Genetical analysis of all Danish patients diagnosed with chronic granulomatous disease.

Chronic granulomatous disease (CGD) is a rare inherited disorder of the innate immune system caused by a defect in NADPH oxidase, leaving the granulocytes unable to kill invading microorganisms. CGD is caused by mutation in one of the five components gp91phox, p22phox, p47phox, p67phox and p40phox, encoded by the X-linked CYBB gene and the autosomal CYBA, NCF1, NCF2 and NCF4 genes respectively. We have collected samples from all Danish patients with known CGD followed in the clinic or newly diagnosed during a 5-year period, a cohort of 27 patients, and characterized them genetically. The cohort includes 10 male patients with X-linked CGD and one female with extremely lyonized expression of a defective CYBB allele. Six patients had mutation in CYBA. Seven of 10 patients with a defect in NCF1 were homozygous for the common GT deletion, one was compound heterozygous for the GT deletion and a splice-site mutation, and two patients were homozygous for a nonsense mutation in exon 7. Three novel mutations were detected, a deletion of exon 6 in CYBA, a duplication of exon 8-13 in CYBB and a splice site mutation in intron 7 of NCF1.

Low thymic size in preterm infants in the neonatal intensive care unit, a possible marker of infection? A prospective study from birth to 1 year of age.

AIM: To study the growth of the thymus in preterm infants. METHODS: Ultrasonographic thymic size (Ti) was studied in 80 preterm infants (gestational age 24-36 weeks) from birth to discharge from the neonatal intensive care unit (NICU). Thirty-three of these infants were followed to 1 year of age. RESULTS: At birth, the median Ti was 5.2 compared with 11.8 in term infants. At discharge, the median Ti was 10.0 and not significantly different from Ti in term infants at birth (p = 0.22). The size of the thymus was significantly associated with postmenstrual age and weight (both p < 0.01). Infections during admission were negatively associated with the size of the thymus (p < 0.01). During the first 3 months after discharge, preterm infants had a significantly higher frequency of infections than did term infants (p = 0.002); hereafter, the preterm infants had significantly fewer infections than term infants (p = 0.002). The median Ti in preterm infants and term infants at 1 year of age was 21.1 and 17.3, respectively. This difference was not statistically significant (p = 0.41). CONCLUSIONS: Growth of thymus was not compromised by preterm birth. Ti is negatively associated with the frequency of infections in preterm neonates submitted to NICU.

Prevention of mother-to-child transmission of HIV in Denmark, 1994-2008.

OBJECTIVES: The aim of this study was to describe trends in the management of pregnancies in HIV-infected women and their outcomes over a 14-year period in Denmark on a national basis. METHODS: The study was a retrospective cohort study of all HIV-infected women in Denmark giving birth to one or more children between 1 June 1994 and 30 June 2008. RESULTS: We identified 210 HIV-infected women with 255 pregnancies, ranging from 7 per year in 1995 to 39 per year in 2006. Thirty per cent of the women were Caucasian and 51% were Black African. Knowledge of HIV status before pregnancy increased from 8% (four of 49) in 1994-1999 to 80% (164 of 206) in 2000-2008. Only 29% (53 of 183) of the women chose to consult an infectious disease specialist when planning pregnancy, while 14% (27 of 199) received assistance with fertility. The proportion of women on antiretroviral therapy (ART) increased from 76% (37 of 49) in 1994-1999 to 98% (201 of 206) in 2000-2008. Vaginal deliveries ranged from 0 in 2003 to 35% of pregnancies in 2007. Mother-to-child transmission (MTCT) of HIV decreased from 10.4% in 1994-1999 to 0.5% in 2000-2008. All women giving birth to an HIV-positive child were diagnosed with HIV during or after delivery and did not receive prophylactic ART. CONCLUSIONS: The annual number of HIV pregnancies increased fivefold during this 14-year period and substantial changes in pregnancy management were seen. No woman treated according to the national guidelines, i.e. ART before week 22, intravenous zidovudine (ZDV) during labour, neonatal ZDV for 4 to 6 weeks and no breastfeeding, transmitted HIV to her child.

Chronic granulomatous disease with partial deficiency of cytochrome b558 and incomplete respiratory burst: variants of the X-linked, cytochrome b558-negative form of the disease.

Five male patients from four different families presented with a clinical record of chronic granulomatous disease (CGD): recurrent infections of the skin and/or respiratory tract with catalase-positive microorganisms, sometimes in combination with granulomata and/or abscesses in various organs. These patients differed from "classical" forms of the disease in that their neutrophils, although deficient in killing in vitro of Staphylococcus aureus, contained a decreased but measurable amount of cytochrome b558 (10-60% of normal on a heme basis), causing weak staining in the nitroblue tetrazolium dye test and a depressed respiratory burst after contact of the cells with fluid or particulate activators of the NADPH:O2 oxidoreductase. In the cell-free activation system, the defect in the patients' cells was localized in the membrane fraction. In each of the four families, the cellular abnormalities showed an X-linked inheritance. Fusion experiments performed with the monocytes from these patients and those from patients with classical X-linked, cytochrome b558-negative (Xb(0)) or autosomal, cytochrome b558-positive (Ab+) CGD showed complementation of NADPH:O2 oxidoreductase activity in the latter but not in the former combination. Thus, the unusual CGD patients represent variant forms of Xb(0) CGD, with mutations in the gene coding for the beta subunit of cytochrome b558 that do not cause complete loss of this protein.

Refractory Pneumocystis carinii infection in chronic granulomatous disease: successful treatment with granulocytes.

Pneumocystis carinii pneumonia developed in an 11-year-old girl with chronic granulomatous disease who had normal cellular and humoral immunity. The patient remained febrile during treatment with sulfamethoxazole-trimethoprim and pentamidine but became afebrile when treated with a series of 12 granulocyte transfusions combined with sulfamethoxazole-trimethoprim. In addition to documenting P carinii infection in chronic granulomatous disease our findings suggest that granulocyte transfusions may be of value in the treatment of severe infections in chronic granulomatous disease.

Fluoroquinolones in the treatment of cystic fibrosis.

Cystic fibrosis patients suffer from recurrent and chronic lung infections mainly caused by Staphylococcus aureus, Haemophilus influenzae and Pseudomonas aeruginosa. The fluoroquinolones, notably ciprofloxacin and ofloxacin, represent an important addition to the therapy of P. aeruginosa infections. They offer the possibility of effective oral treatment for early colonisation as well as chronic infections, even in children. They are associated with only few and mild adverse effects. Development of resistance represents an increasing problem.

Endotoxemia and enhanced generation of oxygen radicals by neutrophils from patients undergoing cardiopulmonary bypass.

Plasma endotoxin concentrations and oxidative burst response of peripheral blood polymorphonuclear leukocytes were examined in 12 patients undergoing coronary artery bypass. The measurements were made just before the operation, 5 minutes after removal of the aortic crossclamp, and 24 hours after the operation. Endotoxin was quantitated by a combination of a sensitive Limulus amebocyte lysate assay and rocket immunoelectrophoresis measuring picogram amounts of endotoxin. Peripheral blood neutrophils were purified by a two-step dextran sedimentation and metrizoate sodium Ficoll (Lymphoprep., Nyegaard, Oslo, Norway) centrifugation. The oxidative burst response of these cells was measured for their ability to generate superoxide anion and was determined by a cytochrome c reduction assay. Preoperatively, all the plasma samples except one were free of endotoxin. The endotoxin levels reached 100 pg/ml 5 minutes after removal of the aortic crossclamp, and except in one sample they had decreased 24 hours after the operation. Studies on the generation of superoxide by neutrophils showed a decline in the response 5 minutes after removal of the aortic crossclamp and an enhancement of the response to f-Met-Leu-Phe by cells obtained from 11 of 12 patients 24 hours postoperatively. In vitro addition of bacterial lipopolysaccharide to blood from healthy individuals also enhanced the superoxide response of the neutrophils. We conclude that during cardiopulmonary bypass the circulating blood is contaminated by endotoxin and the neutrophils are primed for enhanced generation of oxygen radicals. The released oxygen radicals may be involved in the tissue damage observed in these patients.

[Tuberculous meningitis in children]. / Tuberkuløs meningitis hos børn.

Medical records of six children with tuberculous meningitis were reviewed. The patients were admitted to the paediatric departments in the city and country of Copenhagen between 1983 and 1997. Interpretation of cerebrospinal fluid findings, and difficulties in early recognition of tuberculous meningitis are discussed.

[Idiopathic pulmonary hemosiderosis. A cause of severe iron deficiency anemia in childhood]. / Idiopatisk pulmonal haemosiderose. En årsag til svoer jernmangelanoemi i barnealderen.

The most common cause of anaemia in childhood is iron deficiency. Anaemia due to poor dietary iron is seen most often between 9-24 months of age in infants being fed with large amounts of milk during periods of high growth rate. After the age of two years one must look for other causes of iron deficiency, particularly blood loss. In the description of three cases of idiopathic pulmonary haemosiderosis, the problems of severe chronic anaemia in childhood are discussed. Idiopathic pulmonary haemosiderosis is a rare disease in children, characterized by iron deficiency, anaemia and pulmonary symptoms. It has a high mortality, mostly due to acute pulmonary haemorrhage. However, early diagnosis is important, as some therapeutical regimens have succeeded in delaying the pulmonary haemorrhages and a few patients have possibly gained complete remission.

[Acute transverse myelitis caused by enterovirus]. / Akut transversel myelitis forårsaget af enterovirus.

Enteroviruses comprise a group of commonly encountered small RNA viruses with genetic similarities. We report a case of transverse myelitis associated with an enterovirus infection. By use of PCR, enterovirus-specific RNA sequences were detected in the cerebrospinal fluid and in swabs from the throat and the rectum at the time of admission. Routine cultures and serology gave no other explanation for the clinical condition. The patient was treated with intravenous immunoglobulin and steroids and improved gradually. She was fully recovered 18 months after onset of disease.

[Occurrence of tuberculosis among children in the area of Copenhagen 1984-1993]. / Tuberkuloseforekomst blandt børn i Københavnsområdet 1984-1993.

This study was undertaken to describe the epidemiology, clinical manifestations and prognosis of childhood tuberculosis in Copenhagen, with special attention to differences between Danish children and children of foreign origin. Medical records for all children with tuberculosis cared for in the hospitals of the Copenhagen area 1984-1993 were reviewed. Sixty-six patients were identified. Sixteen of 20 Danish patients (80%) and 67% of foreign children had respiratory tuberculosis. Tuberculosis located in cervical lymph nodes was found only in children of foreign origin. Five patients had meningitis. The high incidence among foreign children reflects the incidence in their home countries, but poorer living conditions among ethnic minorities in Denmark may facilitate transmission of tuberculosis. Severe manifestations of tuberculosis still occur, even in a low incidence country.

In vitro effect of R 17934, a new drug with antitubulin activity, on neutrophil granulocyte locomotion and orientation.

R 17934 is a new synthetic antimicrotubule drug, which binds to tubulin at the same site as does colchicine. The interaction of this drug with human neutrophil granulocyte locomotion and orientation was examined, using a modified reversible Boyden chamber and a gradient chamber offering a direct visual assay of cell orientation. R 17934 at concentrations as low as 5 x 10(-6) M was found to inhibit chemokinesis to an equal degree in gradient chambers, and in Boyden chambers using checkerboard experiments, in which the absolute concentrations and the concentration gradients of the chemotactic agent were varied. At higher concentrations (10(-5) M) R 17934 also inhibited chemotaxis in Boyden chambers. Much lower concentrations of R 17934 (10(-7) M) were found to affect the ability of neutrophils to orient in gradient chambers. This finding may show that filters form a more suitable substrate for oriented pseudopod formation of microtubule-deficient cells than do glass or plastic slides. In conclusion this study has provided evidence that in the absence of functioning microtubules neutrophils can still orient and migrate directionally towards the source of a concentration gradient, but that they do so less precisely. The finding that R 17934 inhibited chemokinesis suggests that this drug may interfere with the mechanism translating a recognition signal into locomotion. The previous finding that colchicine also had this effect supports the hypothesis that this process may be microtubule-dependent.

In vitro effect of colchicine on neutrophil granulocyte locomotion. Assessment of the effect of colchicine on chemotaxis, chemokinesis and spontaneous motility, using a modified reversible Boyden chamber.

The effect of colchicine on human neutrophil granulocyte chemotaxis, chemokinesis and spontaneous motility was examined, using a modified reversible Boyden chamber. Colchicine was shown to inhibit the attraction of neutrophils to casein and to a bacterial chemotactic factor at concentrations as low as 10(-7) M. Experiments in which the absolute concentrations and the concentration gradients of the chemotactic agent were varied, revealed that colchicine inhibited chemokinesis rather than chemotaxis. The spontaneous motility measured in the absence of chemotactic agents was not inhibited by colchicine. Pre-incubation of the cells with a bacterial chematactic factor did not change the sensitivity of the cells to colchicine. It is concluded that the integrity of microtubule function is not necessary for the ability of the cells to discern a concentration gradient or to react to this with directional locomotion. Thus the inhibitory effect of colchicine on neutrophil granulocyte chemokinesis may not depend on its inhibition of microtubule function. It is suggested that colchicine may block the still unidentified membrane mechanism involved in the translation of the recognition signal into an appropriate locomotory cell response.

Neutrophil granulocyte chemotaxis in vitro. Comparison of the response to casein and a bacterial chemotactic factor, and evaluation of an automatic method for counting cells on a membrane filter surface.

A method is described for evaluating neutrophil granulocyte chemotaxis in vitro, using a modified reversible Boyden chamber. The variation in cell detachment from the attractant surface of the filter after the migration through the filter was shown to be eliminated by reversing the chambers during the incubation period. In unreversed chambers, the loss of cells from the bottom surface of the filter was much higher on attraction with casein than with a bacterial chemotactic factor (BCF). The finding of a different kinetic response to casein and to BCF may suggest differing affinity to various chemoattractants within circulating neutrophils. The results obtained after introducing an automatic quantitative image analysis system for the counting of the cells at the filter surface are found to be very well correlated to those with conventional direct microscope counting.

Chemotaxis, spreanding nd oxidative metabolism of neutrophils: influence of albumin in vitro.

Studies on polymorphonuclear leukocyte (PMN) migration in vitro have shown that albumin increases the speed of the cells without affecting their orientation, i. e. chemokinesis. In order to analyze this phenomenon the effect of albumin on PMN migration in a Boyden chamber towards various chemotactic agents was examined. On attraction with low molecular weight chemotactic factors (a culture filtrate of E. coli (BCF)) or the synthetic peptide N-Formyl-Methionyl-Leucyl-Phenylalanine (F-Met-Leu-Phe) albumin enhanced the migration of PMN. In contrast, albumin did not enhance the migration towards casein, a high molecular weight chemotactic factor, except at very low concentrations. In a direct visual assay, albumin was found to impair PMN spreading on a polystyrene Petri dish. In the presence of BCF or F-Met-Leu-Phe cell spreading remained inversely related to the concentration of albumin, while addition of casein again eliminated the influence of albumin. The effect of albumin on the interaction of PMN with a substrate was studied by measuring the superoxide anion (02-) release by PMN sedimenting on a micropore filter. The 02- release triggered by this stimulus of the PMN membrane was inversely correlated to the concentration of albumin. These results show that one mechanism of the enhancing effect of albumin on PMN migration is to diminish adhesion of the cells to the substratum, so that it remains reversible. It is suggested that the term chemokinesis is equivocal. The chemokinetic activity of chemotactic factors would be the result of specific stimulation of PMN locomotion. In contrast, the chemokinetic activity of albumin is due to changes of the physico-chemical environment which support PMN locomotion.

Intrauterine supraventricular tachycardia.

Four cases of intra-uterine ectopic supraventricular tachycardia are described. In three there were none or only minor symptoms immediately after delivery and subsequently. The fourth baby having a congenital W-P-W-syndrome was born with severe hydrops fetalis and was asphyxiated. From these cases and from data reported in the literature it is concluded that intra-uterine heart failure is a significant risk when the fetal heart rate persistently exceeds 230 per minute. It is suggested that digoxin administered to the mother may be beneficial to the fetus in cases of intra-uterine ectopic tachycardia.