Spectrum, and clinical and functional implications of UNC13D mutations in familial haemophagocytic lymphohistiocytosis.

OBJECTIVE: Familial haemophagocytic lymphohistiocytosis (FHL) is a fatal disorder of immune dysregulation with defective cytotoxic lymphocyte function. Disease-causing mutations have been identified in the genes encoding perforin (PRF1), syntaxin-11 (STX11), and Munc13-4 (UNC13D). We screened for UNC13D mutations and studied clinical and functional implications of such mutations in a well defined patient cohort. METHODS: Sequencing of UNC13D was performed in 38 FHL patients from 34 FHL families in which PRF1 and STX11 mutations had been excluded. RESULTS: We identified six different mutations affecting altogether 9/38 individuals (24%) in 6/34 (18%) unrelated PRF1/STX11-negative families. Four novel mutations were revealed; two homozygous nonsense mutations (R83X and W382X), one splice mutation (exon 28), and one missense mutation (R928P). In addition, two known mutations were identified (R214X and a deletion resulting in a frame-shift starting at codon 782). There was considerable variation in the age at diagnosis, ranging from time of birth to 14 years (median 69 days). Three of nine patients (33%) developed central nervous system (CNS) symptoms. Natural killer (NK) cell activity was impaired in all four patients studied. Defective cytotoxic lymphocyte degranulation was evident in the two patients investigated, more pronounced in the patient with onset during infancy than in the patient with adolescent onset. CONCLUSIONS: Biallelic UNC13D mutations were found in 18% of the PRF1/STX11-negative FHL families. Impairment of NK cell degranulation was less pronounced in a patient with adolescent onset. FHL should be considered not only in infants but also in adolescents, and possibly young adults, presenting with fever, splenomegaly, cytopenia, hyperferritinaemia, and/or CNS symptoms.

Spectrum and clinical implications of syntaxin 11 gene mutations in familial haemophagocytic lymphohistiocytosis: association with disease-free remissions and haematopoietic malignancies.

OBJECTIVE: To determine the frequency and spectrum of mutations in the gene encoding syntaxin 11 (STX11) in familial haemophagocytic lymphohistiocytosis (FHL), a rare autosomal recessive disorder of immune dysregulation characterised by a defect in natural killer cell function. METHODS: Mutational analysis of STX11 by direct sequencing was done in 28 FHL families that did not harbour perforin mutations, previously identified in some FHL patients. A detailed investigation of clinical features of these patients was also undertaken. RESULTS: Two different STX11 mutations were identified, one nonsense mutation and one deletion, affecting six of 34 children in four of 28 unrelated PRF1 negative families. Both mutations have been reported before. Three patients experienced long periods (> or = 1 year) in remission without specific treatment, which is very uncommon in this disease. Despite the milder phenotype, some children with STX11 mutations developed severe psychomotor retardation. Two of the six patients harbouring STX11 gene defects developed myelodysplastic syndrome (MDS) or acute myelogenous leukaemia (AML). CONCLUSIONS: STX11 gene mutations were found in 14% of the PRF1 negative FHL families included in the present cohort. These results suggest that STX11 gene mutations may be associated with secondary malignancies (MDS/AML), and that there is segregation of specific clinical features in FHL patients with an underlying genotype.

Reversible inhibition of lambda exonuclease with high pressure.

The hydrolytic activity of lambda exonuclease, a highly processive enzyme, is inhibited by high pressure. When assayed at 67 MPa, the activity is 87% of the atmospheric rate; increasing the pressure to 336 MPa causes a greater than 99% inhibition of the enzyme activity. Decreasing the pressure from 336 MPa to 67 MPa at 20 degrees C reverses the inhibition. The use of hydrostatic pressure to control the activity of lambda exonuclease has potential practical application in DNA sample preparation, analysis, and sequencing.

Cloning, nucleotide sequence, and engineered expression of Thermus thermophilus DNA ligase, a homolog of Escherichia coli DNA ligase.

We have cloned and sequenced the gene for DNA ligase from Thermus thermophilus. A comparison of this sequence and those of other ligases reveals significant homology only with that of Escherichia coli. The overall amino acid composition of the thermophilic ligase and the pattern of amino acid substitutions between the two proteins are consistent with compositional biases in other thermophilic enzymes. We have engineered the expression of the T. thermophilus gene in Escherichia coli, and we show that E. coli proteins may be substantially removed from the thermostable ligase by a simple heat precipitation step.

Agreement between subject and collateral verbal reports of alcohol consumption in older adults.

The accurate measurement of alcohol consumption in natural environments is a focal empirical issue in studies of the determinants of drinking. Four studies investigated agreement between subject and collateral questionnaire reports of drinking by adults over age 60 years (N = 83), and three of these studies also assessed agreement between daily subject and collateral prospective reports of subject drinking over 4 (Study 2) or 12 (Studies 3 and 4) week intervals. Subject and collateral questionnaire reports were significantly correlated. Excellent agreement was found for the group of subjects for self-monitoring reports of the number of drinking days and quantity of alcohol consumption per day, including reports of the temporal patterning of consumption, but variability in agreement existed across subject-collateral pairs. The discussion focuses on the conditions conductive to accurate measurement and on the importance of investigating predictor variables of individual differences in the accuracy of verbal reports of alcohol consumption.

Cell patterns in the surface of rabbit articular cartilage revealed by the backscatter mode of scanning electron microscopy.

To study the distribution of cells in the surface layer of articular cartilage, rabbit hip and knee specimens were stained with silver and studied by scanning electron microscopy (SEM). The cartilage was treated en bloc using the Gomori methenamine silver technique, which stains the nuclei of exposed cells with reduced silver. The intact surface was then studied with a binocular microscope and SEM in the backscatter mode Only those cells within 30 microns of the surface stained, permitting that population to be imaged selectively. Depressions in the surface were related to groups of cells in clusters or rows bounded by collagen fibers. This study demonstrates the effectiveness of backscatter imaging in the study of chondrocytes. The relationship between surface contours and underlying cells is more complex than previously described.

Preventive aspects of mobility and functional disability.

Among bone and joint diseases, frequent causes of disability include (a) fractures from falls and osteoporosis, (b) osteoarthritis of hips, knees, and lumbar spine, (c) painful feet, (d) back pain, and (e) intractable rheumatoid arthritis. Risk factors for falling may arise from factors intrinsic to the patient and from hazards in the environment. The prevention and management of osteoporosis are controversial. Most experts endorse the use of estrogens in postmenopausal women and the need for continuing physical activity, particularly walking, in both elderly men and women. To preserve independent living, conservative management of osteoarthritis of hip, knee, and low back consists of properly balancing rest, non-weight-bearing exercises, and the use of walking aids. In rheumatoid arthritis, the clinician should emphasize (a) exercises to maintain range of motion and (b) muscle strength, both to prevent flexion contractures and to maintain the ability to carry on activities of daily living.

Predicting the drinking behavior of older adults from questionnaire measures of alcohol consumption.

The relationship between three questionnaire measures of alcohol consumption and self-monitoring reports of drinking in the natural environment was investigated in 83 normal drinkers over 60 years of age. Following questionnaire administration subjects recorded their alcohol consumption each day for up to three months. Collaterals verified subjects' self-monitoring reports. Questionnaire scores and self-monitoring reports of frequency and quantity of alcohol consumption were generally well correlated. Regression analyses showed the Questionnaire Measure of Habitual Alcohol Use to be the best predictor of both drinking frequency and quantity. Measurement issues related to the use of verbal reports of alcohol consumption with elderly drinkers are discussed.

Thin-film characterization using a scanning laser acoustic microscope with surface acoustic waves.

The use of surface acoustic waves in a scanning laser acoustic microscope for the characterization of the mechanical or acoustic properties of thin films deposited on piezoelectric substrates is demonstrated. Quantitative measurements of mass loading effects of 5000-A-thick tungsten films deposited on lithium niobate substrates were obtained using 100-MHz surface acoustic waves. No information about the tungsten film could be obtained using 100-MHz compressional waves. Methods of generating surface waves on nonpiezoelectric materials so that this technique could be used on arbitrary substrates are discussed.

Isolation of two forms of carboxylester lipase (cholesterol esterase) from porcine pancreas.

Carboxylester lipase (cholesterol esterase, EC 3.1.1.13) has been purified to homogeneity from porcine pancreas. The enzyme is isolated in two molecular mass forms, a monomer of 74 kDa and a dimer of 167 kDa. The dimer consists of two catalytically-active subunits which have molecular masses approximately 9 kDa greater than the monomers. The difference in size was not attributable to carbohydrate or lipid content. The catalytic properties of the two forms are comparable on a weight basis, the amino acid compositions are quite similar, and the N-terminal sequences are nearly identical for 24 residues. These similarities suggest a possible precursor-product relationship between the two carboxylester lipase forms.

Premature rupture of the membranes. A review.

Premature rupture of the membranes remains a significant problem area in obstetrics. When the fetal lung maturity is inadequate and there is no evidence of intrauterine infection, a delay in delivery is recommended. When fetal lung maturity is expected or documented, lengthy delays in delivery should be avoided. Assays for phosphatidylglycerol in amniotic fluid collected from the vagina are most helpful in determining the maturity status. Therapeutic agents to accelerate lung maturity, stop labor or prevent infections must be considered investigational when used during the latency phase of premature rupture of the membranes.

Prevention of endomyometritis using antibiotic irrigation during cesarean section.

After double-blind controlled studies demonstrated cefamandole nafate irrigation of the uterus during cesarean section to be effective in reducing the rate of endomyometritis, antibiotic irrigation was adopted as a standard procedure at Tripler Army Medical Center. The present study analyzes the outcome in patients undergoing cesarean section before (comparison group) and after (treatment group) routine use of antibiotic irrigation began. The incidence of endomyometritis in 100 patients from the comparison group was 20% and in 298 patients from the treatment group 1.7% (P less than .0001). Serum analysis for cefamandole nafate demonstrated little systemic absorption of the antibiotic. Cefamandole nafate intrauterine irrigation at cesarean section effectively prevents endomyometritis.