be the first sign of disc space infection. Because a neurologic deficit may occur before any change is visible, follow-up examinations may be required if epidural infection is suspected on clinical grounds. CONCLUSIONS: Magnetic resonance imaging is the appropriate method for diagnostic work-up of progressive neurologic deficit resulting from epidural infection.     

Antibodies in human sera specific to hypervariable region 1 of hepatitis C virus can block viral attachment

It has been postulated that antibodies specific to the hypervariable region 1 (HVR1) within the putative envelop protein E2 of hepatitis C virus (HCV) can neutralize virus. We studied such antibodies in sera of patients who were infected in a single-source outbreak by a contaminated anti-D immunoglobulin preparation (HCV-AD78). The nucleotide sequences of cDNAs encoding HVR1 of HCV-AD78 were determined. The four major variants (HVR1.A, B, C, and D) were expressed as fusion proteins in Escherichia coli. Sixty-seven percent of sera contained antibodies to HVR1.A. Sera unrelated to infection of the outbreak also recognized HVR1.A but to a lesser extent (15%), suggesting that not all HVR1-specific antibodies are absolutely isolate-specific. Antibodies directed against individual variants of HVR1 were found in sera obtained early postinfection (p.i.) (< or = 1 year) but also in sera obtained several years later. An in vitro binding assay of HCV to tissue culture cells was employed to further characterize these sera. Five of seven sera that were obtained early p.i. prevented binding of HCV to cells. Preincubation of such sera with HVR1-specific fusion proteins restored binding of HCV to cells in four of five sera. These findings suggest that the majority of neutralizing antibodies are directed against HVR1.     

Unitas, Libertas, Caritas: the meaning of science for general practice

OBJECTIVE: Fear of surgery affects recovery in the emotional and behavioral state that leads the child to call the nurse more often or to ingest more sedatives after the operation. The present work evaluated the effects of psychological preparation for surgery during the pre-surgical period on the post-surgical recovery during the hospital stay in order to evaluate the effects that this preparation had on the recovery of the child. PATIENTS AND METHODS: The sample was composed of 60 pediatric patients of both sexes between 7 and 14 years of age admitted for minor scheduled surgery. Fear and five indicators of recovery were evaluated during the post-surgical period (call to the nurse, ingestion of sedatives and liquids, miction and sleep). The children were assigned randomly to one of the following four groups: filmed modeling, coping skills, filmed modeling plus coping skills, and control. RESULTS: The results indicated that the prepared children showed less fear, called the nurses less often, were administered fewer doses of sedatives and slept better after the operation. Analysis of the size of the effect showed that the programs that included coping skills training obtained the most benefits in the post-surgical recovery.     

Testing cord blood human chorionic gonadotropin as a surrogate marker for early identification of human immunodeficiency virus-1 infection in children

We measured human chorionic gonadotropin (hCG) in cord sera of 22 infants born to women infected with the human immunodeficiency virus-1 (HIV-1). hCG was also determined in cord sera from 173 infants born at a suburban hospital to HIV-1-seronegative women. The findings indicate that 16 (9%) of 173 HIV-1-seronegative samples had hCG levels greater than 90 IU/L (values were distributed as a Poisson curve). In contrast, 8 (36%) of the 22 infants born to HIV-1-infected women had hCG levels in excess of 90 IU/L, and 7 (88%) of these were shown to be HIV-infected. The remaining 14 infants born to HIV-1-infected women had low hCG levels, and 3 (21%) of the 14 had HIV infection. Mean follow-up time for HIV-uninfected infants was 17.5 months (range 9 months to 3 years). A statistically significant association between maternal-fetal HIV-1 transmission and hCG levels > or = 90 IU/L in cord sera was observed (p = 0.02). The difference between CD4 counts among mothers who transmitted HIV and those who did not was also statistically significant (p = 0.025). On the basis of this study's findings, we propose that cord blood hCG may serve as a surrogate marker for HIV-1 infection. Testing hCG levels in cord sera is an inexpensive and readily available screening test for early identification of infants at increased risk for getting HIV-1 from their mothers.     

Absence of pericentromeric heterochromatin (9qh-) in a patient with bilateral retinoblastoma

The diagnosis of HIV infection is based on screening of HIV antibodies and confirmed by a more specific supplementary test. The most common confirmation test is Western blot, which is expensive, time consuming and subject to technical skill. The present study was carried out to evaluate whether the anti-HIV-1 antibody titer is valid as a supplementary test for diagnosis of HIV-1 infection. Anti-HIV-1 antibody titers of 2,414 anti-HIV-1 positive sera determined by the particle agglutination (PA) method were analysed in comparison with the Western blot analysis. The Western blot negative result was found in 11 of 2,414 (0.46%) anti-HIV-1 positive sera, these sera also gave negative anti-HIV by ELISA. The PA titers of these sera were found in the range of 16 to 64. Seventeen samples (0.70%) with anti-HIV-1 in the titer range of 16 to 256 showed indeterminate Western blot analysis. The rest, 2,386 of these 2,414 sera (98.84%), were shown to be positive by Western blot. However, all of the 2,356 sera with antibody titers > or = 512 (97.6%) demonstrated positive Western blot results. Five cases among the 17 (29.4%) indeterminate sera were examples of early seroconversion of HIV infection, which were confirmed in follow up specimens. The results suggest that only the samples with antibody titers < 512 are required to be confirmed for HIV infection by Western blot. It is possible that early seroconversion may be inferred from anti-HIV titers. Therefore, in order to reduce time and cost, the PA anti-HIV titer can be used as an alternative supplementary test for diagnosis of HIV-1 infection in most positive screened anti-HIV samples. Western blot is needed for testing in only a few cases.     

Radioimmunoassay detects the frequent occurrence of autoantibodies to the Mr 65,000 isoform of glutamic acid decarboxylase in Japanese insulin-dependent diabetes

Glutamic acid decarboxylase antibodies (GAD65Ab) are common in new onset Caucasian insulin-dependent diabetic (IDDM) patients but it is unclear if this marker is also prevalent in patients of other ethnic backgrounds. We determined antibodies against human recombinant GAD in Japanese diabetic patients using a radioimmunoassay with competition between in vitro translated 35S-GAD65 and non-labelled recombinant human GAD65 (rhGAD65). GAD67 antibodies (GAD67Ab) were similarly analyzed but without antigen competition. In 73 Japanese diabetic patients, GAD65Ab were found in 11/16 (69%) of patients with short-duration (less than 5 yrs) IDDM, 6/23 (26%) with long-duration (5 or more yrs) IDDM and 10/20 (50%) with slowly progressive diabetes. High GAD65Ab levels were associated with concomitant autoimmune diseases (p = 0.021). GAD67Ab were found in 4/16 (25%) of patients with short-duration IDDM, 3/23 (13%) with long-duration IDDM and 2/20 (10%) with slowly progressive diabetes. In 14 non-insulin dependent diabetic (NIDDM) patients, GAD65Ab and GAD67Ab were not found (0/14) and 1/50 (2%) healthy controls were positive in either assay. Among the GAD67Ab-positive samples, 8/9 (88%) were also high level GAD65Ab positive, 7/9 (77%) were displaced by an excess of rhGAD65 and the antibody levels correlated (r2 = 0.573; p = 0.003). Our data are consistent with a strong association of GAD65Ab also in Japanese IDDM, and suggest that, when present, GAD67Ab are frequently directed to epitope(s) common to GAD65 and GAD67.     

A descriptive study of U.S. OSHA penalties and inspection frequency for musculoskeletal disorders in the workplace

In an attempt to examine in vivo the early metabolic consequences of severe acute head injury, 1H MRS was performed in four patients from 8 to 25 h (mean 15 h) following trauma. In three of these patients, decompressive surgery was performed 4-5 h prior to the MRS. High levels of lactate (area of lactate peak >50% of the mean areas of the NAA, choline-containing, and creatine-containing compound peaks) were found at 8 h posttrauma in the one patient who was not operated on and at 10 h posttrauma in one of the patients who underwent surgery. In the other two postoperative patients, at 18 and 25 h after trauma, lactate levels were found to be low (lactate peak <20% of the mean area of the other three peaks). In the one patient who had a follow-up at 6 days and who had the largest initial lactate levels, these remained high. These findings suggest that high levels of lactate may not be an inevitable consequence of severe head injury and that similar MRS studies should be performed on each individual patient before therapies to reduce lactate are considered. There appeared to be no correlation between the relative amounts of lactate and outcome.     

Fc receptors are not required for antibody-mediated protection against lethal malaria challenge in a mouse model

The mechanisms by which Abs mediate protection during blood-stage malaria infections is controversial, with some evidence pointing to the direct effect of Abs on parasite invasion and growth, while other studies suggest that Abs act in cooperation with monocytes to achieve parasite inhibition. To determine whether the effector phase of protection in vivo to the rodent parasite Plasmodium yoelii yoelii requires Fc receptor bearing cells, we passively transferred immune sera into FcR gamma-chain knockout mice. Inflammatory macrophages from these knockout mice were unable to mediate phagocytosis or Ab-dependent cell-mediated cytotoxicity (ADCC) through Fc gamma RI, Fc gamma RII, or Fc gamma RIII. Passive transfer of either P. y. yoelii hyperimmune sera or anti-GST-PYC2 sera directed to the major merozoite surface protein (MSP-1) of this parasite enabled both BALB/cByJ mice and FcR gamma-chain-deficient mice to resist lethal P. y. yoelii 17XL (Py17XL) challenge. mAb302, a protective IgG3 Ab, also passively protected both strains of mice. Most of these samples contain Ab isotypes that would not be able to protect mice if their protective effects required Ab-dependent cell-mediated cytotoxicity. These results establish that, in this infection, protection is directly mediated by Abs and does not require the participation of Fc receptors.     

Observations on the relationship between verbal explicit and implicit memory and density of neurons in the hippocampus

The relationship between neuronal density and verbal memory in left and right hippocampal subfields was investigated in patients who underwent surgery for alleviation of temporal lobe epilepsy. The surgery consisted of unilateral partial removal of the hippocampus along with the anterior temporal lobe and amygdala. Study 1 looked at post-surgical explicit vs implicit verbal memory for lists of words while Study 2 looked at pre- and post-surgical explicit memory for word pairs. Left subfield CA1 appeared to be the most consistently involved in explicit and implicit memory. The results of the two studies confirm presence of hemispheric asymmetry in verbal memory. The notion that hippocampal control of memory is most apparent in post-surgical performance is discussed.     

Early development and progression of lymphocyte-stimulatory cross-reactive idiotypes expressed on antibodies to soluble egg antigens during Schistosoma mansoni infection of mice

Idiotypes (Id) that stimulate immunoregulatory anti-Id T lymphocyte proliferation are expressed on murine and human antibodies (Ab) to soluble egg antigens (SEA) of Schistosoma mansoni. Kinetics of early expression of these stimulatory Id have now been studied using immunoaffinity-purified serum anti-SEA Ab from mice infected with S. mansoni for 6, 7, 8, 12, or 16 weeks. Rabbit anti-Id Ab specific for mouse anti-SEA Id expressed at 8 weeks post-infection (anti-8WkId) demonstrated the strongest interactions with Id present at 7 and 8 weeks post-infection by competitive enzyme-linked immunosorbent assay. Anti-8WkId Ab reacted progressively less well with 12 WkId, 6WkId, and 16WkId. Splenocytes from mice infected for 8 weeks demonstrated the highest blast transformation responses in vitro to anti-SEA Id from mice infected for 6 weeks, while 7, 8, 12, and 16 weeks post-infection Id preparations stimulated progressively less proliferation. These data indicate that although eventual Id-associated immunoregulatory events contribute to chronicity in this disease, production of anti-SEA Ab that express stimulatory cross-reactive immunoregulatory Id comprises a substantial portion of the initial, acute anti-SEA response in mice infected with Schistosoma mansoni. Furthermore, either this particular Id-expressing response is not maintained, or its proportional presence is greatly diminished by the cumulative production of other multiple anti-SEA Ab during the establishment of chronicity, perhaps in response to its immunoregulatory influence very early in infection.     

Human diploid cell culture rabies vaccine (HDCV) and purified chick embryo cell culture rabies vaccine (PCECV) both confer protective immunity against infection with the silver-haired bat rabies virus strain (SHBRV)

The demonstration of extensive differences in the antigenic makeups of the silver-haired bat rabies virus (SHBRV) and canine rabies virus (COSRV) strains raised concerns as to whether current licensed rabies vaccines are sufficiently protective against SHBRV. NIH mouse protection test results show that both the human diploid cell culture rabies vaccine (HDCV) and the purified chicken embryo cell rabies vaccine (PCECV) protected against lethal infection with SHBRV as well as the canine rabies strain COSRV. However, in this investigation, the potencies of both vaccines in mice were found to be significantly higher for COSRV than for SHBRV. The in vivo protection data are confirmed by in vitro virus neutralizing antibody (VNA) test results which demonstrate that mice immunized with HDCV or PCECV develop significantly higher VNA titres against COSRV than against SHBRV. In contrast, VNA tests of sera from individuals immunized with HDCV or PCECV showed that humans, as opposed to mice, develop significantly higher VNA titres against SHBRV than against COSRV. These data suggest that HDCV and PCECV will protect humans against infection with the silver-haired but rabies virus strain in addition to canine rabies virus strains.     

Evaluation of butanone, carbon dioxide, and 1-octen-3-OL as attractants for mosquitoes associated with north central Florida bay and cypress swamps

In the sera of (NZB x NZW)F1 (B/W) mice that develop a lupus-like syndrome, increased levels of IgG antibodies (Ab) reacting with TNP have been detected before the appearance of IgG anti-DNA Ab and clinical symptoms. A single injection of trinitrophenyl-bovine serum albumin (TNP/BSA) in physiological saline into a young B/W mouse (3 months old), followed by fusion of its splenocytes 3 days later, gave rise to hybridomas simultaneously secreting IgM and IgG Ab with anti-TNP reactivity. Both mu and gamma chains were detected in culture supernatants by ELISA, and double isotype-producing cells were labeled by immunofluorescence. Molecular analysis of two of these double isotype-producing hybridomas showed the presence of mRNA coding for both mu and gamma chains of Ig, and this gamma mRNA could be translated in vitro into a gamma heavy (H) chain. Comparison of the H chain variable-region sequences of IgM and IgG revealed 100% homology between mu and gamma V(H) genes in one clone, while mu and gamma V(H) genes showed only 80% homology in the other clone. Both clones produced a single kappa light (L) chain. These two hybridomas, isolated from a B/W mouse, thus represent two different mechanisms of double isotype expression: the first one corresponds to an IgM to IgG switch, while the second one reflects a lack of allelic exclusion.     

IgD-receptor up-regulation on human peripheral blood T cells in response to IgD in vitro or antigen in vivo correlates with the antibody response to influenza vaccination

Aged individuals (more than 65 years) were classified as antibody (Ab) responders on the basis that they showed increases to more than or = 1:40 in serum Ab titers to all influenza virus strains present in the trivalent influenza vaccine within 4 weeks after immunization. The peripheral blood mononuclear cells (PBMC) from pre-immunization samples of blood taken from seven Ab-responders and seven Ab-nonresponders were examined for their ability to exhibit up-regulation of IgD-receptor (IgD-R) after exposure for 2 h to immobilized cross-linked IgD, as shown by rosetting with IgD-coated ox erythrocytes. The responsiveness to IgD was found to be predictive of the ability to produce Ab responses to viral protein Ag: the IgD-R up-regulation was greater than 5% in all Ab-responders and less than 4% in all the Ab-nonresponders. In addition, there was an excellent correlation between mean Ab titers (to the three viruses in sera collected 4 weeks after immunization) and the percentage of IgD-R+ cells obtained in response to IgD in PBMC from the same individual prior to immunization: p = 0.894. Injection of influenza vaccine itself also induced IgD-R on PBMC in vivo. The percentage of IgD-R+ cells peaked after 24 h, was still detectable above background by day 7 or 14, and returned to pre-injection levels by day 28 in young subjects and aged Ab-responders, but not in Ab-nonresponders. Similarly, purified peripheral blood T cells obtained from aged Ab-responders exhibited IgD-R upon immunization in vivo. These findings suggest that Ag injection causes rapid up-regulation of IgD-R by cross-linking IgD in humans as well as in mice as shown previously. In analogy with results in mice, the present data are consistent with a role for IgD-R+ T cells in the humoral response in man. Proliferative responses to influenza proteins in peripheral blood T cells from vaccinated individuals were found to peak on day 7 and were higher in Ab-responders than in Ab-nonresponders.     

Dorothy and The Wizard. Intergenerational issues in mental health and aging

Among patients undergoing maintenance hemodialysis, a high prevalence of hepatitis C virus (HCV) infection can be observed. In a prospective study, sera of 273 patients were examined for the presence of HCV infection by serological tests and by PCR. Thirty-five patients (12.8%) were HCV antibody positive, and in 31 of them HCV RNA could be detected by PCR. Among the 238 seronegative patients HCV infection was detected in 12 cases (5.0%) exclusively by PCR. Only in 1 of these patients seroconversion could be observed within the 18-month follow-up period. These findings demonstrate that in hemodialysis patients PCR is necessary for the diagnosis of HCV infection.     

Detection of the 70-kilodalton histoplasma capsulatum antigen in serum of histoplasmosis patients: correlation between antigenemia and therapy during follow-up

Histoplasmosis is an important systemic fungal infection, particularly among immunocompromised individuals, who may develop a progressive disseminated form which is often fatal if it is untreated. In such patients, the detection of antibody responses for both diagnosis and follow-up may be of limited use, whereas the detection of Histoplasma capsulatum var. capsulatum antigens may provide a more practical approach. We have recently described an inhibition enzyme-linked immunosorbent assay (ELISA) for the detection in patients' sera of a 69- to 70-kDa H. capsulatum var. capsulatum-specific antigen which appears to be useful in diagnosis. To investigate its potential for the follow-up of histoplasmosis patients during treatment, antigen titers in the sera of 16 patients presenting with different clinical forms of histoplasmosis were monitored at regular intervals for up to 80 weeks. Sera from four of five patients with the acute form of the disease showed rapid falls in antigenemia, becoming antigen negative by week 14 (range, weeks 10 to 16). Sera from four patients with disseminated histoplasmosis showed falls in antigen levels; three of them became antigen negative by week 32; the fourth patient became negative by week 48. In contrast, antigen titers in four of six AIDS patients with the disseminated form of the disease remained positive throughout follow-up. Sera from only one patient who presented with the chronic form of the disease were analyzed, and this individual's serum became antigen negative by week 9. The inhibition ELISA is shown to be of particular use in the monitoring of non-AIDS patients with the acute and disseminated forms of the disease and may complement existing means of follow-up.     

High prevalence of antibodies to glutamic acid decarboxylase in comparison to islet cell antibodies in patients with long-standing insulin-dependent diabetes mellitus

To elucidate the clinical significance of antibodies to glutamic acid decarboxylase (GAD Ab) compared to islet cell antibodies (ICA) in recent-onset and long-standing insulin-dependent diabetes mellitus (IDDM). We examined GAD Ab and ICA in 29 recent-onset and 85 long-standing patients with IDDM. GAD Ab was detected by a radioimmunoassay kit using purified pig brain GAD as an antigen. The prevalence of GAD Ab in the recent-onset diabetic patients was 55.2%, slightly lower than that of ICA (65.5%). In contrast, the prevalence of GAD Ab in long-standing diabetic patients was 42.4%, which was significantly higher than that of ICA (23.5%) (p < 0.01). GAD Ab were consistently detected in approximately 40% of patients with long-standing disease, while ICA decreased according to duration of disease. The GAD Ab titer in ICA-positive patients (mean +/- SD, 1588.2 +/- 6755.1; range, 6-38574) was significantly higher than that in ICA-negative patients (mean +/- SD, 13.4 +/- 17.9; and range, 5-72 units) (p < 0.001). These findings suggest that GAD Ab are more useful than ICA to know participation of immune disorders in long-standing patients with IDDM.