Responsible design and implementation of technologies for the prevention of infectious diseases: towards a values-based assessment framework for the Dutch government.

OBJECTIVES: The Dutch government implemented the apps 'CoronaMelder' and 'CoronaCheck' to prevent the transmission of SARS-CoV-2. They faced many questions on how to responsibly implement such technologies. Here, we aim to develop an assessment framework to support the Dutch national government with the responsible design and implementation of technologies for the prevention of future infectious diseases. STUDY DESIGN: Three-stage web-based Delphi process. METHODS: The assessment framework was developed through two research phases. During the Initial Design phase, a conceptual version of the assessment framework was developed through a scoping review and semistructured interviews with a scientific board. The Consensus phase involved a three-stage web-based Delphi process with an expert community. RESULTS: The final assessment framework consists of five development phases, 10 values, and a total of 152 questions. CONCLUSIONS: Technology assessment frameworks help policymakers to make informed decisions and contribute to the responsible implementation of technologies in society. The framework is now available for the Dutch government and other stakeholders to use in future pandemics. We discuss the possibilities of using the framework transnationally.

Novel paradigm enables accurate monthly gestational screening to prevent congenital toxoplasmosis and more.

Background: Congenital toxoplasmosis is a treatable, preventable disease, but untreated causes death, prematurity, loss of sight, cognition and motor function, and substantial costs worldwide. Methods/Findings: In our ongoing USA feasibility/efficacy clinical trial, data collated with other ongoing and earlier published results proved high performance of an Immunochromatographic-test(ICT) that enables accurate, rapid diagnosis/treatment, establishing new paradigms for care. Overall results from patient blood and/or serum samples tested with ICT compared with gold-standard-predicate-test results found ICT performance for 4606 sera/1876 blood, 99.3%/97.5% sensitive and 98.9%/99.7% specific. However, in the clinical trial the FDA-cleared-predicate test initially caused practical, costly problems due to false-positive-IgM results. For 58 persons, 3/43 seronegative and 2/15 chronically infected persons had false positive IgM predicate tests. This caused substantial anxiety, concerns, and required costly, delayed confirmation in reference centers. Absence of false positive ICT results contributes to solutions: Lyon and Paris France and USA Reference laboratories frequently receive sera with erroneously positive local laboratory IgM results impeding patient care. Therefore, thirty-two such sera referred to Lyon's Reference laboratory were ICT-tested. We collated these with other earlier/ongoing results: 132 of 137 USA or French persons had false positive local laboratory IgM results identified correctly as negative by ICT. Five false positive ICT results in Tunisia and Marseille, France, emphasize need to confirm positive ICT results with Sabin-Feldman-Dye-test or western blot. Separate studies demonstrated high performance in detecting acute infections, meeting FDA, CLIA, WHO ASSURED, CEMark criteria and patient and physician satisfaction with monthly-gestational-ICT-screening. Conclusions/Significance: This novel paradigm using ICT identifies likely false positives or raises suspicion that a result is truly positive, rapidly needing prompt follow up and treatment. Thus, ICT enables well-accepted gestational screening programs that facilitate rapid treatment saving lives, sight, cognition and motor function. This reduces anxiety, delays, work, and cost at point-of-care and clinical laboratories. Author's Summary: Toxoplasmosis is a major health burden for developed and developing countries, causing damage to eyes and brain, loss of life and substantial societal costs. Prompt diagnosis in gestational screening programs enables treatment, thereby relieving suffering, and leading to > 14-fold cost savings for care. Herein, we demonstrate that using an ICT that meets WHO ASSURED-criteria identifying persons with/without antibody to Toxoplasma gondii in sera and whole blood with high sensitivity and specificity, is feasible to use in USA clinical practice. We find this new approach can help to obviate the problem of detection of false positive anti- T.gondii IgM results for those without IgG antibodies to T.gondii when this occurs in present, standard of care, predicate USA FDA cleared available assays. Thus, this accurate test facilitates gestational screening programs and a global initiative to diagnose and thereby prevent and treat T.gondii infection. This minimizes likelihood of false positives (IgG and/or IgM) while maintaining maximum sensitivity. When isolated IgM antibodies are detected, it is necessary to confirm and when indicated continue follow up testing in ∼2 weeks to establish seroconversion. Presence of a positive ICT makes it likely that IgM is truly positive and a negative ICT makes it likely that IgM will be a false positive without infection. These results create a new, enthusiastically-accepted, precise paradigm for rapid diagnosis and validation of results with a second-line test. This helps eliminate alarm and anxiety about false-positive results, while expediting needed treatment for true positive results and providing back up distinguishing false positive tests.

Chromosomal damage in human leukocytes induced by lysergic acid diethylamide.

Addition of lysergic acid diethylamide to cultured human leukocytes resulted in a marked increase of chromosomal abnormalities. The distribution of chromosome breaks deviated significantly from random, with an accumulation of aberrations in chromosome No. 1. Cytogenetic investigation of a patient extensively treated with this drug over a 4-year period for paranoid schizophrenia showed a similar increase in chromosomal damage.

Sphingosine kinase 1 overexpression induces MFN2 fragmentation and alters mitochondrial matrix Ca2+ handling in HeLa cells.

Sphingosine kinase 1 (SK1) converts sphingosine to the bioactive lipid sphingosine 1-phosphate (S1P). S1P binds to G-protein-coupled receptors (S1PR1-5) to regulate cellular events, including Ca2+ signaling. The SK1/S1P axis and Ca2+ signaling both play important roles in health and disease. In this respect, Ca2+ microdomains at the mitochondria-associated endoplasmic reticulum (ER) membranes (MAMs) are of importance in oncogenesis. Mitofusin 2 (MFN2) modulates ER-mitochondria contacts, and dysregulation of MFN2 is associated with malignancies. We show that overexpression of SK1 augments agonist-induced Ca2+ release from the ER resulting in increased mitochondrial matrix Ca2+. Also, overexpression of SK1 induces MFN2 fragmentation, likely through increased calpain activity. Further, expressing putative calpain-cleaved MFN2 N- and C-terminal fragments increases mitochondrial matrix Ca2+ during agonist stimulation, mimicking the SK1 overexpression in cells. Moreover, SK1 overexpression enhances cellular respiration and cell migration. Thus, SK1 regulates MFN2 fragmentation resulting in increased mitochondrial Ca2+ and downstream cellular effects.

Signaling mediated by the cytosolic domain of peptidylglycine alpha-amidating monooxygenase.

The luminal domains of membrane peptidylglycine alpha-amidating monooxygenase (PAM) are essential for peptide alpha-amidation, and the cytosolic domain (CD) is essential for trafficking. Overexpression of membrane PAM in corticotrope tumor cells reorganizes the actin cytoskeleton, shifts endogenous adrenocorticotropic hormone (ACTH) from mature granules localized at the tips of processes to the TGN region, and blocks regulated secretion. PAM-CD interactor proteins include a protein kinase that phosphorylates PAM (P-CIP2) and Kalirin, a Rho family GDP/GTP exchange factor. We engineered a PAM protein unable to interact with either P-CIP2 or Kalirin (PAM-1/K919R), along with PAM proteins able to interact with Kalirin but not with P-CIP2. AtT-20 cells expressing PAM-1/K919R produce fully active membrane enzyme but still exhibit regulated secretion, with ACTH-containing granules localized to process tips. Immunoelectron microscopy demonstrates accumulation of PAM and ACTH in tubular structures at the trans side of the Golgi in AtT-20 cells expressing PAM-1 but not in AtT-20 cells expressing PAM-1/K919R. The ability of PAM to interact with P-CIP2 is critical to its ability to block exit from the Golgi and affect regulated secretion. Consistent with this, mutation of its P-CIP2 phosphorylation site alters the ability of PAM to affect regulated secretion.

Proteases during the growth of Ehrlich ascites tumor. II. The kallikrein-kinin system.

Ascitic fluid and ascites tumor cells from Swiss mice bearing Ehrlich ascites tumor were assayed for components of the kallikrein-kinin system at various times during tumor growth. Changes in component levels were correlated with those in the plasma. Ascitic fluid contained an acetone-activated prekallikrein that increased in concentration during tumor growth and reached peak levels during the 7th-10th day post transplant. No free kinin activity was present in the ascitic fluid. During tumor growth, kininogen levels increased in parallel with prekallikrein levels. The ascitic fluid also contained a kinin-destroying activity that was initially high during the early phase of tumor growth. Tumor cell fractions, prepared by ultracentrifugal techniques, had no kinin-forming activity while possessing kinin-destroying activity that was localized in the soluble protoplasmic protein and nuclear fractions. The kinin-forming activity of the ascitic fluid resembled that of the plasma with respect to pH optima, kinetics of kinin formation, and effect of protease inhibitors. The kininase activity of both ascitic fluid and plasma differed from that of the tumor cell fractions.

Proteases during the growth of Ehrlich ascites tumor. I. The fibrinolysin system.

Component levels of the fibrinolysin system in the plasma and ascitic fluid of Swiss mice bearing Ehrlich ascites tumor cells were determined during a 15-day tumor growth time phase. During tumor growth, the concentration of plasminogen in the ascitic fluid decreased inversely to the total packed cell volume. Free plasmin was not present in the ascitic fluid nor was there any measurable plasminogen activator activity. Both antiplasmin activity and fibrinogen levels present in the fluid decreased during tumor growth. The nuclear and mitochondrial-microsomal subcellular fractions of the tumor cell exhibited plasminogen activator activity. No significant changes in the above parameters occurred in the plasma during the tumor growth period we studied.

Monoclonal antibodies to bradykinin inhibit smooth muscle contractile action of bradykinin.

Four hybridoma cell lines have been established that secrete monoclonal antibodies to nonapeptide bradykinin. Bradykinin coupled to ovalbumin, using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide as coupling agent, was used to immunize BALB/c mice. Spleen cells from the immunized animals were fused to P3-X63-AG8-653 mouse myeloma cells. The resultant hybrid cells were screened by enzyme-linked immunoassay for production of antibodies to bradykinin. Hybrids from four positive wells were subcloned by limiting dilution and expanded as ascites tumor into pristane-primed mice. All the four hybrids secreted monoclonal antibodies of IgG1 (k) isotype. Unlabeled peptides bradykinin, lysyl-bradykinin (kallidin) and methionyl-lysyl-bradykinin competed with the radiolabeled [Tyr1]kallidin for monoclonal antibody binding sites. These antibodies recognized preferentially either NH2- or COOH-terminals of the nonapeptide bradykinin and can distinguish between des-Arg1-bradykinin and des-Arg9-bradykinin. Bradykinin fragments smaller than eight residues were not recognized by these antibodies. Monoclonal antibodies BK-D6A5, BK-B6C9 and BK-A3D9 neutralized the smooth muscle contractile activity of bradykinin. An enzyme-linked immunoassay developed using these monoclonal antibodies showed the effective range of bradykinin determination between 5 and 150 ng.

The carbohydrate structure of the asparagine-linked oligosaccharides of rat plasma thiostatin.

The complete carbohydrate structure of the asparagine-linked oligosaccharides of rat plasma thiostatin was elucidated through chemical and enzymatic methods including gas chromatography-mass spectrometry (GC-MS) and lectin affinity chromatography. Pronase digestion of thiostatin yielded a major glycopeptide fraction with asparagine the most abundant amino acid present. Based on one mole of aspartic acid, the following molar ratios obtained for the four major amino acids: aspartic acid (1.0), threonine (0.53), glycine (0.48) and serine (0.30). Neutral sugar analysis yielded a 3:2 molar ratio for mannose to galactose based on an assigned value to mannose of 3. On this basis, the fraction also contained 3 residues of sialic acid and, on average, 0 to 1 residue of fucose. GC-MS of partially methylated alditol acetates from the glycopeptide fraction identified the presence of biantennary and triantennary structure. Analyses of the neutral sugar and amino-acid composition, together with methylation data, support a biantennary N-linked structure for this major glycopeptide fraction and a triantennary N-linked structure as a lesser component. Sequencing of the desialyated 14C-labelled glycopeptide fraction by sequential exoglycosidase digestion and lectin affinity chromatography uncovered the following saccharide order: terminal galactose, N-acetylglucosamine and pentasaccharide inner core. This sequence is consistent with the N-linked glycan structures demonstrated by methylation and compositional analyses.

The effect of detergents on bovine cytochrome c oxidase: a kinetic approach.

(1) Investigation of the relationship between the detergent concentration and steady-state and pre-steady-state kinetics of cytochrome c oxidase proved to be a valid approach in the study of protein-detergent interaction. (2) Laurylmaltoside, sodium cholate and Triton X-100 influenced the kinetics of cytochrome c oxidase cooperatively at detergent concentrations near their critical micelle concentration. This mode of interaction reflects disaggregation of the oxidase as a result of cooperative binding of the detergent. (3) Addition of increasing concentrations of Tween-80 to the aggregated enzyme caused a more gradual decrease in aggregation of the oxidase, which did not result in a change in activity of the enzyme. This suggests that aggregation of cytochrome c oxidase occurs in a highly regular manner in which no catalytic sites are shielded off. (4) Oxidase aggregates present at detergent concentrations below the critical micelle concentration of laurylmaltoside and Triton X-100 showed considerable activity. Their kinetics were equal to those of the oxidase in Tween-80, suggesting that the protein molecules are aligned in a similar way in all oligomers. Aggregates present in low concentrations of sodium cholate showed turnover rates that were twice as low as those observed with other aggregates. (5) Solubilisation of the oxidase by sodium cholate or Triton X-100 resulted in almost complete inhibition of enzymic activity, whereas the association rate of ferrocytochrome c was almost equal to that found for monomeric oxidase in laurylmaltoside. These results are in agreement with a mixed-type inhibition.

Hypertrophic cardiomyopathy caused by a novel alpha-tropomyosin mutation (V95A) is associated with mild cardiac phenotype, abnormal calcium binding to troponin, abnormal myosin cycling, and poor prognosis.

BACKGROUND: We report hypertrophic cardiomyopathy (HCM) in a Spanish-American family caused by a novel alpha-tropomyosin (TPM1) mutation and examine the pathogenesis of the clinical disease by characterizing functional defects in the purified mutant protein. METHODS AND RESULTS: HCM was linked to the TPM1 gene (logarithm of the odds [LOD] score 3.17). Sequencing and restriction digestion analysis demonstrated a TPM1 mutation V95A that cosegregated with HCM. The mutation has been associated with 13 deaths in 26 affected members (11 sudden deaths and 2 related to heart failure), with a cumulative survival rate of 73+/-10% at the age of 40 years. Left ventricular wall thickness (mean 16+/-6 mm) and disease penetrance (53%) were similar to those for the ss-myosin mutations L908V and G256E previously associated with a benign prognosis. Left ventricular hypertrophy was milder than with the ss-myosin mutation R403Q, but the prognosis was similarly poor. With the use of recombinant tropomyosins, we identified several functional alterations at the protein level. The mutation caused a 40% to 50% increase in calcium affinity in regulated thin filament-myosin subfragment-1 (S1) MgATPase assays, a 20% decrease in MgATPase rates in the presence of saturating calcium, a 5% decrease in unloaded shortening velocity in in vitro motility assays, and no change in cooperative myosin S1 binding to regulated thin filaments. CONCLUSIONS: In contrast to other reported TPM1 mutations, V95A-associated HCM exhibits unusual features of mild phenotype but poor prognosis. Both myosin cycling and calcium binding to troponin are abnormal in the presence of the mutant tropomyosin. The genetic diagnosis afforded by this mutation will be valuable in the management of HCM.

Development and evaluation of an HIV-1 transfection-neutralization assay.

We developed a transfection-neutralization assay for human immunodeficiency virus type 1 (HIV-1) infectious molecular clones. In this assay CD4 negative adherent cells, transfected in microtiter plates with fixed amounts of proviral DNA of molecular HIV-1 clones, are cocultivated with CD4 positive T cell lines or primary peripheral blood mononuclear cells (PBMC) in the presence of anti-HIV-1 sera or monoclonal antibodies (MAbs). Results obtained with this technique were reproducible and compared favorably with a conventional cell-free infection inhibition assay. The transfection-neutralization assay obviates the need for virus stock preparation and, therefore, is particularly suitable for the evaluation of HIV-1 clones with slow replication kinetics and of recombinant chimeric HIV-1 clones inclined to undergo additional mutations during stock preparation. The potential value of this assay for the analysis of the specificity of neutralizing sera and MAbs was demonstrated in experiments with V3 chimeric molecular clones.

Immunologic methods for quantitative estimation of small peptides and their application to bradykinin.

A simple strategy was developed for the immunologic quantitative determination of small, biologically active peptides utilizing bradykinin (BK) as the model peptide prototype. Methods were developed for the preparation of a peptide-carrier complex suitable for immunization and for immobilization of peptides onto the plastic surface of enzyme-linked immunosorbent assay (ELISA) plates. An avidin-bound biotinylated peptide complex was used for raising peptide antibodies with high titers (1:4000) in the rabbit. The peptide BK was coupled to synthetic polymeric carriers poly-D-lysine (PL) and poly-D-lysine-succinylated (PLS) via the BK carboxy and amino terminus, respectively, with the aid of a water soluble carbodiimide. These carriers with antigen peptide side chains as well as avidin-biotinyl-peptide complexes were efficient surface immobilizing reagents for microwell plastic plates used in the detection of kinins by ELISA. Monoclonal antibodies reacted competitively with kinins in plates coated with either PL-BK or PLS-BK. In contrast, rabbit (polyclonal) antibodies reacted specifically in the plates coated with PLS-BK but only a non-specific reaction could be obtained with the PL-BK coated plates (i.e., could not be displaced with BK). Based on results using synthetic BK analogues, the carboxy terminal half of the BK molecule appears to be the stronger antigenic determinant in both mouse and rabbit systems. The polyclonal antibodies demonstrated a greater affinity to bradykinin compared to the monoclonal antibodies. Their use improved the sensitivity of the ELISA for kinin determination by one order of magnitude. Kinin levels determined in plasma tryptic digests by ELISA with the polyclonal antibodies and PLS-BK system were in agreement with published values.

Frequent and early HIV-1MN neutralizing capacity in sera from Dutch HIV-1 seroconverters is related to antibody reactivity to peptides from the gp120 V3 domain.

The temporal development of HIV-1 neutralizing activity and antibodies to the gp120-V3 neutralization domain were studied in sera from 20 Dutch HIV-1-infected individuals followed from seroconversion on. Serum neutralizing capacity was assessed with three T cell line-tropic isolates: HIV-1MN, HIV-1HXB2, and the patient isolate HIV-1(320). Neutralizing activity to HIV-1MN developed in 18 individuals (90%) within 0 to 10 months after seroconversion. Parallel evolution of IgG reactivity to V3 peptides of United States/European type variants, and the capability of such peptides to completely inhibit HIV-1MN neutralization in four of five tested sera (taken 1-2 years after seroconversion), indicate that a large proportion of HIV-1MN neutralizing antibodies is directed to V3. The early appearance and high frequency of HIV-1MN neutralizing activity in the Dutch study group indicate the close relationship of HIV-1MN to HIV-1 variants circulating in the Netherlands. Neutralizing activity to HIV-1HXB2 (in 15 of 20 individuals) developed several months after that to HIV-1MN in all individuals (average, 10 months after seroconversion) and was not seen in the absence of HIV-1MN neutralizing activity. Neutralizing activity to the Dutch isolate HIV-1(320) (found in 11 of 18 tested individuals) emerged simultaneously with that to HIV-1MN in 4 individuals but appeared later in 7. In most individuals, HIV-1HXB2 neutralization was not accompanied by reactivity to a V3 peptide from this strain, indicating that the extension of neutralizing activity to more divergent strains, which takes place at later stages, must be attributed to non-V3-directed antibodies.

Structural and functional analysis of negatively charged milk proteins with anti-HIV activity.

Several polyanionic reagents such as dextran sulfates, heparin sulfates, and negatively charged proteins have been reported to exhibit anti-HIV activity in vitro. Particularly potent inhibition has been reported for the milk protein beta-lactoglobulin (betaLG) on modification by 3-hydroxyphthalic anhydride (3HP). The introduction of multiple negatively charged carboxyl groups along the polypeptide backbone obviously leads to repulsion within the protein molecule and this is likely to affect the specific tertiary, and perhaps also secondary, structure of the protein. We used several biophysical techniques to probe the structural changes that occur on 3HP modification of betaLG. The results suggest that the protein becomes largely unstructured on chemical modification. Although a profound anti-HIV activity was measured for 3HP-betaLG, similar antiviral effects were observed with two other 3HP-modified milk proteins, alpha-lactalbumin and alpha(S2)-casein, but not with the unmodified proteins. Most potent inhibition of HIV-1 replication was obtained with 3HP-modified alpha-lactalbumin, which also demonstrated the least cytotoxicity. These combined results indicate that HIV inhibition is a general property of negatively charged polypeptides and do not support a model in which the negatively charged 3HP-betaLG protein interacts in a structure-specific manner with the CD4 cell surface receptor for HIV-1 entry.

Quantitative determination of kinins released by trypsin using enzyme-linked immunosorbent assay (ELISA) and identification by high-performance liquid chromatography (HPLC).

Rapid ELISA and HPLC procedures were developed for quantitation and identification of various natural kinins. Antibradykinin mouse monoclonal antibodies were used to determine kinin levels in the range of 20-200 ng. Bradykinin coupled to bovine serum albumin was used to coat the plates in a 3- to 4-hr ELISA. Synthetic kinin standards isoleucine-seryl-bradykinin (Ileu-Ser-BK), methionyl-lysyl-bradykinin (Met-Lys-BK), tyrosine-bradykinin (Tyr-BK) and bradykinin (BK) yielded almost identical curves with a mixture of A5 and D9 monoclonal antibodies. [Tyr5]-BK, [Tyr8]-BK and des-arginine9 bradykinin (des-Arg9-BK) showed negligible amounts of cross-reactivity. ELISA-compatible trypsin digestion developed for release of kinins from plasma of normal humans, rats and turpentine-treated rats gave values of 3.2, 6.9 and 70 micrograms/ml plasma, respectively. High performance liquid chromatography methods were developed for complete resolution of kinins on a C-18 reversed phase mu-Bondapak column before and after derivatization with phenyl isothiocyanate (PITC). The simple PITC derivatization procedure yielded good quantitation above 20 pmol. The ELISA and HPLC methods were used in a complementary fashion to assay and identify kinins in biological fluids as well as during the course of kininogen purification.

Rodent kinin-forming enzyme systems--I. Purification and characterization of plasma kininogen.

Low molecular weight (LMW) kininogen was purified 70-fold with a 16% yield from fresh rat plasma by DEAE-Sephadex chromatography, ammonium sulfate precipitation, Sephadex G-200 gel filtration, SP-Sephadex chromatography, CM-cellulose chromatography, and Sephadex G-200 gel filtration. Ferguson plots of polyacrylamide gel electrophoretic patterns revealed four bands with relative molecular weights of 64,000, 123,500, 252,436 and 357,900 (ratio of 1:2:4:6). Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis provided a single protein band with a molecular weight of 72,000, suggesting that the four kininogen bands had been caused by the aggregation of a single oligomeric protein. The purified LMW rat kininogen Fraction B (3.9 micrograms bradykinin/mg) was used to elicit an antiserum in the rabbit. Monospecificity of the antiserum was demonstrated by immunoelectrophoresis (Laurell rocket and Grabar methods) and, thus, the homogeneity of the kininogen was also. The purified kininogen (both Fractions A and B) formed kinin with human urinary kallikrein, rat urinary kallikrein and hog pancreatic kallikrein. Murphy-Sturm lymphosarcoma acid protease also formed kinin when incubated with the kininogen at pH 3.0. The isoelectric point for both fractions was at pH 4.3. Amino acid analyses showed the two kininogen fractions to be rich in acidic amino acids and to have a total carbohydrate content of 8.5% consisting of galactose (1.2 to 1.5%), mannose (1.9 to 2.1%), N-acetylglucosamine (4.3 to 5.1%), N-acetylgalactosamine (0.3%), and sialic acid (0.68%).

Rodent kinin-forming enzyme systems--II. Purification and characterization of an acid protease from Murphy-Sturm lymphosarcoma.

An acid protease from the rat Murphy-Sturm lymphosarcoma (MSLS) tumor was purified 640-fold by extraction of the tumor tissue, acid precipitation with glacial acetic acid, ammonium sulfate precipitation, DEAE-Sephadex A-50 batch adsorption, QAE-Sephadex A-50 column chromatography, Sephadex G-200 gel filtration, and CM-32 cellulose chromatography. The protease hydrolyzed bovine hemoglobin and formed vasopeptide kinins when incubated with purified rat plasma kininogen. Two protease fractions obtained by Sephadex G-200 gel filtration had identical molecular weights of 39,500-41,000 and were similar in other physico-chemical and kinetic characteristics. The purified enzyme showed three major isozymic forms (alpha, beta and gamma) with isoelectric points (pI) of 5.2, 5.5 and 5.8, respectively, and nearly identical amino acid compositions. The enzyme had a high moles percent of both aspartic and glutamic acids. The carbohydrate moiety of the enzyme contained 2 moles of N-acetylglucosamine and 8 moles of mannose per mole of enzyme. The pH optimum for the digestion of bovine hemoglobin was approximately 3.0 with a sharp decline of activity on either side of the pH optimum. The protease activity was very stable above pH 3.4. The Km values for the purified enzyme fractions A and B were 31.17 and 31.19 microM, respectively, and the corresponding Vmax values were 6.17 and 5.5 microM tyrosine per mg per min at 37 degrees and pH 3.0. The enzyme was inhibited strongly by pepstatin (Ki = 31 X 10(-9)M and alpha = 0.1). The acid protease released kinin from purified rat plasma kininogen at an initial rapid rate which plateaued at 460 ng bradykinin equivalents/mg substrate after a 2-hr incubation at 37 degrees.

Control of methicillin-resistant Staphylococcus aureus in a neonatal intensive-care unit: use of intensive microbiologic surveillance and mupirocin.

OBJECTIVE: To describe the epidemiology and the interventions used to control two methicillin-resistant Staphylococcus aureus (MRSA) epidemics involving 46 infants with two fatalities in a neonatal intensive care unit (NICU). SETTING: A 50-bed, level III NICU in a university hospital. INTERVENTIONS: After traditional interventions failed to stop the first epidemic, an intensive microbiologic surveillance (IMS) program was developed. Cultures were obtained on all infants each week, and those colonized with MRSA were isolated. When an infant was found to be colonized with MRSA, cultures immediately were obtained on all surrounding infants. This was continued until no MRSA-colonized infants were found in the area. During the first epidemic, mupirocin was used in an attempt to eradicate the organism from the unit. RESULTS: All infants, colonized and noncolonized, and parents of and personnel working with colonized infants were treated simultaneously with 5 days of mupirocin. This failed to eradicate MRSA in colonized infants. The spread of MRSA ceased in the unit, but a second epidemic occurred 4 months later. This time, IMS alone was successful in quickly containing the epidemic, and MRSA disappeared from the unit after all colonized infants were discharged. Plasmid analysis demonstrated that the same strain was responsible for both outbreaks. CONCLUSIONS: IMS and isolation are effective in containing the spread of MRSA in an NICU. The use of mupirocin failed to eradicate the organism.

New instrument for automatic recording of clot lysis.

An experimental instrument that determines and records automatically the end-point of clot lysis is described. The instrument is unique in that it measures the end product of lysis directly and quantitatively, unlike currently available indirect methods that rely on the optical or mechanical changes indicative of lysis. Data showing that the instrument is capable of yielding reliable, reproducible results in a variety of experimental conditions are presented.