Rapid Semiautomated Screening and Processing of Urine Specimens

A rapid urine culture procedure was evaluated in which positive urines were detected by using light-scatter photometry (Autobac). Specimens were analyzed at 3, 5, and 6 h. Specimens detected as positive at 3 h were then further evaluated by a direct 3-h susceptibility procedure (Autobac) and by a 4-h identification procedure (Micro-ID). Of 949 specimens, 175 had >10⁵ colony-forming units per ml by colony count. Of these latter specimens, 75.4% had been detected by 3 h, and 95.4% were detected by 6 h. Of specimens positive by Autobac at 3 h, 96% (95.7%) had >10⁵ colony-forming units per ml. If pure by Gram stain, those positive specimens were inoculated to direct susceptibility and identification systems. When direct Autobac susceptibilities were compared with the standard Autobac method done from the plate the following day, discrepancy rates were 1.3% very major, 2.1% major, and 7.4% total. The direct identifications were 94% (94.2%) correct when using the Micro-ID manual and a collection of octal patterns unique to this system, in which urine/broth culture inoculum was employed instead of the usual organism colony suspension. Those urine specimens negative after screening at 3 h were evaluated at 5 and 6 h, and an additional 126 specimens were detected as positive. These were then processed by routine plate inoculation, due to the limitations of the work day. By 6 h, 95.4% of specimens with >10⁵ colony-forming units per ml were detected. The 4.6% false-negative results consisted of patients on antibiotics, or slowly growing bacteria suspected of being distal urethral contaminants. Thus, 83.5% of the urine cultures received by 9:00 a.m. (10.6% 3-h positives and 72.9% negative at 6 h) could be evaluated and reported within one 8-h work day.     

Isolation of low-frequency class-switch variants from rat hybrid myelomas

Class-switch variants have been isolated from rat-rat hybrid myelomas by sib selection using a simple assay based on red cell-labelled antiglobulins. The variants detected are consistent with the gene order deduced from molecular cloning. They appear to arise spontaneously at a rate approximately ten-fold lower than for mouse cell lines but the rate of switching back to the parental isotype is substantial in comparison. An IgG2b variant antibody having the same specificity as CAMPATH-1 for human lymphocytes and monocytes is active in antibody-dependent cell-mediated killing (unlike the parental IgG2a) and may prove to be a valuable therapeutic antibody for immunosuppression and treatment of leukaemia and lymphoma.     

Effect of HLA class I or class II incompatibility in pediatric marrow transplantation from unrelated and related donors

The degree of histoincompatibility that can be tolerated, and the relative importance of matching at individual HLA class I and class II locus in bone marrow transplantation (BMT) has not been established. We hypothesized that matching for HLA-DR may not be more important than matching for HLA-A or HLA-B in selection of a donor for successful BMT. We retrospectively analyzed the outcomes of 248 consecutive pediatric patients who received allogeneic BMT from related donors (RD, n = 119) or unrelated donors (URD, n = 129). HLA-A and HLA-B were serologically matched, and HLA-DRB1 were identical by DNA typing in 69% of donor-recipient pairs. Most patients (89%) had hematologic malignancies; the rest had aplastic anemia or a congenital disorder. One HLA-A antigen mismatch was associated with a decrease in survival (p = 0.003) and a delay in granulocyte engraftment (p = 0.02) in recipients of RD marrow; as well as a decrease in survival (p = 0.02) and the development of severe acute graft-versus-host disease (GVHD) (p = 0.03) in recipients of URD marrow. One HLA-B antigen mismatch was associated with a decrease in the survival (p = 0.05) and the development of severe GVHD (p = 0.0007) in recipients of RD marrow. One HLA-DRB1 allele mismatch was associated only with a decrease in the survival (p = 0.0003) of recipients of RD marrow. Results of this study suggest that disparity in HLA-A and HLA-B antigens may not be better tolerated than disparity in HLA-DR allele in allogeneic BMT. Further studies are warranted to confirm our results.     

Incidence and outcome of adenovirus disease in transplant recipients after reduced-intensity conditioning with alemtuzumab.

Adenoviruses are emerging as a major cause of infectious complications after allogeneic transplantation. We evaluated the incidence and outcome of symptomatic adenovirus infection or adenovirus disease after alemtuzumab-based reduced-intensity conditioning in 86 consecutive patients. The overall probability of adenovirus disease was 18.4% (11/86 patients). Five patients died of progressive adenovirus disease, and this was the most important infectious cause of mortality in this cohort. The probability of nonrelapse mortality was 49% in patients with adenovirus disease compared with 25.5% in those without (P=.007). The severity of lymphocytopenia and continuation of immunosuppressive therapy were the most important risk factors for progressive adenovirus disease and death. In contrast, patients who were not receiving immunosuppressive therapy or had had it reduced or withdrawn cleared the virus. We also detected a correlation between the lack of preemptive anti-cytomegalovirus (CMV) therapy for CMV reactivation and the risk of progressive adenovirus disease (P=.05). Our findings highlight the emergence of adenovirus as an important posttransplantation pathogen even after reduced-intensity conditioning and demonstrate the effect of the severity of lymphocytopenia, anti-CMV prophylaxis, and immunosuppressive therapy on the outcome of adenovirus disease.     

Detergent-resistant membrane microdomains facilitate Ib oligomer formation and biological activity of Clostridium perfringens iota-toxin

Clostridium perfringens iota-toxin consists of two separate proteins identified as a cell binding protein, iota b (Ib), which forms high-molecular-weight complexes on cells generating Na(+)/K(+)-permeable pores through which iota a (Ia), an ADP-ribosyltransferase, presumably enters the cytosol. Identity of the cell receptor and membrane domains involved in Ib binding, oligomer formation, and internalization is currently unknown. In this study, Vero (toxin-sensitive) and MRC-5 (toxin-resistant) cells were incubated with Ib, after which detergent-resistant membrane microdomains (DRMs) were extracted with cold Triton X-100. Western blotting revealed that Ib oligomers localized in DRMs extracted from Vero, but not MRC-5, cells while monomeric Ib was detected in the detergent-soluble fractions of both cell types. The Ib protoxin, previously shown to bind Vero cells but not form oligomers or induce cytotoxicity, was detected only in the soluble fractions. Vero cells pretreated with phosphatidylinositol-specific phospholipase C before addition of Ib indicated that glycosylphosphatidyl inositol-anchored proteins were minimally involved in Ib binding or oligomer formation. While pretreatment of Vero cells with filipin (which sequesters cholesterol) had no effect, methyl-beta-cyclodextrin (which extracts cholesterol) reduced Ib binding and oligomer formation and delayed iota-toxin cytotoxicity. These studies showed that iota-toxin exploits DRMs for oligomer formation to intoxicate cells.     

Local bioactive tumour necrosis factor (TNF) in corneal allotransplantation

The aim of this study was to examine the kinetic profile of bioactive TNF levels in aqueous humour of rabbit eyes undergoing corneal allograft rejection and to investigate the effect of locally blocking TNF activity after corneal transplantation. In a rabbit corneal transplantation, endothelial allograft rejection was identified and correlated with increase in central graft thickness. Samples of aqueous humour obtained on alternate days following transplantation were tested for TNF mRNA and bioactive TNF protein. To investigate the effect of locally blocking TNF activity in allograft recipients, the fusion protein TNFR-Ig was administered by injections into the anterior chamber after transplantation. Pulsatile increases in levels of this cytokine were found in 14 of 15 allograft recipients. Peaks of TNF bioactivity preceded by varying intervals the observed onset of rejection in allograft recipients. TNF levels were not elevated in aqueous humour from corneal autograft recipient controls or in serum of allografted animals. mRNA levels were elevated before onset of and during clinically observed allograft rejection. In three of seven animals receiving TNFR-Ig injections on alternate days from day 8 to day 16 post-transplant, clear prolongation of corneal allograft survival was demonstrated. Bioactive TNF is present in aqueous humour following rabbit corneal allotransplantation. Rather than correlating directly with endothelial rejection onset, pulsatile peak levels of TNF precede and follow the observed onset of endothelial rejection. Blockade of TNF activity prolongs corneal allograft survival in some animals, indicating that this cytokine may be a suitable target in local therapy of corneal allograft rejection.     

Shigella Infection of Henle Intestinal Epithelial Cells: Role of the Host Cell

The process of Henle 407 embryonic intestinal epithelial cell infection by Shigella flexneri 2a M42-43 was studied in an in vitro model system. The role of the Henle cell was assessed. It was established that entry of S. flexneri into cells was suppressed by reagents which inhibit uptake of particles by phagocytic cells. The compounds tested included cytochalasin B, dibutyryl-cyclic adenosine monophosphate, choleragen (Vibrio cholera enterotoxin), iodoacetate, and dinitrophenol. Cytochalasin B inhibited infection at concentrations of 1.0 mug/ml or greater. Dibutyryl-cyclic adenosine monophosphate at concentrations of 1 mM and choleragen at 0.1 mug/ml caused significant suppression of infection. Iodoacetate or dinitrophenol, at 0.1 mM concentrations, inhibited internalization of virulent shigellae, and a combination of these compounds inhibited infection at 0.01 mM concentrations. Preincubation of Henle cell monolayers with the combination of iodoacetate and dinitrophenol (0.05 mM) also inhibited infection markedly. The data suggest that infection of epithelial cells by S. flexneri in vitro is accomplished by an endocytic process induced by virulent bacteria. The process appears to be similar to uptake of particles by phagocytic cells. Ultrastructural analysis by transmission electron microscopy provided corroborative evidence of phagocytosis of shigellae by Henle cells in that intracellular bacteria were often observed within membrane-limiting vacuoles resembling phagosomes.