Amidolytic assay of human factor XI in plasma: comparison with a coagulant assay and a new rapid radioimmunoassay

The traditional coagulant assay for plasma factor XI suffers from a relatively high coefficient of variation, the need for rare congenitally deficient plasma, and a poor correlation between precision and sensitivity. We have developed a simple functional amidolytic assay for factor XI in plasma using the chromogenic substrate PyrGlu-Pro-Arg- p-nitroanilide (S-2366). After inactivation of alpha 1-antitrypsin, CI inhibitor, and other plasma protease inhibitors with CHCI3, plasma was incubated with kaolin, in the absence of added calcium, which limited the enzymes formed to those dependent on contact activation. Soybean trypsin inhibitor was used to minimize the action of kallikrein on the substrate. Once the reaction was complete, corn trypsin inhibitor was used to inactive factor XIIa, the enzyme generated by exposure of plasma to negatively charged surfaces, which had activated the factor XI. The assay is highly specific for factor XI, since plasma totally deficient in that zymogen yielded only 1%-3% of the enzymatic activity in normal plasma under identical conditions. The requirements for complete conversion of factor XI to XIa in plasma within 60 min were, respectively, factor XII, 0.6 U/ml, and high molecular weight kininogen, 0.2 U/ml. Prekallikrein was not an absolute requirement for complete activation but did accelerate the reaction. The intraassay coefficient of variation was 3.4%, and the mean of 35 normal plasmas was 1.00 U +/- 0.24 SD. In addition, a new rapid radioimmunoassay was devised using staphylococcal protein A as the precipitating agent for a complex of factor XI antigen with monospecific rabbit antibody. The mean was 1.01 U +/- 0.30 SD. The correlation coefficients for amidolytic versus coagulant and amidolytic versus radioimmunoassay were r = 0.95 for the former and 0.96 for the latter. Thus, a simple, accurate amidolytic assay and a radioimmunoassay have been devised for measuring factor XI in plasma that correlate well with the coagulant activity of factor XI, as determined in our laboratory.     

Characteristics of dysphagia in children with cerebral palsy

Videofluoroscopic modified barium swallow (VMBS) examinations may provide clinically relevant information regarding deglutition in children with cerebral palsy and dysphagia. A retrospective review of clinical evaluations and VMBS studies on 90 consecutive children with cerebral palsy and dysphagia was completed. Most children were referred because of concerns regarding airway protection during oral feedings. Most children had multiple disabilities and 93% were nonambulatory. The majority of children were totally dependent for oral feedings (80%). Oral and pharyngeal phase abnormalities were present in almost all patients. Abnormalities of deglutition were observed only while swallowing specific food textures in the majority of patients. Aspiration of specific food textures was significantly more common than aspiration of all food textures (p<0.0001). Finally, aspiration was silent in 97% of the patients. VMBS studies can provide clinicians with valuable information regarding the most appropriate food textures and rates of oral feeding for children with cerebral palsy and dysphagia.     

Emergence and control of methicillin-resistant Staphylococcus aureus in a children's hospital and pediatric long-term care facility.

BACKGROUND: After a 6-year quiescence, methicillin-resistant Staphylococcus aureus (MRSA) was isolated from 30 patients in a children's hospital and a pediatric long-term care facility from November 1987 through April 1989. After six nosocomial cases had occurred at the children's hospital, increased infection control measures directed at MRSA were initiated in August 1988. Because MRSA had been identified in three patients in the pediatric long-term care facility within 24 hours of their admission to the children's hospital, other patients transferred from the pediatric long-term care facility to the children's hospital were isolated and screened for MRSA. METHODS: We reviewed the medical records of these patients and evaluated their response to therapy with rifampin alone or in combination with trimethoprim-sulfamethoxazole. RESULTS: In the 8-month period after initiation of infection control measures, MRSA was identified in 10 residents of the pediatric long-term care facility; there was also one nosocomial children's hospital case. Phage typing showed that one MRSA strain predominated in patients at the pediatric long-term care facility but did not implicate this strain as the source for MRSA introduction into the children's hospital. Of 16 patients with MRSA who completed therapy and were available for follow-up, 13 (81%) had elimination of colonization. CONCLUSION: Prompt institution of MRSA surveillance, barrier isolation, and therapy to eliminate colonization should be considered in hospitals with a new introduction of MRSA.     

Isolation of Mycoplasma pneumoniae from synovial fluid samples in a patient with pneumonia and polyarthritis

A patient with a long history of arthritis developed pneumonia. Two weeks into her hospital course, the patient developed effusions in her knee and wrist that yielded cultures positive for Mycoplasma pneumoniae. To our knowledge, this is the third reported case of M pneumoniae isolation from a joint and the first report of isolation of M pneumoniae from two joints in a patient without hypogammaglobulinemia. The evidence suggests that in individuals with atypical pneumonia and joint effusions, M pneumoniae should be considered as a source of infection.     

Ringversuch zum Nachweis genomischer Veränderungen bei Non-Hodgkin-Lymphomen mittels In-situ-Hybridisierung

Background

The detection of characteristic genomic aberrations by fluorescence in situ hybridization (FISH) has a high diagnostic impact on lymphomas according to the World Health Organization (WHO). To investigate the reproducibility of non-isotopic ISH results a multicenter trial was carried out involving eight institutes for hematopathology.

Material and methods

Analyses were performed on two diffuse large B-cell lymphomas (DLBCL) without known aberrations, on one follicular lymphoma with a IGH/BCL2 translocation and BCL6 split and on two B-cell lymphomas intermediate between DLBCL and Burkitt’s lymphoma with c-MYC and BCL2 rearrangements, one with an additional BCL6 split. Break-apart probes for BCL6 and c-MYC, as well as fusion probes for the c-MYC/IGH and the IGH/BCL2 translocations were used.

Results

All aberrations were correctly detected by all centres and no false positive or false negative results were obtained. The numbers of positive cells varied from 25% to 94%. Pearson’s correlation coefficient between the centres was always >?0.8.

Conclusions

The ISH analysis of recurrent genomic aberrations in formalin-fixed paraffin-embedded (FFPE) tissue is a highly reproducible technique which yields substantial additive help for lymphoma diagnostics.     

Rare Pathogenic Variants in Mitochondrial and Inflammation-Associated Genes May Lead to Inflammatory Cardiomyopathy in Chagas Disease

Abstract

Cardiomyopathies are an important cause of heart failure and sudden cardiac death. Little is known about the role of rare genetic variants in inflammatory cardiomyopathy. Chronic Chagas disease cardiomyopathy (CCC) is an inflammatory cardiomyopathy prevalent in Latin America, developing in 30% of the 6 million patients chronically infected by the protozoan Trypanosoma cruzi, while 60% remain free of heart disease (asymptomatic (ASY)). The cytokine interferon-γ and mitochondrial dysfunction are known to play a major pathogenetic role. Chagas disease provides a unique model to probe for genetic variants involved in inflammatory cardiomyopathy.

Methods

We used whole exome sequencing to study nuclear families containing multiple cases of Chagas disease. We searched for rare pathogenic variants shared by all family members with CCC but absent in infected ASY siblings and in unrelated ASY.

Results

We identified heterozygous, pathogenic variants linked to CCC in all tested families on 22 distinct genes, from which 20 were mitochondrial or inflammation-related – most of the latter involved in proinflammatory cytokine production. Significantly, incubation with IFN-γ on a human cardiomyocyte line treated with an inhibitor of dihydroorotate dehydrogenase brequinar (enzyme showing a loss-of-function variant in one family) markedly reduced mitochondrial membrane potential (ΔψM), indicating mitochondrial dysfunction.

Conclusion

Mitochondrial dysfunction and inflammation may be genetically determined in CCC, driven by rare genetic variants. We hypothesize that CCC-linked genetic variants increase mitochondrial susceptibility to IFN-γ-induced damage in the myocardium, leading to the cardiomyopathy phenotype in Chagas disease. This mechanism may also be operative in other inflammatory cardiomyopathies.