Reduction of the Carbapenemase Inactivation Method (CIM) assay time by real-time PCR.

Carbapenemase Inactivation Method (CIM) is a test to detect presence of the carbapenemase in Gram-negative bacteria. Determination of the carbapenemase production by inactivation of meropenem requires that a zone of control E. coli inhibition be measured approximately 6-24 h after plating. We have modified the CIM test by developing a rapid method which instead measures the growth of E. coli indicator strain ATCC 25922 using real-time PCR, referred to as a nucleic acid testing CIM (natCIM). Our natCIM, therefore reduces the detecting time from 6 to 24 h to approximately 4 h.

Additive effects of the dopamine D2 receptor and dopamine transporter genes on the error-related negativity in young children.

The error-related negativity (ERN) is a negative deflection in the event-related potential that occurs approximately 50 ms following the commission of an error at fronto-central electrode sites. Previous models suggest dopamine plays a role in the generation of the ERN. We recorded event-related potentials (ERPs) while 279 children aged 5-7 years completed a simple Go/No-Go task; the ERN was examined in relation to the dopamine D2 receptor (DRD2) and dopamine transporter (DAT1) genes. Results suggest an additive effect of the DRD2 and DAT1 genotype on ERN magnitude such that children with at least one DRD2 A1 allele and children with at least one DAT1 9 allele have an increased (i.e. more negative) ERN. These results provide further support for the involvement of dopamine in the generation of the ERN.

Multicenter evaluation of the Clostridium difficile TOX A/B TEST.

Clostridium difficile, the primary cause of nosocomial diarrhea in the United States and many other industrialized countries, is recognized as a major health concern because of its ability to cause severe intestinal disease leading to complications such as relapses and infections due to vancomycin-resistant enterococci. The disease results from two toxins, toxins A and B, produced by this pathogen. In this study, we evaluated the TOX A/B TEST, a new 1-h enzyme immunoassay (EIA) that detects toxins A and B. We compared the test with the tissue culture assay, which is recognized as the "gold standard" for C. difficile testing. Evaluations were performed in-house at TechLab, Inc. (Blacksburg, Va.) and off-site at four clinical laboratories. Of 1,152 specimens tested, 165 were positive by the TOX A/B TEST and tissue culture and 973 were negative by both tests. The sensitivity and specificity were 92.2 and 100%, respectively. The positive and negative predictive values were 100 and 98.6%, respectively, and the correlation of the TOX A/B TEST with tissue culture was 98.8%. When discrepant samples were resolved by culture, the sensitivity and specificity were 93.2 and 98.9%, respectively. The positive and negative predictive values were 100 and 98.8%, respectively, with a correlation of 99.0%. There were no specimens that were positive by the TOX A/B TEST and negative by tissue culture. Fourteen specimens were negative by the TOX A/B TEST but positive by tissue culture. Of these, two were negative by toxigenic culture, five were positive by toxigenic culture, and seven were not available for further testing. There were no indeterminate results, since the test does not have an indeterminant zone. In a separate study, 102 specimens that were positive by tissue culture and the TOX A/B TEST were examined in toxin A-specific EIAs. Two specimens that presumptively contained toxin A-negative, toxin B-positive (toxA-/toxB+) isolates were identified. One specimen was from a patient with a clinical history consistent with C. difficile infection. Isolates obtained from these specimens by selective culture on solid media and in broth tested toxA-/toxB+ when grown in brain heart infusion dialysis flasks, which stimulate in vitro production of both toxins. Our findings show that the TOX A/B TEST is suitable as a diagnostic aid for C. difficile disease because it correlates well with tissue culture and detects isolates that may be missed with toxin A-specific EIAs.

Anti-HIV type 1 cytotoxic T lymphocyte effector activity and disease progression in the first 8 years of HIV type 1 infection of homosexual men.

Cytotoxic T lymphocytes (CTL) may play an important role in host defense against HIV-1 infection. In this study, we examined the responses of circulating effector CTL (CTLe) specific for Gag, Pol, Env, and Tat in 57 HIV-1-infected men, 49 of whom were asymptomatic and had documented time since seroconversion of < 8 years. CTLe responses to at least one of the four HIV-1 gene products were detected in 83% of the subjects. The magnitude and prevalence of the anti-Tat responses were significantly less than the responses to Gag, Pol, and Env. Cell depletion studies indicated that the lytic activity against the HIV-1 structural proteins was mediated by CD8+ T cells, although 30% of Env-specific lysis was mediated by CD16+ natural killer cells. Anti-HIV-1 CTLe responses against Gag and Pol were significantly less in subjects infected for over 6 years as compared to those infected for shorter periods of time. We found no correlation, however, between anti-HIV-1 CTLe responses and either CD4+ or CD8+ T cell counts, rates of CD4+ T cell loss, HIV-1 infectious viral load, use of antiviral medications, or subsequent progression to AIDS. Our results indicate that anti-HIV-1 CTLe activity is relatively stable in asymptomatic subjects infected < 6 years, and is not an early marker for risk of disease progression.

Effects of adoptive immunotherapy with autologous CD8+ T lymphocytes on immunologic parameters: lymphocyte subsets and cytotoxic activity.

CD8+ cytotoxic T lymphocytes (CTL) may be an important parameter of host resistance to HIV infection. The present study determined whether CD8+ cells could be purified and propagated in vitro to enhance anti-HIV CTL activity, and the immunologic effects of infusion of these cells into autologous, HIV-infected patients as a potential immunotherapy for AIDS and AIDS-related complex (ARC). CD8+ lymphocytes from five AIDS and ARC patients were purified from leukapheresis preparations in cell culture flasks coated with CD8-specific monoclonal antibodies and propagated in vitro for 3 weeks. The ex vivo propagated cells were 98% (+/- 1%) CD8+ and 43% (+/- 6%) HLA-DR+. The majority of the CD8+ cell preparations had increased lytic activity against autologous B lymphoblastoid cells infected with vaccinia virus vectors expressing HIV-IIIb structural proteins gag, pol, or env, relative to that of fresh blood mononuclear cells tested prior to purification and culture. The results also show for the first time that CD8+ CTL from HIV-infected patients can lyse cells expressing the HIV regulatory protein, tat. Enhanced expression of CD56 (natural killer cell marker) and lytic activity against vaccinia virus control vector-infected, autologous targets were also noted in the CD8+ cell preparations. Infusion of the CD8+ CTL into autologous patients was well-tolerated and resulted in low but discernible, temporal increases in circulating cytotoxic activity against the HIV gene-expressing targets.

Generation and characterization of ex vivo propagated autologous CD8+ cells used for adoptive immunotherapy of patients infected with human immunodeficiency virus.

Cytolytic T lymphocytes play an important role in host defense against viral infections, including human immunodeficiency virus (HIV). In a phase I clinical trial (protocol 080 of the AIDS Clinical Trials Group), generation of CD8+ effector cells from peripheral blood of patients with acquired immunodeficiency syndrome (AIDS)-related complex (ARC) or AIDS and safety of autologous adoptive transfer of these cells were evaluated. For therapeutic infusions, CD8+ T cells were purified by positive selection on anti-CD8 monoclonal antibody-coated flasks from leukapheresed peripheral blood of seven patients. These CD8+ T cells were cultured in the presence of interleukin-2 and phytohemagglutinin for up to 3 weeks to obtain cells sufficient for therapeutic infusions (10(8) to 10(10)). All 31 cell cultures established from the seven patients and used for therapy were highly enriched in CD8+ (mean, 97%), CD8+HLA-DR+ (50%), cytotoxic CD8+CD11b- (82%), and memory CD29+ (78%) T lymphocytes. In vitro expanded CD8+ cells had excellent cytotoxic function at the time they were used for therapy, including HIV-specific activity against autologous targets infected with vaccinia vectors expressing HIV-IIIb antigens, gag, pol, and env. Anti-HIV activity of cultured CD8+ cells was significantly higher than that of autologous fresh peripheral blood lymphocytes. Our results show that CD8+ T lymphocytes obtained from peripheral blood of symptomatic HIV-infected patients can be purified, cultured to obtain large numbers of cells with enhanced anti-HIV activity, and safely infused into patients with AIDS as a form of immunotherapy.

Cell-mediated immunity and immunosuppression in herpes simplex virus infection.

Innate and adaptive forms of cellular immunity have important, interactive roles in host resistance to herpes simplex virus (HSV) infection. Hence, suppression of non-HSV specific and anti-HSV specific cellular immune responses can predispose the host to severe HSV infection. Studies using depletion and adoptive transfer of selected subpopulations of NK cells, macrophages, and CD4+ and CD8+ T lymphocytes indicate that each of these is of significance in protection against infection with HSV. Further evidence suggests that cytokines such as interferons alpha and gamma, interleukin 2 and leukocyte migration inhibition factor also have central roles in these cell functions during HSV infection. Of importance is that HSV itself can result in transient suppression of several innate and adaptive cellular immune responses during acute episodes of infection in normal adults. Mechanisms by which HSV may mediate this immune dysfunction include enhanced activity of suppressor T cells and soluble suppressor factors, decreases in cytokine production, decreases in expression of major and minor histocompatibility antigens and direct inhibition of cytotoxic effector cell function. Knowledge of anti-HSV cellular immunity and of the immunosuppressive properties of HSV are of importance in the development of appropriate treatment and vaccine strategies for this herpesvirus.

Repeated dose administration of desmopressin acetate in uncomplicated cardiac surgery: a prospective, blinded, randomized study.

The effects of single or repeated doses of desmopressin on blood loss were examined in uncomplicated cardiac surgery, while assessing the potential for thrombogenic side effects. Seventy patients undergoing elective coronary artery bypass grafting (CABG) were studied. Patients were randomized into three blinded groups: Group I received DDAVP (0.3 micrograms/kg), IV, after cardiopulmonary bypass (CPB) and 12 hours later in the Intensive Care Unit (ICU); Group II, DDAVP (0.3 micrograms/kg), IV, after termination of CPB and saline (placebo) 12 hours later in the ICU; Group III, saline (placebo) IV after CPB and 12 hours later in the ICU. Blood loss and bleeding time decreased for Group I at 24 hours (P < 0.04) when compared to Group III; however, blood product replacement, as well as intraoperative and total blood loss at 36 hours, were not different among treatment and control groups. There were four myocardial infarctions recorded in Group I, two in Group II, and one in Group III. These differences were not found to be statistically significant. It is concluded that in routine CABG the prophylactic use of single or repeat dose DDAVP does not effectively decrease blood loss or blood product replacement.

HLA-restricted lysis of herpes simplex virus-infected monocytes and macrophages mediated by CD4+ and CD8+ T lymphocytes.

Freshly isolated human peripheral blood monocytes and in vitro monocyte-derived macrophages were infected with HSV type 1 and used as target cells in a cell-mediated cytotoxicity assay. PBMC from both HSV-immune and non-immune donors were stimulated in vitro for 5 days with UV-inactivated HSV Ag and used as effector cells. Effectors from HSV-immune donors mediated virus-specific lysis of both monocyte and macrophage targets, whereas effectors from non-immune donors failed to mediate target cell lysis. Mean virus-specific lysis of autologous monocytes was (8.5 +/- (+/- 2.0)%) compared to a threefold greater virus-specific lysis of autologous macrophages (24.7 (+/- 4.3)%). More than 70% of this lysis was mediated by CD16- T lymphocytes. Further analysis demonstrated that the majority of the lysis against autologous and allogeneic targets was HLA-DR-restricted and mediated by CD4+ CTL. However, CD8+ CTL also contributed to the lysis of autologous targets as well as allogeneic targets having a common HLA-A and/or -B determinant. The HLA-restricted cytotoxicity was virus-specific as HSV-infected, but not CMV-infected, cells were lysed. CTL-mediated lysis of HSV-infected monocytes and macrophages may be of significance in the anti-viral and immunoregulatory host response.

Association of alpha interferon production with natural killer cell lysis of U937 cells infected with human immunodeficiency virus.

Mononuclear leukocytes from human immunodeficiency virus (HIV)-seronegative and -seropositive homosexual men lysed HIV-infected U937 cells to a significantly greater degree than uninfected U937 cells. Depletion of cell subsets with monoclonal antibodies and complement indicated that the effector cells were primarily of the CD16+ phenotype. Acid-stable alpha interferon (IFN-alpha) production induced by the HIV-infected cells correlated with, although was not an absolute requisite for, preferential lysis of the infected targets. The activity of these CD16+, natural killer (NK) cells decreased in relation to the duration of HIV infection and the presence of acquired immunodeficiency syndrome. Pretreatment of peripheral blood mononuclear cells from HIV-seronegative subjects, but not HIV-seropositive men, with IFN-alpha or recombinant interleukin-2 enhanced lysis of both uninfected and HIV-infected U937 cells. These results suggest that IFN-alpha-associated, NK-like mechanisms are active in the cytotoxic response against HIV-infected cells and that HIV infection results in an early and progressive depression of such responses. Prospective investigations may be useful in determining the role of this NK cell response in the natural history and pathogenesis of HIV infection and the efficacy of therapeutic modalities.

HLA-DR-restricted cytotoxicity of cytomegalovirus-infected monocytes mediated by Leu-3-positive T cells.

Mononuclear leukocytes from 14 cytomegalovirus (CMV)-seropositive and six CMV-seronegative normal healthy donors were treated with soluble CMV antigen for 5 days to generate cytotoxic T lymphocyte (CTL) activity. CMV-antigen-stimulated lymphocytes from CMV-seropositive but not CMV-seronegative donors lysed autologous peripheral blood monocyte targets infected with CMV in 13 of 14 donors (mean percentage of virus-specific lysis = 19.0 +/- 4.5%, effector to target ratio of 50:1). Freshly donated, unstimulated lymphocytes displayed little or no lysis of CMV-infected monocytes. Lysis was virus specific in that CMV-stimulated CTL did not kill herpes simplex virus-infected monocytes. The mean level of lysis of CMV-infected autologous targets was equivalent to that of HLA-DR-matched targets (20.0 +/- 8.0%), and was significantly greater than that of HLA-A/B-matched targets (6.3 +/- 2.5%, p less than 0.035) and HLA-mismatched targets (3.3 +/- 2.5%, p less than 0.01). Enrichment for T cell subsets with the use of selective depletion methods with monoclonal antibodies showed that CTL activity against autologous and HLA-DR-matched allogeneic targets was present predominantly in Leu-3-positive T lymphocytes. These results show for the first time that short term stimulation of heterogeneous lymphocytes from CMV-seropositive donors with CMV antigen can generate CMV-specific, Leu-3-positive CTL that are primarily restricted in their activity to autologous and class II, HLA-DR-matched targets. Our findings suggest a role for Leu-3-phenotypic CTL in immunity to CMV, and provide a model for analysis of this antiviral effector function during immunodeficient states.