Clinical and physiologic studies of two siblings with prekallikrein (Fletcher factor) deficiency.

Two siblings with hereditary Fletcher factor (prekallikrein) deficiency were studied for alterations of fibrinolysis, platelet function, skin inflammatory responses, permeability factor (PF/dil) formation and leukocyte chemotaxis. In vivo stimulation of fibrinolytic activity was normal; the bleeding time and platelet functions (adhesivity, aggregation, release reaction) were also normal. Both immediate (wheal-flare reaction to histamine, bradykinin, prostaglandin E1, physical agents) and delayed sensitivity skin test reactions were within normal limits. Migration of subjects' leukocytes to attractants in skin windows and in Boyden-type chambers was the same as that of control leukocytes. Serum complement components were essentially normal. One subject's leukocytes showed normal tissue factor production on stimulation by endotoxin, although prekallikrein deficiency did impair the endotoxin-stimulated generation of serum procoagulant activity. PF/dil caused increased vessel permeability in human skin; in vitro generation of PF/dil required both the Hageman factor and prekallikrein. The Fletcher factor-deficient subjects responded in a normal manner to PF/dil. Based on the Fletcher factor-coagulation assay, the biologic half-disappearance time of prekallikrein (after the transfusion of normal plasma in one of the subjects) was estimated at 35 hours. Therefore, these studies suggest that severe prekallikrein (Fletcher factor) deficiency in man is not associated with any clinically significant impairment in hemostasis, fibrinolysis, inflammatory responses or leukocyte function.

Desquamative interstitial pneumonia and antigen-antibody complexes in two infants with congenital rubella.

Desquamative interstitial pneumonia was observed in two infants with the late-onset congenital rubella syndrome. In both infants this unusual lung disease was associated with circulating immunoglobulin M complexes and interstitial pulmonary deposits of IgM by immunofluorescence. Both infants had IgG deficiency. The first child recovered with a reduction in IgM complex levels and synthesis of rubella-specific IgG. The second infant died during the acute phase of his illness at which time there were high serum concentrations of IgM complexes and slightly increased levels of IgG complexes. Delayed maturation of the immune response in congenital rubella may predispose to persistent antigenemia and pulmonary deposition of rubella antigen-containing IgM complexes resulting in an acute form of interstitial pneumonia.

Tissue factor pathway inhibitor (TFPI) levels in the plasma and urine of children with meningococcal disease.

Tissue factor pathway inhibitor (TFPI) is a potent inhibitor of the TF-dependent coagulation system. In meningococcal disease, up-regulation of tissue factor expression on blood monocytes and possibly on endothelial cells has the potential to trigger the activation of the TF-dependent pathway of coagulation. Intravascular coagulation is considered to be a major pathogenic factor in meningococcal disease. We postulated that imbalance between TF expression and TFPI concentration might lead to uncontrolled coagulation in meningococcal disease. The aim of this study was to assess the levels of total TFPI in the plasma of patients with meningococcal disease and assess whether increased leaking of the TFPI was occurring. TFPI antigen levels and activity were measured in the plasma of 54 patients with meningococcal disease, and 13 healthy control children. TFPI antigen level were also determined in the urines of 14 of the 54 and 9 healthy control children. Plasma TFPI activity was reduced in the meningococcal diseased patients (mean of 0.503 +/- 0.341 U/ml; control, 1.010 +/- 0.199 U/ml: p <0.0001), as was the TFPI antigen levels (mean of 54.85 +/- 35.05 ng/ml; Control, 94.51 +/- 11.44 ng/ml; p <0.0001). In contrast, TFPI antigen levels were increased in the urine of these patients when compared to the levels found in the urine of the healthy control children (mean of 12.96 +/- 5.392 ng/mmol creatinine; Control, 0.239 +/- 0.191 ng/mmol creatinine; p <0.035). A lack of correlation between TFPI-activity and TFPI-antigen plasma levels was observed (r = 0.002, p = 0.85). This data is consistent with the hypothesis that in meningococcal disease there is increased inactivation of plasma TFPI by the up regulation of tissue factor expression but in addition increased clearance of TFPI in urine is occurring.

Suppression of human monocyte tissue factor synthesis by antisense oligodeoxynucleotide.

Tissue factor (TF) is a cellular receptor and cofactor for factor V11/V11a which initiates the blood coagulation cascade. An understanding of TF cell biology is therefore of critical importance to the pathophysiology of many disorders. We have utilized an antisense oligodeoxynucleotide (aODN), directed against human blood monocyte TF mRNA, to investigate the feasibility of inhibiting the synthesis of TF. CD14 receptor-mediated endocytosis was used as a means of delivering TF aODN to monocytes. This DNA carrier system consists of the fab portion of anti-CD14 antibody covalently linked to poly (L-lysine). Co-treatment of monocytes with TF aODN and endotoxin resulted in 80.4 +/- 2.2% suppression of TF activity when compared with control endotoxin stimulated cells. Control experiments with TF sense ODN, mismatched aODN, and an irrelevant aODN were performed to exclude nonspecific inhibitory effects. The cytotoxicity of the DNA carrier complex was also evaluated. These results demonstrate that this TF mRNA antisense ODN specifically suppressed the synthesis of biologically active monocyte TF and that antisense ODNs might therefore represent a useful tool in the investigation of monocyte/macrophage TF function.

Co-localization of tissue factor protein and its mRNA in human blood monocytes.

Triggering of both the extrinsic and intrinsic coagulation pathways is mediated by the cell surface receptor Tissue Factor (TF). The ability to observe the cell surface TF protein and its mRNA at the single cell level would facilitate our understanding of the cellular biology of TF in health and diseased states. Employing the methods of immuno-gold silver staining and in situ hybridization using non-isotopically labelled oligoprobes, tissue factor antigen and mRNA were detected simultaneously in endotoxin stimulated human peripheral blood monocytes.

Production of tissue factor by monocyte progenitor cells.

Tissue factor (TF) is known to be produced by monocytes from human peripheral blood. However the production of this factor by haematopoietic progenitor cells is not yet known. We thus studied human monocyte progenitor cells isolated from bone marrow of normal and diseased individuals. These cells were non-adherent, monocytic and able to phagocytose particles ranging from 0.3-1 microns. Unactivated partial thromboplastin time clotting assay demonstrated procoagulant activity consistent with TF function, which was blocked by a neutralizing anti-TF monoclonal antibody, G12. The production of TF messenger RNA was demonstrated on dot blot and northern blot analysis utilizing an oligonucleotide probe.

Group B streptococcal colonisation and the outcome of pregnancy.

Vaginal colonisation of pregnant women with group B streptococci (GBS) was not related to age, parity or blood group. There were marked differences between racial groups, Asians having a low colonisation rate and Negroes a high rate. Vaginal GBS colonisation was associated with intrapartum pyrexia, but not with preterm labour, premature rupture of membranes or other complications in labour. Group B streptococci may be an important cause of bacteriuria in pregnancy and their effect on the outcome of pregnancy as urinary pathogens needs further evaluation.

The cardiovascular and oncotic effects of albumin infusion in premature infants.

We determined the oncotic and cardiovascular effects of a standardised infusion of human albumin (1.2 g/kg over 2 h as a 20% solution) in 12 premature infants on 18 occasions when hypovolaemia was suspected on clinical grounds. Blood volume increased by a median value of 15.5%, and fell to preinfusion values by 3 h post infusion in all but four cases. Albumin concentration and colloid osmotic pressure rose during infusion and remained raised even when blood volume had fallen to preinfusion levels. Blood pressure rose in 3 cases only and heart rate fell by greater than 5 beats/min in 6 cases. Indices of long- and short-term heart rate variability were unchanged, but blood pressure variability fell in the second hour of infusion (P = 0.03), an effect which was independent of changes in lung inflation. No changes in blood gases or oxygenation occurred during infusion and no evidence of pulmonary oedema was found. There were wide variations in oncotic and cardiovascular responses to the standardised infusion both between and within subjects. When human albumin is infused in this manner some protection against respiration-induced variability in blood pressure can result, but the circulatory response may prove difficult to predict in the individual.

Coagulation changes associated with a high haematocrit in the newborn infant.

Sequential blood coagulation studies were performed on samples from three newborn infants with venous haematocrit values of over 65%. Combinations of thrombocytopaenia, circulating fibrin monomer and evidence of intravascular thromboplastic activity were found. Reduction of the haematocrit value by partial exchange transfusion in each infant was followed by improvement of the abnormal coagulation findings. Some possible mechanisms for the origin of the coagulation abnormalities are considered.

Decision making in the neonatal intensive care environment.

Consideration as to whether withdrawal of intensive care support might be a more appropriate line of action than to continue with full intensive care has become a part of the life and death decision making process undertaken in neonatal intensive care units. After outlining the moral objectives of delivery of health care, the arguments for taking quality of life and its various components into account during these deliberations are presented. The circumstances in which the appropriateness of continuing care should be considered are highlighted and the care options presented. The crucial importance of allowing time for parents to come to terms with the situation is emphasised as is the need for giving clear guidelines to junior staff over resuscitation issues. Finally, an environment for providing optimal family support during the process of withdrawal is suggested.

Studies on tissue factor activity and production by leukocytes of human umbilical cord and adult origin.

Human adult and umbilical cord-derived leukocytes were shown to be capable of generating tissue factor activity on exposure to endotoxin and to reduced pH. Blood for leukocyte separation was collected from normal adults and from newly delivered sections of umbilical cord and mixed leukocyte preparations were obtained by separation over methyl cellulose Hypaque. The coagulant activity of the cell suspension was assayed using a one-stage or two-stage method. Cord-derived leukocytes were shown to develop greater coagulant activity than adult-derived leukocytes when stimulated by endotoxin in vitro at 2,000 cells/mm-3. This response to endotoxin was partially inhibited by prior exposure of the cells to prostaglandin (PG) E1 an to L-epinephrine. Acetylcholine stimulated the production of coagulant activity in the absence of endotoxin. Both cord and adult-derived leukocytes (20,000/mm-3) developed coagulant activity when exposed to pH reduction by lactic or hydrochloric acids and this activity was shown to be tissue factor.

Neonatal candida septicaemia: diagnosis on buffy smear.

We report a case of neonatal candida septicaemia diagnosed by examination of a buffy smear. This would seem to be a fairly simple test for a disease where a good prognosis is dependent on rapid diagnosis and isolation using standard culture techniques is notoriously unforthcoming.

Defect of platelet function associated with chronic hypoglycaemia.

Two patients are described with chronic hypoglycaemia; the first having glucose-6-phosphatase deficiency (type I glycogen storage disease), and the second fructose 1:6-diphosphatase deficiency. Both cases were associated with a bleeding diathesis, a defect of platelet aggregation, and a deficiency of platelet adenine nucleotides. The effect on the platelet abnormalities of a period of normoglycaemia was studied in both patients. Correction of the platelet abnormalities occurred rapidly after stabilization of the blood glucose within the normal range. Normal function persisted for the duration of the normoglycaemia, facilitating diagnostic liver biopsy and surgical procedures. A biochemical explanation for the nucleotide deficiency is suggested.

The endotoxin-induced coagulant activity of human monocytes.

Leucocyte suspensions, exposed to endotoxin in vitro, develop coagulant activity which has been identified as tissue factor. Pure suspensions of polymorphonuclear (PMN) neutrophils, lymphocytes and monocytes were exposed to endotoxin and tested for tissue factor activity after 4 h incubation at 37 degrees C. The results indicate that the monocyte is the cell primarily responsible for the endotoxin-induced coagulant activity of mixed leucocyte suspensions, the small amount of activity demonstrated in PMN neutrophil and in lymphocyte suspensions at high cell concentrations being accounted for by monocyte contamination of less than 1%.

Low dose intraventricular fibrinolytic treatment to prevent posthaemorrhagic hydrocephalus.

Posthaemorrhagic ventricular dilatation (PHVD) is thought to be due to clots from intraventricular haemorrhage obstructing cerebrospinal fluid pathways involved in reabsorption. Over 60% of infants with progressive PHVD have gone on to require surgical shunt placement. Previous treatments all have major problems. The object of this pilot study was to achieve enough fibrinolysis to restore pathways of cerebrospinal fluid reabsorption and so avoid shunt surgery. Nine preterm infants with progressive PHVD were treated with intraventricular infusion of streptokinase for 12-72 hours. All the infants survived and surgical shunting was required in only one case. A 200% increase in fibrinolytic activity was demonstrated in both ventricular and spinal fluid during streptokinase treatment. There were no cases of infection. Minor rebleeding occurred in one case and was not a serious problem. This represents the first direct therapeutic approach to the pathology of PHVD.

The expression of surface tissue factor apoprotein by blood monocytes in the course of infections in early infancy.

The expression of surface tissue factor procoagulant activity and its shedding by blood monocytes can be induced by several stimuli. Few of these defined situations, other than the presence of bacteria and their toxins, are commonly present in the young human infant. In this study, measurements were made of the percentage of monocytes expressing surface tissue factor apoprotein (TFA) in blood taken from babies in the early weeks of life. Mononuclear cells were separated from blood in an environment free of detectable endotoxin. After exposure to a polyclonal rabbit antibody raised to purified brain TFA and subsequent exposure to a fluorescin-labeled murine anti-rabbit IgG, the cell fluorescent activity was analyzed by flow cytometry. The percentage of monocytes showing strong fluorescence was determined. In every instance when systemic bacterial infection was present, more than 60% of the monocytes examined showed fluorescence indicative of the presence of surface TFA. In a single case of fungal Candida septicemia, none of the monocytes was positive. More than 60% of cells were found to be positive in certain instances where infection was highly probable but not proven. Positive cells were found in three cases of isoimmune hemolytic disease of the newborn, as had been anticipated from previous studies, whereas less than 25% of monocytes derived from babies in the absence of discernible infection or isoimmune hemolytic disease expressed surface TFA (p less than 0.001). These findings provide insight into a possible mechanism of coagulation activation in sepsis and may prove to be a useful predictor of the presence of infection or endotoxemia in young infants.

The development of anti-HLA antibodies in multiply transfused preterm infants.

The development of antihuman leucocyte antigen antibodies (aHLAA) in response to multiple transfusions in preterm infants was studied prospectively. Fifty seven infants requiring a minimum of two blood transfusions were recruited after obtaining informed written parental consent. They were randomised to receive either whole blood or blood that had been passed through a leucocyte filter. Anti-HLAA were sought in maternal and cord blood so as to ensure that any aHLAA detected after transfusion had not been passively transferred antenatally, and in 1 ml samples drawn monthly from the baby, at least 10 days from a previous transfusion, until discharge from hospital. Anti-HLAA were detected by microlymphocytotoxicity assay. Results were obtained in 42 babies, 19 in the filter and 23 in the no filter group. Fifteen babies had to be excluded because of protocol violation or because they died. None of the babies receiving filtered blood developed aHLAA, but seven babies in the no filter group developed aHLAA. In conclusion, multiply transfused preterm infants have the ability to elaborate antibodies to HLA and leucocyte filters may prevent this.