Delineation by use of specific monoclonal antibodies of the T-cell receptor and major histocompatibility complex interaction sites on the superantigen toxic shock syndrome toxin 1.

Murine monoclonal antibodies (MAbs) specific for toxic shock syndrome toxin 1 (TSST-1), a bacterial superantigen, showed the ability either to detect TSST-1 bound to histocompatibility locus antigen (HLA)-DR molecules or to inhibit TSST-1 binding to HLA-DR. A MAb capable of detecting DR-bound TSST-1 could also inhibit the toxin-induced activation of a T-cell receptor Vbeta15-expressing murine T-cell hybridoma. Alternatively, MAbs with specificity for the HLA-DR association site could present TSST-1 in vitro, stimulating CD4+ human T cells to proliferate. These functional activities correlated directly with with MAb specificity for HLA-DR versus T-cell receptor Vbeta interaction sites on TSST-1 as determined by reactivity with a panel of recombinant TSST-1 mutant molecules. Therefore, these MAbs discriminate the superantigen functional sites on the TSST-1 molecule and constitute reagents with the property of being potent modulators of the toxic activity of TSST-1.

Slipped capital femoral epiphysis. The prevalence of late contralateral slip.

We evaluated the prevalence of slipped capital femoral epiphysis in the contralateral hip of 169 children who had been managed with pinning in situ and thirty who had been managed with immobilization in a spica cast. Only children who had initially been seen with a unilateral slip and had been followed for a minimum of two years or until skeletal maturity were included in the study. The average duration of follow-up was 3.6 years (range, 0.5 to 9.5 years) for the group that had been managed with a cast and 2.8 years (range, 1.0 to 8.3 years) for the group that had been managed operatively. In sixty-one (36 per cent) of the 169 patients who had had operative treatment and two (7 per cent) of the thirty who had been managed with a spica cast, a slip subsequently developed in the contralateral hip; this difference was significant (p = 0.001). On the basis of these findings, we recommend that closer attention be paid to the potential development of a slip in the contralateral hip after pinning.

Detecting alerts, notifying the physician, and offering action items: a comprehensive alerting system.

We developed and evaluated a system to automatically identify serious clinical conditions in inpatients. The system notifies the patient's covering physician via his pager that an alert is present and offers potential therapies for the patient's condition (action items) at the time he views the alert information. Over a 6 month period, physicians responded to 1214 (70.2%) of 1730 alerts for which they were paged; they responded to 1002 (82.5% of the 1214) in less than 15 minutes. They said they would take action in 71.5% of the alerts, and they placed an order directly from the alert display screen in 39.4%. Further study is needed to determine if this alerting system improves processes or outcomes of care.

Identification of class II major histocompatibility complex and T cell receptor binding sites in the superantigen toxic shock syndrome toxin 1.

Superantigens, in association with class II major histocompatibility complex (MHC) molecules, activate T cells bearing particular beta chain variable domains of the T cell receptor (TCR). Unlike conventional peptide antigens, superantigens bind as intact proteins to TCR and MHC molecules outside their peptide binding sites. To characterize these interactions at the molecular level, random point mutations were generated in the gene encoding toxic shock syndrome toxin 1, a bacterial superantigen associated with toxic shock syndrome. Functionally impaired mutants were identified based on their lack of murine and human T cell stimulatory activities, and experiments analyzing binding to human histocompatibility leukocyte antigen-DR molecules differentiated residues involved in MHC from TCR binding. The results showed that the great majority of mutations are clustered in two distinct regions of the toxic shock syndrome toxin 1 molecule. The class II MHC binding site is located in the hydrophobic region of the NH2-terminal domain, and the TCR binding site is primarily in the major central groove of the COOH-terminal domain. These studies provide insight into the interactions necessary for superantigen-mediated disease in humans.

Assembly of the bacteriophage T4 replication machine requires the acidic carboxy terminus of gene 32 protein.

The acidic carboxy-terminal 89-amino acid fragment of bacteriophage T4 gene 32 protein was expressed in Escherichia coli to high levels from an inducible plasmid construct. Infection of induced cells by wild-type T4 phage results in impaired phage DNA synthesis. The time at which DNA synthesis begins and the diminution in DNA synthesis rates correlate with the amount of carboxy-terminal peptide that accumulates intracellularly prior to infection. Correspondingly, when induced cells are infected with viable phage containing a small deletion near the carboxy-terminus of 32 protein (delta PR201), the inhibition of phage DNA synthesis was much more severe. The mutant 32 protein competes less well against overproduced wild-type acid peptide than does wild-type 32 protein. The purified acid peptide, when used as the attached ligand for affinity chromatography, binds several T4 proteins from phage-infected cells, including 43 protein (T4 DNA polymerase), Dda protein (a DNA helicase), and UvsX protein (a Rec-like recombination protein). Furthermore, at 50- to 100-fold molar excess of acid peptide over intact 32 protein, phage DNA synthesis was specifically inhibited at the initiation step in an in vitro 5-protein DNA replication experiment. We propose that one or more phage replication proteins are titrated as non-productive protein-protein complexes at a site away from the DNA template. This implies that the carboxy-terminal domain of 32 protein is involved in an obligate step of replication machine assembly when the protein is properly attached to ssDNA in the vicinity of a primer-template junction. The assembly defect we observe is strikingly similar to the repression, or "squelching", of the activity of certain eukaryotic transcriptional activators.

Purification of the mosquitocidal and cytolytic proteins of Bacillus thuringiensis subsp. israelensis.

Two proteins from parasporal crystals of Bacillus thuringiensis subsp. israelensis were purified to electrophoretic homogeneity by gel filtration and anion-exchange chromatography. The larger of the two proteins (molecular weight, 68,000) was not cytolytic, whereas the smaller protein (molecular weight, 28,000) was highly cytolytic when assayed against rat erythrocytes. When these proteins were assayed against larvae of the yellow fever mosquito, Aedes aegypti, the larger protein was at least 100-fold more toxic than the smaller protein. Although proteolytic activity was not detected in solubilized crystals nor in purified protein preparations, the toxin (molecular weight, 68,000) was readily degraded to smaller, nontoxic molecules, even when maintained at 4 degrees C. Mixtures of the two purified proteins were significantly more toxic to mosquito larvae than was either protein alone. Thus, it is likely that both the mosquitocidal and the cytolytic protein play roles in the overall insecticidal action of the parasporal crystal produced by this bacterium.

Separation of the cytolytic and mosquitocidal proteins of Bacillus thuringiensis subsp. israelensis.

The cytolytic and mosquitocidal proteins of Bacillus thuringiensis subsp. israelensis were isolated from parasporal crystals and subsequently separated from each other. The proteins were separated by gel filtration chromatography and their molecular weights were estimated by both gel filtration chromatography and SDS-polyacrylamide gel electrophoresis. The apparent molecular weights of the mosquitocidal protein and the cytolytic protein were estimated to be 65,000 daltons and 28,000 daltons, respectively.