Detection of Staphylococcal Superantigenic Toxins by a CD69-Specific Cytofluorimetric Assay Measuring T-Cell Activation

The presence of staphylococcal superantigenic toxins in the supernatants of liquid cultures was detected by an easy and rapid method assessing the activation of T lymphocytes by cytofluorimetric measurement of CD69 expression. Staphylococcus aureus cells were grown in Eagle’s minimum essential medium supplemented with 5% heat-inactivated fetal calf serum. Supernatant fluids from all S. aureus strains producing superantigen-related toxins, including enterotoxins A to E, toxic shock syndrome toxin, and exfoliative toxins A and B, induced CD69 expression in a significantly higher number of T cells than a cutoff of 2%. This CD69 assay might be used for initial detection of superantigens from S. aureus strains isolated in the context of staphylococcal toxemia or related chronic human diseases such as atopic dermatitis or Kawasaki syndrome.     

Assessment of Resolution and Intercenter Reproducibility of Results of Genotyping Staphylococcus aureus by Pulsed-Field Gel Electrophoresis of SmaI Macrorestriction Fragments: a Multicenter Study

Twenty well-characterized isolates of methicillin-resistant Staphylococcus aureus were used to study the optimal resolution and interlaboratory reproducibility of pulsed-field gel electrophoresis (PFGE) of DNA macrorestriction fragments. Five identical isolates (one PFGE type), 5 isolates that produced related PFGE subtypes, and 10 isolates with unique PFGE patterns were analyzed blindly in 12 different laboratories by in-house protocols. In several laboratories a standardized PFGE protocol with a commercial kit was applied successfully as well. Eight of the centers correctly identified the genetic homogeneity of the identical isolates by both the in-house and standard protocols. Four of 12 laboratories failed to produce interpretable data by the standardized protocol, due to technical problems (primarily plug preparation). With the five related isolates, five of eight participants identified the same subtype interrelationships with both in-house and standard protocols. However, two participants identified multiple strain types in this group or classified some of the isolates as unrelated isolates rather than as subtypes. The remaining laboratory failed to distinguish differences between some of the related isolates by utilizing both the in-house and standardized protocols. There were large differences in the relative genome lengths of the isolates as calculated on the basis of the gel pictures. By visual inspection, the numbers of restriction fragments and overall banding pattern similarity in the three groups of isolates showed interlaboratory concordance, but centralized computer analysis of data from four laboratories yielded percent similarity values of only 85% for the group of identical isolates. The differences between the data sets obtained with in-house and standardized protocols could be the experimental parameters which differed with respect to the brand of equipment used, imaging software, running time (20 to 48 h), and pulsing conditions. In conclusion, it appears that the standardization of PFGE depends on controlling a variety of experimental intricacies, as is the case with other bacterial typing procedures.The use of electric field pulsing techniques in conjunction with agarose gel electrophoresis for discrimination of large DNA molecules was introduced by Schwarz and Cantor in 1984 (9). During the past decade the methodology has been adapted and improved by various research groups to the point that pulsed-field gel electrophoresis (PFGE) for bacterial strain typing is now utilized with relative ease in a variety of laboratories (1). The combination of contour-clamped homogeneous field electrophoresis and PFGE for the molecular analysis of Staphylococcus aureus has been reported since the late 1980s (7, 19). At present, PFGE is considered to have both the reproducibility and resolving power of a standard technique for the epidemiological typing of bacterial isolates (10, 15).Molecular typing systems can identify different strains within a species, generating data useful for taxonomic or epidemiologic purposes (10, 14). A frequently observed shortcoming of typing systems in general is their lack of reproducibility: most typing systems do not provide a definitive strain identification, which is usually due to the variability of the technique and the lack of large databases containing fragment patterns from a wide variety of organisms to which unknowns can be compared. These problems were recently described in detail for two molecular typing systems. A multicenter study on random amplification of polymorphic DNA for discrimination of S. aureus strains revealed a lack of interlaboratory reproducibility among the banding patterns generated by the participating centers, although the epidemiological interpretation of the data was similar for all the centers involved (16). For PFGE, a similar lack of interlaboratory reproducibility of patterns was observed, although the interpretation of the experimental data also differed per participating center (2). The latter study analyzed 12 different methicillin-resistant S. aureus (MRSA) strains with different techniques optimized in each center and different sources and types of equipment. Since interlaboratory discrepancies with respect to classification of the strains were observed, the study concluded that there is a clear need for standardization of the technique, including the construction of a panel of reference strains to assist the individual researcher in the optimization of the PFGE protocol.The aim of the present study was to compare the fragment patterns of a well-defined collection of MRSA isolates in 12 laboratories using in-house and a standard set of PFGE parameters to determine whether standardization of experimental parameters (DNA preparation and switching protocols) would improve intercenter reproducibility of PFGE analysis.     

Bladder acellular matrix as a substrate for studying in vitro bladder smooth muscle-urothelial cell interactions

The objective of this study was to evaluate the ability of bladder acellular matrix (BAM) to support the individual and combined growth of primary porcine bladder smooth muscle (SMC) and urothelial (UEC) cells. An in vitro co-culture system was devised to evaluate the effect of UEC on (i) SMC-mediated contraction of BAM discs, and (ii) SMC invasiveness into BAM. Cells were seeded onto BAM discs under 4 different culture conditions. Constructs were incubated for 1, 7, 14 and 28 days. Samples were then harvested for evaluation of matrix contraction. Immunohistochemistry (IHC) was utilized to examine cellular organization within the samples and conditioned media supernatants analyzed for net gelatinase activity. BAM contraction was significantly increased with co-culture. The same side co-culture configuration lead to a greater reduction in surface area than opposite side co-culture. IHC revealed enhanced SMC infiltration into BAM when co-culture was utilized. A significant increase in net gelatinase activity was also observed with the co-culture configuration. Enhanced infiltration and contractile ability of bladder SMCs with UEC co-culture may, in part, be due to an increase in gelatinase activity. The influence of bladder UECs on SMC behaviour in vitro indicates that BAM may contain some key inductive factors that serve to promote important bladder cell-cell and cell-matrix interactions.     

Use of HLA marker associations and HLA haplotype linkage to estimate disease risks in families with gluten-sensitive enteropathy

Based on a two-locus, double recessive model, we derive formulas for the risks that relatives of individuals with gluten-sensitive enteropathy (GSE) will also develop the disease. The calculations take advantage of: the linkage between the HLA locus and one of the two proposed GSE loci, and the preferential association of the HLA-DR3 and DR7 alleles with the GSE disease allele that occupies the HLA-linked locus. We use Bayes' rule to quantitate the strength of the association between the GSE disease allele and the HLA marker allele. This method predicts that siblings of the proband have an overall 10% risk for GSE, which is consistent with observed family data. This predicted risk rises to 30% when siblings are HLA-identical to the proband (also consistent with observed data) or when the sibling has the DR3 allele in the HLA haplotypes not shared with the proband. In those populations where DR7 also is associated with GSE, siblings of probands have a 10% predicted risk for GSE when only one HLA haplotype is shared with the proband and DR7 is included in the unshared haplotype. Other DR alleles are associated with much lower disease risks. By separating individuals into high and low risk groups, HLA typing identifies those individuals who would benefit from further diagnostic procedures. This general strategy should be applicable to other multilocus, marker-associated diseases.     

Comparison of a Multiplexed Fluorescent Covalent Microsphere Immunoassay and an Enzyme-Linked Immunosorbent Assay for Measurement of Human Immunoglobulin G Antibodies to Anthrax Toxins

Recently, the Centers for Disease Control and Prevention reported an accurate, sensitive, specific, reproducible, and quantitative enzyme-linked immunosorbent assay (ELISA) for immunoglobulin G (IgG) antibodies to Bacillus anthracis protective antigen (PA) in human serum (C. P. Quinn, V. A. Semenova, C. M. Elie et al., Emerg. Infect. Dis. 8:1103-1110, 2002). The ELISA had a minimum detectable concentration (MDC) of 0.06 μg/ml, which, when dilution adjusted, yielded a whole-serum MDC of 3.0 μg of anti-PA IgG per ml. The reliable detection limit (RDL) was 0.09 μg/ml, while the dynamic range was 0.06 to 1.7 μg/ml. The diagnostic sensitivity of the assay was 97.6% and the diagnostic specificity was 94.2% for clinically verified cases of anthrax. A competitive inhibition anti-PA IgG ELISA was also developed to enhance the diagnostic specificity to 100%. We report a newly developed fluorescence covalent microbead immunosorbent assay (FCMIA) for B. anthracis PA which was Luminex xMap technology. The FCMIA MDC was 0.006 μg of anti-PA IgG per ml, the RDL was 0.016 μg/ml, and the whole-serum equivalent MDC was 1.5 μg/ml. The dynamic range was 0.006 to 6.8 μg/ml. Using this system, we analyzed 20 serum samples for anti-PA IgG and compared our results to those measured by ELISA in a double-masked analysis. The two methods had a high positive correlation (r² = 0.852; P < 0.001). The FCMIA appears to have benefits over the ELISA for the measurement of anti-PA IgG, including greater sensitivity and speed, enhanced dynamic range and reagent stability, the use of smaller sample volumes, and the ability to be multiplexed (measurement of more than one analyte simultaneously), as evidenced by the multiplexed measurement in the present report of anti-PA and anti-lethal factor IgG in serum from a confirmed clinical anthrax infection.     

Monoclonal small (well-differentiated) lymphocytic proliferations of the gastrointestinal tract resembling lymphoid hyperplasia: A neoplasm of uncertain malignant potential

Three histologically benign-appearing or diagnostically equivocal small lymphocytic proliferations of the gastrointestinal tract were examined by fresh-frozen section immunohistologic techniques. In one case, a dense infiltrate in the small intestine, consisting of small lymphocytes with round nuclei, was limited almost entirely to the mucosa. In another case, a localized colonic polyp was formed by mucosal and submucosal lobules of benign-appearing lymphoid aggregates with centrally located germinal centers. The third case, a penetrating gastric ulcer, was surrounded by histologically hyperplastic lymphoid tissue which included germinal centers. The small lymphocytes in all three cases were strongly positive for B-cell-associated antigens (B1, B2, BA-1), and all exhibited monoclonal light-chain restriction. Even though treatment consisted only of surgical resection of the lesions, no patient has had progressive disease during follow-up periods ranging from 24 to more than 50 months. We believe that the infiltrates in these cases are analogous to the morphologically benign monoclonal small lymphocytic proliferations common to the lung and orbit and that they have an uncertain, but probably low, malignant potential.     

Elimination of B-lineage leukemia and lymphoma cells from bone marrow grafts using anti-B4-blocked-ricin immunotoxin

Bone marrow is the primary site of disease in patients with acute lymphoblastic leukemia (ALL) and is frequently involved in patients with non-Hodgkin's lymphoma (NHL). At the time of autologous bone marrow transplantation, marrow grafts from patients with leukemia and lymphoma are often still contaminated by malignant cells, even when such patients achieve complete clinical remission. In this study, we evaluated the potential of anti-B4-blocked-ricin (anti-B4-bR) immunotoxin to eliminate residual ALL and NHL cells from bone marrow. Anti-B4-bR binds to the CD19 antigen, which is B-lineage specific, and, at concentrations of 5×10^–9 M or greater, could eliminate more than 3 logs of CD19+ Nalm-6 or Namalwa cells in a 20-fold excess of normal irradiated bone marrow after only 5 hr of incubation. This activity was abrogated by the addition of anti-B4 but not by the presence of galactose, which is the natural ligand for native ricin. Also, when used at these high concentrations, anti-B4-bR showed little nonspecific toxicity against normal hematopoietic progenitors. In conclusion, a single short exposure to anti-B4-bR is capable of inducing high levels of depletion of CD19+ leukemia and lymphoma cells without significant nonspecific toxicity against normal marrow progenitors. Therefore, anti-B4-bR offers an interesting approach to the elimination of B-lineage malignant cells prior to autologous bone marrow transplantation.     

Breast cancer risk associated with ovulation-stimulating drugs

BACKGROUND: Despite the recognized role of hormones in the aetiology of breast cancer, there has been little evaluation of hormonal preparations used to treat infertility. METHODS: A retrospective cohort study of 12,193 women evaluated for infertility between 1965 and 1988 at five clinical sites identified 292 in situ and invasive breast cancers in follow-up through 1999. Standardized incidence ratios (SIRs) compared breast cancer risks with those of the general population. Analyses within the cohort estimated rate ratios (RRs) associated with medications after adjustment for other breast cancer predictors. RESULTS: Infertile patients had a significantly higher breast cancer risk than the general population [SIR = 1.29, 95% confidence interval (CI) 1.1-1.4]. Analyses within the cohort showed adjusted RRs of 1.02 for clomiphene citrate and 1.07 for gonadotrophins, and no substantial relationships to dosage or cycles of use. Slight and non-significant elevations in risk were seen for both drugs after > or = 20 years of follow-up (RRs = 1.39 for clomiphene and 1.54 for gonadotrophins). However, the risk associated with clomiphene for invasive breast cancers was statistically significant (RR = 1.60, 95% CI 1.0-2.5). CONCLUSIONS: Although there was no overall increase in breast cancer risk associated with use of ovulation-stimulating drugs, long-term effects should continue to be monitored.     

The tunel assay in the diagnosis of graft-versus-host disease: caveats for interpretation

Acute graft-versus-host disease (GVHD) is a significant cause of morbidity and mortality following bone marrow transplantation, and early detection is important to allow effective therapy. Since the presence of apoptotic keratinocytes (dyskeratotic bodies) has been suggested as a useful diagnostic criterion for GVHD, attention has focused on the use of the TUNEL assay to detect apoptosis in clinical specimens. We reviewed clinical specimens upon which TUNEL had been performed for possible artifacts that might interfere with accurate evaluation for GVHD. Several distinct types of artifact were found and could be re-created in experimental systems. Artifacts in TUNEL staining generally resulted from the lack of specificity of this reaction for apoptotic cell death. Artifacts were found resulting from inadequate fixation, over-exposure of the TUNEL reaction, and proximity to the section edge. In addition, a novel artifact, apparently resulting from DNA shearing during the sectioning process, was noted and confirmed using confocal microscopy of experimental specimens. The TUNEL assay must therefore must be interpreted with caution in the clinical setting. In our laboratory, we consider TUNEL-positive cells as apoptotic only when accompanied by apoptotic morphology. Although these criteria clearly miss some cells in the early stages of apoptosis, they provide the highest specificity for apoptotic cell death.     

Characterization of somatic cell hybrids exhibiting extinction of AFP,Albumin and an AFP-HPRT Transgene

We utilized an AFP-HPRT transgene, i.e. the HPRT coding sequences under the regulation of AFP enhancer and promoter sequences, to localize the AFP extinguisher locus in intertypic somatic cell hybrids (hepatoma X fibroblast). This hybrid gene construct, which directly links AFP regulation to a reversibly selective gene, enabled the selection of stably transfected cells which express AFP, as well as cells showing extinction of AFP. Mouse hepatoma cells stably transfected with and expressing the transgene were fused to human fibroblasts, and the resulting somatic cell hybrids were characterized using Southern, Northern and karyotypic analyses. That several hybrids exhibited the proper extinction of AFP, AFP-HPRT and albumin suggests coregulation of these genes by an extinguisher. Segregant lines derived from these hybrids were selected for the loss of extinguisher activity and for reexpression of the transgene. Karyotypic analysis of hybrid and segregant lines, exhibiting proper AFP, albumin and AFP-HPRT phenotypes, revealed that the presence of human chromosome 7 was most closely associated with the AFP-extinguished state. The hybrids generated in these studies now make it possible to isolate the sequences responsible for AFP and albumin extinction.