Investigation of an electronic emergency department information system as a data source for respiratory syndrome surveillance.

Emergency department syndromic surveillance may provide early warning of disease outbreaks due to bioterrorism or natural phenomena. The purpose of this investigation was to explore how an electronic emergency department information system could be used as a data source for respiratory syndrome surveillance. The process of data collection, entry, and transmission is described, and then a subset of data elements with potential epidemiological value is selected. The quality of the data contained in the system was evaluated by conducting a retrospective analysis of emergency department visits recorded in the system during 2001 and by reviewing clinical charts of cases with respiratory diagnoses. Diagnosis codes, discharge disposition, and demographic data were relatively complete; additional clinical data were not. Diagnosis codes were rapidly and reliably recorded. Data available in the system allows a description of emergency department visits for respiratory syndrome in terms of age, gender, location, severity of illness, and distribution in time. Encrypted data were transmitted every four hours to the health department without added work for emergency department personnel. Although significant obstacles remain, electronic emergency department information systems such as this may provide rapid, reliable data for syndromic surveillance.

Security aspects of electronic data interchange between a state health department and a hospital emergency department.

Electronic emergency department reporting provides the potential for enhancing local and state surveillance capabilities for a wide variety of syndromes and reportable conditions. The task of protecting data confidentiality and integrity while developing electronic data interchange between a hospital emergency department and a state public health department proved more complex than expected. This case study reports on the significant challenges that had to be resolved to accomplish this goal; these included application restrictions and incompatibilities, technical malfunctions, changing standards, and insufficient dedicated resources. One of the key administrative challenges was that of coordinating project security with enterprise security. The original project has evolved into an ongoing pilot, with the health department currently receiving secure data from the emergency department at four-hour intervals. Currently, planning is underway to add more emergency departments to the project.

Concept and progress of a regional effort to improve blood lead reporting to six Western States by incorporating electronic laboratory reporting.

While electronic laboratory reporting (ELR) has the potential to be both more timely and complete than non-electronic data transmission and direct electronic data transfer can also reduce data input errors, these benefits are often underutilized. A survey of states in HHS Regions IX and X (Alaska, Arizona, California, Hawaii, Oregon, Washington) led to collaborative efforts to maximize ELR benefits on a regional scale. Collaboration outcomes included the ratification of a regional blood lead HL7 message format and the formation of a multi-state committee to address reporting discrepancies by the large regional labs to multiple states in the regions.

Microflora of partially processed lettuce.

Bacteria, yeasts, and molds isolated from partially processed iceberg lettuce were taxonomically classified. The majority of bacterial isolates were gram-negative rods. Pseudomonas, Erwinia, and Serratia species were commonly found. Yeasts most frequently isolated from lettuce included members of the genera Candida, Cryptococcus, Pichia, Torulaspora, and Trichosporon. Comparatively few molds were isolated; members of the genera Rhizopus, Cladosporium, Phoma, Aspergillus, and Penicillium were identified.

Inhibition of T-lymphocyte mitogenic responses and effects on cell functions by bovine herpesvirus 1.

The mitogenic response of bovine peripheral blood mononuclear cells stimulated by concanavalin A (ConA) was suppressed by infectious bovine herpesvirus 1 (BHV-1). Proliferation in response to interleukin-2 (IL-2) by IL-2-dependent lymphocyte cultures was also inhibited by BHV-1. Although inhibition of mitogenesis approached 100%, less than 1 cell in 1,000 was productively infected by BHV-1 in ConA-stimulated cultures. Neither conditioned medium from mitogen-stimulated peripheral blood mononuclear cell cultures nor human recombinant IL-2 reversed suppression by the virus. Infection by BHV-1 did not influence the expression of IL-2 or IL-2 receptor mRNA in ConA-stimulated cultures, nor did it affect the cytolytic capabilities of lymphocytes. The data suggest that the inhibition of T-lymphocyte proliferation is the result of a nonproductive BHV-1 infection.

Role of second metal ion in establishing active conformations of concanavalin A.

The stoichiometry of Mn2+ binding to concanavalin A was found to be influenced by temperature, pH, and the presence or absence of saccharide. Demetalized concanavalin A binds one Mn2+ (S1 site) at 5 degrees C, pH 6.5, and two Mn2+ at 25 degrees C (S1 and S2 sites). The association constants for Mn2+ are 6.2 x 10(5) and 3.7 x 10(4) M-1 for the S1 and S2 sites, respectively, at 25 degrees C. Concanavalin A with one Mn2+ bound per monomer remains in an open conformation and exhibits a relatively high water proton relaxation rate. Concanavalin A with two Mn2+ ions remains in a closed conformation characterized by a lower relaxation rate. The rate of binding of the second Mn2+ to concanavalin A as determined by ESR and the rate of conversion of open form to closed form (folding over) as determined by proton relaxation rate measurements gave an identical rate constant of 80.0 +/- 5.8 M-1 h-1 at 17 degrees C. Ca2+, Sr2+, and high levels of methyl alpha-D-mannopyranoside also induce folding of concanavalin A. Ca2+ is not catalytic but stoichiometric in causing the folding. Mn2+ in the S1 site can be displaced by Ni2+, Co2+, and Zn2+, and Mn2+ in the S2 site can be displaced by Ca2+ and Sr2+. Concanavalin A with Ni2+, Co2+, Zn2+, or Mn2+ in the S1 site and Ca2+ or Sr2+ in the S2 site has a higher affinity for methylumbelliferyl alpha-D-mannopyranoside than Ni-Mn-, Co-Mn-, Zn-Mn-, and Cd-Cd-concanavalin A.

Suppression of lymphocyte proliferation by proteins secreted by cultured Sertoli cells.

Secreted proteins from cultured rat Sertoli cells were assessed for effects on phytolectin-stimulated rat splenic lymphocytes. Sertoli cell proteins (SCP) suppressed DNA, RNA and protein synthesis in stimulated rat splenic lymphocytes whether added at 0, 4, 24 and 48 h after culture initiation. SCP preparations were not toxic to cells. SCP suppressive activity was heat stable but was not associated with the carbohydrate component of SCP preparations. SCP also suppressed the proliferation of lymphoid and non-lymphoid cell lines from several different animal species but did not inhibit proliferation-independent lysis of YAC-1 target cells by rat natural killer cells. These results suggest that Sertoli cells synthesize inhibitory factors that might be secreted into seminal plasma. Furthermore, our results demonstrate that one mode of action of these factors is suppression of cell proliferation.

Evidence for two discrete phases of IL-2 production in bovine lymphocytes.

Regulation of IL-2 production in Con A-stimulated bovine lymph node cells was studied by following the time course of IL-2 synthesis and secretion over a 24-h period. A biphasic time course curve of IL-2 secretion was observed after stimulation with Con A alone or with Con A plus PMA. Both phases of IL-2 production were confirmed by three separate biochemical techniques: IL-2 protein bioassay, mRNA Northern blots, and anti-IL-2 antibody detection. Bovine IL-2 transcripts were detected as early as 2 h after Con A stimulation. The early phase of IL-2 protein secretion was initially detected 3 h after Con A stimulation, and the late phase occurred between 10 and 16 h. Both phases of IL-2 production were enhanced when PMA was added to the Con A stimulation. Each phase of IL-2 protein secretion was preceded by the accumulation of IL-2 mRNA.

Cloning of cDNA for the bovine IL-2 receptor (bovine Tac antigen).

We have cloned the Tac analog of the bovine IL-2 receptor (IL-2R) cDNA. Using mouse and human cDNA probes, we isolated five bovine IL-2R clones from a lambda gt11 bovine long-term lymphocyte cDNA library. Three of the clones had inserts of 2600 base pairs (bp), the same size as the bovine IL-2R mRNA visualized on Northern blots. The full-length cDNA contain a 190-bp 5' untranslated region, followed by a 825-bp coding region, and a 3' untranslated region that contain 1600 bp. Comparison of the bovine and human IL-2R-coding sequences revealed 71% homology at the nucleotide level. The 3' and 5' non-coding regions were not as homologous, apart from a specific site in the 5'-untranslated region that contained a 5'-upstream start codon. In this region, 24 of 26 nucleotides were identical for the human and bovine cDNAs. Further analysis of the bovine IL-2R sequence also revealed the following: (i) the hydrophobic domains of the IL-2R protein were more conserved between species than the hydrophilic domains, (ii) the predominant site of intracellular IL-2R phosphorylation in mouse and human was a conserved Ser which was not conserved in the bovine sequence, and (iii) there exists a statistically significant amino acid homology with the AIDS gag protein.

Bovine interleukin 2: regulatory mechanisms.

A cDNA clone of the bovine interleukin-2 (IL-2) gene has been isolated and demonstrated to produce a functional bovine IL-2 protein when transfected into either CV-1 or COS-1 monkey cells. Homology comparisons of both the nucleotide and predicted amino acid sequences of bovine IL-2 with those of the human and mouse show extensive regions of sequence conservation between the species. The amino acid sequence of the mature bovine IL-2 protein shares about 60-63% homology with those of the human and mouse, but the 3' untranslated regions of the human and mouse gene share as much, if not greater, sequence homology with the 3' untranslated regions of the human and mouse genes. In particular, a tandemly repeated sequence (TATT), n, found in the 3' untranslated tail of the bovine IL-2 clone is also found in the 3' untranslated region of a large group of cytokine genes and other inducible genes of the lymphoid and immune response systems. This sequence may serve a specific regulatory function in the immune system.

Comparison of basement membrane matrix degradation by purified proteases and by metastatic tumor cells.

We have examined the nature of biochemical degradation of an isolated basement membrane matrix (bovine lens capsule) using different methodologies. The first strategy was quantitation of the release of surface-bound 125I and a second the documentation by SDS-PAGE of the appearance of putative cleavage products and the loss of high-molecular-weight components from the matrix. Basement membrane matrix bands resolved on SDS-PAGE were identified by their protease sensitivities as well as by Western immunoblots using monoclonal antibodies developed for this study. Radioiodinated components were found predominantly at positions on the gel equivalent to 160-200 kd and 400 kd proteins. Since these labeled moieties were sensitive to bacterial collagenase digestion and stained with anticollagen type IV antibodies, they were determined to represent various configurations of collagen type IV. Several other lower-molecular-weight bands also stained with the anticollagen IV antibodies. Monoclonal antibodies reactive with laminin exhibited a complex staining pattern on the gels, which included the expected 200 and 400 kd components. We confirmed that lens capsule basement membrane contained only a single heparan sulfate glycosaminoglycan species, and tumor cell-induced glycosaminoglycan degradation within the basement membrane matrix was detected using cellulose acetate electrophoresis. Distinctive putative cleavage products were resolved on SDS-PAGE gels from matrices subjected to digestion by a variety of purified proteases as well as by metastatic tumor cells or their conditioned media. Tumor cells of different histiotypes produced different characteristic cleavage patterns, suggestive of the existence of several pathways of matrix degradation. Overall, primary tumor cells exhibited a greater degradative activity towards the basement membrane matrix than did long-term tissue culture-passaged cells. The same tumor cell line could exhibit considerably different patterns of both protein and glycosaminoglycan degradation depending on recent culture history. The relevance of these biochemical studies to the pathogenesis of malignant neoplasms is shown by: 1) the evaluation of degradative activities of B16 tumor cell populations exhibiting enhanced lung-colonizing phenotypes, and 2) the ability of a known antimetastatic moiety with antiprotease activity (Haementeria leech species salivary gland extract) to protect matrix components from degradation by tumor cell-conditioned medium.

Trypanosoma brucei: a fluorescence and spin label study of the membranes of the bloodstream form.

Fluidity of the plasma membrane of Trypanosoma brucei brucei has been examined with fluorescence and electron spin resonance spectroscopy. Fluorescent probes 1,6-diphenyl-1,3,5-hexatriene and 8-anilino-1-naphthalene sulfonate and the spin label probe 5-doxyl stearate have been employed to examine fluidity under a variety of conditions. The temperature dependence of 8-anilino-1-naphthalene sulfonate polarization and of the order parameter S for 5-doxyl stearate reveals phase alterations near 30 C. 1,6-Diphenyl-1,3,5-hexatriene polarization shows that proteolysis of the surface glycoprotein with trypsin increases fluidity but treatment with human serum which is trypanocidal produces no detectable change in membrane fluidity.

Molecular cloning of a functional bovine interleukin 2 cDNA.

A cDNA clone of the bovine interleukin 2 (IL-2) gene has been isolated and demonstrated to be functional in the production of secreted bovine IL-2 protein when transfected into monkey cells. The bovine IL-2 clone is 791 base pairs in length and contains an open reading frame of 474 base pairs coding for a bovine IL-2 precursor polypeptide of 158 amino acids with an estimated molecular weight of 17,884. The putative hydrophobic leader or signal sequence of the precursor protein is 23 amino acid residues long, suggesting that, after removal by processing, the mature secreted bovine IL-2 protein contains 135 amino acids and has a molecular weight of 15,464. Comparisons of both the nucleotide sequence and the predicted amino acid sequence of bovine IL-2 with those of the human and mouse IL-2 show extensive regions of sequence conservation between the species, interspersed with other regions of less similarity. The 3' untranslated region of the bovine IL-2 gene shares as much, if not greater, sequence homology with the 3' untranslated regions of the human and mouse genes as do the transcribed coding regions of these genes, suggesting an involvement of this region in regulation. In particular, a tandemly repeated sequence, (TATT)n, found in the 3' untranslated tail of the bovine IL-2 clone is also found in the 3' untranslated region of the other known interleukin and interferon genes, as well as in similar regions of many other inducible genes of the lymphoid and immune response systems, suggesting a cell or tissue-specific regulatory function for these evolutionarily conserved sequences.

The role of tumor-cell surface carbohydrate in experimental metastasis.

Quantitative studies on the binding of concanavalin A (Con A) and wheat-germ agglutinin (WGA) to a series of rat hepatocarcinoma metastatic variants revealed a positive correlation between the amount of cell-surface-bound lectin and lung colonization potential. Scatchard analysis of Con A and WGA binding to 10 individual clones isolated from a subcutaneous (s.c.) tumor transplant and to tumor-cell isolates from 10 individual spontaneous lung metastases from the same animal showed diverse binding characteristics for these cell populations. Nevertheless, the expression of Con A receptor sites accurately predicted the lung colonization potential of 3 isolates from the lung metastases. Higher lectin binding curves were observed for the clones from the subcutaneous tumor than for the isolates from lung metastases. These data suggest that a high Con-A binding potential is indicative of a high lung colonization potential for these hepatocarcinoma cells, but that this phenotype may be rapidly lost during tumor outgrowth in the lungs. The binding of tumor cells to vascular endothelial cell monolayers was inhibited in the presence of Con A; however, no inhibition was observed with 2 other lectins. Attachment of tumor cells to endothelial cell monolayers was also inhibited by the monosaccharides methyl alpha-D-mannopyranoside and N-acetyl-D-galactosamine. Other monosaccharides tested did not alter the attachment of tumor cells to endothelial cell monolayers.

Metal ion binding to tetrameric lima bean lectin.

The binding of Mn2+ and Ca2+ to tetrameric lima bean lectin has been examined by equilibrium dialysis and magnetic resonance techniques. Demetalized lectin prepared by acid treatment binds either 1 Mn2+ or 2 Ca2+/monomer. When demetalized lectin is presaturated with Ca2+, only 1 Mn2+ binds per dimer. Water proton relaxation rate enhancements and Mn2+ electron spin resonance spectra were used to monitor metal ion association processes. Following Mn2+ binding to demetalized lectin, a conformational change with activation energy of 16 kcal/mol was detected; this is similar in magnitude to that observed for a conformational change with the lectin concanavalin A. The pH dependence suggests that a histidine residue is involved. ESR spectroscopy shows clearly that 1 Mn2+ binds to each demetalized subunit, but that Ca2+ induces dissociation of half the Mn2+; this result is in agreement with the equilibrium dialysis studies.

Development and maintenance of bovine cytotoxic lymphocytes with recombinant human interleukin-2.

Long-term bovine lymphocyte cultures were initiated by stimulation with alloantigens and maintained in continuous culture using medium containing recombinant human interleukin-2 (rh IL-2). The development of specific and lectin-dependent killing was monitored following primary alloantigen challenge. Cytolytic activity was barely detectable after 7 days of culture, but gradually increased with peak activity occurring after 21 days of culture. A panel of monoclonal antibodies (MoAb) was used to determine whether a shift in the antigen phenotype of the cell population occurred during culture. The primary cell type that grew in culture was of the T-cell lineage with minimal or no expression of class II antigens. The activities of adenosine deaminase (ADA), purine nucleotide phosphorylase (PNP), adenosine kinase (AK), deoxyadenosine kinase (dAK), deoxycytidine kinase (dCK), 5'-nucleotidase (5'-N), AMP deaminase, hypoxanthine-guanine phosphoribosyl transferase (HGPRT or HPRT), and adenine phosphoribosyl transferase (APRT) were measured by microassay in resting peripheral blood lymphocytes (PBL) and in cells from long-term cultures. Large increases in the activities of PNP and HPRT with a decrease in the activity of ADA were observed. The data show that long-term cultures of lymphocytes can be readily generated, and that sequential changes in antigenic phenotype and function can be monitored and correlated with quantitative changes in enzyme activity.

Inhibition of rat peritoneal mast cell histamine secretion by protoporphyrin and incandescent light.

Rat peritoneal mast cells (RPMC) exposed to protoporphyrin (PP) and incandescent light (IL) become refractory to the stimulatory effects of compound 48/80. Once initiated, this refractory state continues to develop even after removal of the light source and is essentially complete within 30 min. While this state of unresponsiveness appears to be relatively permanent in the dark, prolonged incubation in the light (greater than 80 min) induces cell lysis. We have shown that the resistant state is not specific for the mast cell stimulator compound 48/80. Mast cells passively sensitized with IgE and illuminated for 30 min in the presence of 100 ng/ml PP also fail to release histamine upon stimulation by anti-rat IgE, anti-rat F(ab')2, concanavalin-A (Con-A), and the calcium ionophore A23187. The inability to respond to immunological stimuli could not be ascribed to the reversible loss of membrane-bound IgE from its receptor. While the binding of either the inducer to IgE or IgE to its receptor may actually be impaired in refractory cells, the significance of such impairments on the development of the resistant state in these cells is precluded by the inability of A23187 to either increase intracellular 45Ca2+ levels or induce histamine release. The data suggest that the RPMC refractory state develops as a result of covalent inter- and/or intramolecular cross-linking of membrane proteins. Furthermore, this cross-linking may involve sulfhydryl or amino groups essential to either stimulus transduction, or the histamine secretory process itself.

Continuous cultivation of equine lymphocytes: evidence for occasional T cell-like maturation events in horses with hereditary severe combined immunodeficiency.

Peripheral blood mononuclear cells (PBMC) from 14 foals with hereditary severe combined immunodeficiency (SCID) were studied to determine the extent of lymphocyte differentiation that occurs in this disorder. PBMC from all 14 horses had the morphologic characteristics of large granular lymphocytes (LGL). Cells from only one of 14 horses were responsive to phytolectin stimulation in a standard blastogenesis assay; however, PBMC from all 14 horses proliferated in continuous culture in the presence of partially purified interleukin 2. Furthermore, there were differences in the growth patterns of these cultured cells that correlated with their ability to respond to phytolectin stimulation. PBMC obtained from the 13 phytolectin-unresponsive foals survived in culture for only 4 to 5 wk, divided very slowly, developed large granules composed primarily of calcium phosphate, and accumulated high concentrations of histamine. In contrast, PBMC from the phytolectin-responsive SCID foal proliferated in continuous culture for over 100 days, divided as rapidly as normal equine PBMC under identical culture conditions, and did not accumulate granules or histamine. These observations indicate that lymphoid cell differentiation occurs in some horses with SCID even though the identity of the LGL is unresolved. Two possibilities are that LGL are products of a pathway separate from that of lymphocytes or that LGL are precursors of mature lymphocytes.

Calorimetric study of carbohydrate binding to concanavalin A.

Flow microcalorimetry has been used to examine the delta H of binding of two types of saccharides, a series of simple monosaccharides and a series of alpha-(1----4)-linked glucosides, to the lectin Concanavalin A. It has been found that the delta H decreases with any change in the stereochemistry of a hydroxyl group relative to methyl alpha-D-mannopyranoside. The data have allowed the calculation of the relative contribution of two of the hydroxyl groups. The delta H's of binding for the alpha-(1----4)-linked glucosides are approximately 31 kJ/mol, and the apparent association constants vary insignificantly with increasing length. This result indicates that only one glucose residue binds to concanavalin A by hydrogen bonds, and that the additional glucose residues have no interaction either by hydrogen bonds or by nonspecific hydrophobic interactions. This result confirms the absence of an extended binding site for alpha-(1----4)-linked glucopyranosides, in contrast to that proposed for alpha-(1----2)-linked mannopyranosides which show an increase in apparent association constants with increasing length.

Production and characterization of bovine interleukin-2.

A lymphokine analogous to interleukin-2 from other species was generated from bovine suprapharyngeal lymph nodes and characterized biochemically. The isolated material can support the long-term growth of concanavalin A (Con-A) or mixed lymphocyte reaction (MLR)-activated bovine T cells. The material elutes from DEAE-Sephadex at a low salt concentration, approximately 0.075 M, and exhibits molecular weight of approximately 25,000 on Sephadex G-100. When subjected to analysis by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), three peaks of activity were observed, corresponding to molecular weights of 14,400, 16,800 and 20,200. Finally, the isolated material exhibited a marked heterogeneity when subjected to chromatofocussing. Three major peaks of activity, with pIs of approximately 5.95, 5.41 and 5.0 are present with peaks of lesser activity at pIs of 5.82, 5.70, 4.73 and 4.20.