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We developed a sensitive tissue factor (TF) chromogenic assay on a limited number of endothelial cells (EC), performed in microtiter plates, and which uses a normal pooled human plasma instead of purified concentrates as a source of coagulation factors. Primary cultures of human umbilical vein EC (HUVEC), both unstimulated and stimulated by lipopolysaccharide (LPS) were incubated with 50 microliters of of diluted normal human plasma (NHP) and 50 microliters of Factor Xa-specific chromogenic substrate (CBS 31-39, Stago, France). Hirudin was added at 4 U/ml to the plasma/CBS 31-39 mixture to inhibit thrombin generation. Optical densities were read at 405 nm and corresponding amounts of generated factor Xa were expressed in mU Xa/well using a standard curve established with purified human Factor Xa. The following parameters were then defined: the number of EC to plate (10(4) EC/well of a 96-well plate), the plasma-test dilution (1:20), the concentration of CBS 31-39 (0.50 mM) and the incubation time of reagents with EC (2 hours). The procoagulant activity (PCA) measured was only dependent on TF since it was no longer detectable either when FVII-deficient plasma was tested instead of normal human plasma or when PCA assays were performed in the presence of a blocking anti-human TF monoclonal antibody. This method allowed detection of a TF-dependent PCA on as few as 1000 EC per well. In addition, TF expression equal to 50% of maximal values was measured with LPS concentrations as low as 1 ng/ml, supporting the high sensitivity of the assay.

Successful pig-to-human xenotransplantation may expose swine endothelium to the human immune system. Since endothelial MHC class II expression is crucial in the genesis of an allogeneic lymphocyte response, the involvement of porcine MHC (SLA) class II molecules in the induction of human lymphocyte proliferation was studied. When cocultured with a confluent monolayer of irradiated porcine aortic endothelial cells (PAEC), human peripheral blood mononuclear cells (PBMC) incorporated tritiated thymidine. Monocyte depletion strongly reduced the magnitude of the lymphocyte proliferative response. Resting cultured PAEC were SLA class II-negative and an induction of these molecules during the xenogeneic mixed lymphocyte endothelial cell culture (XMLEC) was not observed. Moreover, the addition of an antibody directed against the SLA-DR molecule was without effect. Lymphocyte proliferation was also studied in response to SLA class II-positive stimulating cells--either human TNF-alpha-stimulated PAEC or porcine splenocytes. Induction of SLA class II molecules on PAEC had no effect on the human PBMC proliferative response. Moreover, human PBMC did not proliferate in response to porcine splenocytes. These results suggest (1) that SLA class II molecules are not involved in the induction of the human lymphocyte proliferative response and (2) that the endothelial nature of the stimulating cells plays a key role in this proliferation.

During pregnancy, important modifications of hemostasis occur resulting in mothers in hypercoagulability and the role of placental cells such as trophoblast cells has been hypothesized. In this study, we first showed that syncytiotrophoblast plasma membranes, isolated from normal human placenta, expressed a strong tissue factor (TF) activity. We then studied TF activity of two continuous trophoblast cell lines (JEG-3 and BeWo) in comparison to human umbilical vein endothelial cells (HUVEC) and transformed human endothelial cells (ECV-304). TF assays were performed on intact detached confluent cells. Unstimulated JEG-3 and BeWo cells exhibited a very high TF activity which slightly increased after 2 to 4 h TNF-alpha stimulation. In contrast, HUVEC and ECV-304 had a lower basal TF activity which was mainly inducible by TNF-alpha, with a maximum effect after 4 to 6 h stimulation. For both cell types, TF activity was decreased to basal value after 16-hour TNF-alpha stimulation. These results support that trophoblast cells are able to express TF but the involvement of this property in the hemostatic physiological changes observed during pregnancy, remains to be demonstrated.

Choriocarcinoma cells (CC) in vitro are resistant to NK lysis but sensitive to lysis by blood or decidual effectors activated by interleukin-2 (IL-2). Because lytic activity requires a step of adhesion, the adhesive properties of the choriocarcinoma cells BeWo, JEG-3, and JAR were examined functionally toward peripheral blood lymphocytes. The adhesion of lymphocytes to choriocarcinoma cells was very low and did not increase after stimulating lymphocytes with IL-2. As demonstrated by cytofluorimetry analysis, choriocarcinoma cells and cytotrophoblast cells prepared from term placenta expressed intercellular adhesion molecule-1 (ICAM-1), whereas only CC expressed CD56. Tumor necrosis factor-alpha or interferon-gamma increased the expression of ICAM-1 on choriocarcinoma cells without modifying the adhesion of lymphocytes to choriocarcinoma cells. These results suggest that resistance of choriocarcinoma cells to lysis by cytotoxic effectors could partially be attributed to the low level of lymphocyte adhesion to these cells.

Background: Cardiac abnormalities, such as myocarditis, pericarditis or verrucous endocarditis (Libman-Sacks endocarditis) occur in about one third of patients with systemic lupus erythematosus. This study describes an unusual aspect of endocardial involvement., Case Reports: Case no. 1: A 14 year-old girl was admitted 3 months after acute hemichorea because of heart failure plus biological inflammatory findings. Echocardiography showed mitral insufficiency with enlargement of the left atrium and ventricle. There was some infiltration involving the endocardium of the left ventricle, the chordae tendinae and the mitral valve. The titres of anti-DNA and anti-nuclear antibodies were elevated while the serum hemolytic complement was depressed. Skin biopsy showed IgG, IgM and C1q deposits along the dermoepidermal junction. Corticosteroids, diuretics and vasodilator drugs failed to completely cure the heart failure; the mitral insufficiency required mitral valve replacement 21 months later. Pathological examination showed extended fibrotic changes of the endocardium. Case no. 2: A 4 year-old boy was admitted for acute heart failure due to mitral insufficiency, associated with biological inflammatory findings. Echocardiography showed mitral insufficiency and enlarged left atrium and ventricle. Anti-DNA and anti-nuclear antibody titres were elevated. The patient was given antibiotics followed by corticosteroids and immunosuppressive drugs. The persistence of the mitral insufficiency required mitral valve replacement 7 months later. Pathological examination showed fibrotic changes of the endocardium. Exacerbation of the inflammatory process was seen 2 months after surgery, with development of diffuse proliferative lupus nephritis. The patient died of kidney failure and neurological complications, 44 months after cardiac surgery., Conclusion: In both patients, the systemic lupus erythematosus was revealed by endocardial involvement, a complication that is usually seen later. The endocardium changes responsible for mitral insufficiency and requiring valve replacement in these two cases were different from those described as verrucous endocarditis in classical forms of the disease.

IL-4 and TNF-alpha increase endothelial cell adhesiveness for PBL by promoting the expression of adhesion molecules. We investigated the intracellular cAMP involvement in the increased endothelial cell adhesivity induced by IL-4 or TNF-alpha. We showed that both IL-4 and TNF-alpha increased intracellular cAMP in endothelial cells (EC). Furthermore, dibutyryl-cAMP and forskolin (which increased intracellular cAMP) increased basic EC adhesivity for PBL. The co-stimulation of EC with cAMP elevating agents and TNF-alpha, but not IL-4, resulted in an additive increase in EC adhesiveness. 2',5' dideoxyadenosine, an inhibitor of adenylate cyclase, decreased PBL adhesion to IL-4- but not TNF-alpha-treated EC. Similarly, HA1004, a protein kinase A inhibitor, totally reversed the IL-4 but not TNF-alpha effect on EC adhesiveness, whereas H7, a protein kinase C inhibitor, did not antagonise cytokine-enhanced EC adhesivity. These results indicate that IL-4, but not TNF-alpha, uses a cAMP-dependent pathway to increase PBL adhesion. Furthermore, we showed that cAMP elevation in EC did not induce vascular cell adhesion molecule 1, the only identified adhesion molecule induced by IL-4, indicating that a rise in cAMP in EC promotes an as yet unidentified adhesion pathway. Our results show that IL-4 increases EC adhesiveness for PBL through activation of protein kinase A by promoting an unidentified adhesion pathway.

We have developed a panel of MoAbs against four separate but overlapping epitopes on endothelial cell (EC) vascular cell adhesion molecule-1 (VCAM-1). Two of the MoAbs (1G11 and 1E5) inhibited T cell adhesion to tumour necrosis factor (TNF)-activated EC, whilst two MoAbs (1.4C3 and 6D9) did not. Using these MoAbs we have identified a circulating form of VCAM-1 (cVCAM-1) which has identical epitope distribution to the EC form, and which is able to support the adhesion of the human lymphoblastoid cell line Jurkat J6 by a VLA-4- and VCAM-1-dependent mechanism when immobilized from plasma. cVCAM-1 isolated by immunoaffinity and size-exclusion chromatographies was shown by SDS-PAGE to have an apparent mol. wt of 85-90 kD. Levels of cVCAM-1 were significantly raised (P < 0.001) in rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) compared with normal individuals. It is possible that cVCAM-1 may be a useful plasma marker for the diagnosis and management of patients with inflammatory diseases. Furthermore, detection of elevated cVCAM-1 levels may act as a guide to the importance of VCAM-1-dependent cell adhesion in different pathological settings.

For many years Doppler ultrasound has helped to identify the cause of renal allograft dysfunction. However, Doppler examinations were often performed after the onset of acute renal failure. In the present study we used Doppler ultrasound during grafting to follow changes in renovascular resistance. As early as 30 min after the renal artery had been unclamped, the calculated resistance index (RI) at the hilar part of the renal artery was significantly higher in the group of patients who developed acute tubular necrosis (ATN) than in the group of patients with early normalization of renal function (P = 0.05). This result did not correlate with raised cold and warm ischemia times and serum creatinine level on discharge in patients who presented with ATN. RI higher than 0.730 min after unclamping allows for an identification of those grafts at greater risk for the development of ATN and should be an indication for the early introduction of intensive therapy.

Interleukin 4 and tumor necrosis factor alpha increase endothelial cell adhesiveness for lymphocytes by promoting adhesion molecules synthesis. We have shown that intracellular mechanisms, which transduce IL 4--but not TNF alpha--signal, involve a protein kinase A.

Lymphocyte adhesion to allogeneic endothelium is a critical step in graft rejection. To further characterize the expression and the regulation of adhesion molecules involved in this process, we stimulated endothelial cells with Interleukin 4 or tumor necrosis factor alpha (TNF alpha) and blocked lymphocyte adhesion to stimulated-endothelial cells with monoclonal antibodies. We demonstrated that lymphocytes bound endothelial cells by at least four adhesion pathways: a) a LFA 1/ICAM 1 dependent pathway on TNF alpha-stimulated endothelial cells, b) a LFA 1 dependent/ICAM 1 independent pathway on unstimulated or IL 4-stimulated endothelial cells, c) a VLA 4/VCAM 1 dependent pathway on unstimulated and stimulated-endothelial cells, d) a CD 2 dependent/LFA 3 independent pathway on IL 4 and TNF alpha-stimulated endothelial cells.

We demonstrated that tumour necrosis factor alpha (TNF-alpha) and interleukin 4 (IL-4) increased endothelial cell (EC) adhesiveness for peripheral blood lymphocytes (PBL) by promoting transcription and protein synthesis. The different kinetics observed with TNF-alpha and IL-4 suggest the involvement of different adhesion molecules. Blocking adhesion assays and immunofluorescence analysis showed that PBL adhesion to endothelial cells involves different pair adhesion molecules. Whereas IL-4 promoted an LFA-1-dependent/ICAM-1-independent adhesion pathway on EC, TNF-alpha stimulated an LFA-1-dependent/ICAM-1-dependent adhesion pathway on EC. In contrast, VLA-4/VCAM-1 molecules were involved in PBL adhesion both to IL-4 and to TNF-alpha-stimulated EC. Finally, we found that a CD2-dependent/LFA-3-independent adhesion pathway was mainly involved in IL-4-stimulated EC.

Seven forms of X-linked (XL) immunodeficiency have been described (XL agammaglobulinemia, XL severe combined immunodeficiency [SCID], Wiskott-Aldrich syndrome, XL chronic granulomatous disease, XL hyper-IgM syndrome with low IgG and IgA, and XL lymphoproliferative syndrome), and properdine deficiency. Although there are (some) phenotypic variants, diagnosis is relatively simple on the basis of clinical, immunological, and genetic characteristics. We studied a family in which several males were affected by severe infections and whose pedigree suggested recessive XL inheritance of an immunodeficiency. Immunologic and genetic studies (X inactivation patterns in females and restriction fragment length polymorphism [RFLP] segregation) were performed in order to characterize the immunodeficiency. The propositus, a 5-yr-old boy, was found to have a severe and progressive T- and B-cell functional immunodeficiency characterized by defective antigen-specific responses. No lymphocyte subsets or membrane anomalies were detected and the immunodeficiency did not correspond to usual XL forms. Studies of DNA from two of the informative females, the mother and one sister revealed nonrandom X chromosome inactivation of T cells and, partially, B cells but not PMN, a pattern similar to that observed in XL SCID carriers. RFLP studies identified a haplotype segregating with the abnormal locus that may be localized in the proximal part of the long arm of the X chromosome. We thus report the characterization of a new XL immunodeficiency that may correspond either to another XL locus or to an attenuated phenotype of XL SCID.

In order to investigate whether deficient immunoglobulin production in common variable immunodeficiency (CVI) patients was related to defective T cells functions, phenotype and proliferative responses to mitogen of peripheral blood mononuclear cells (PBMC) were investigated in 9 patients with CVI. The results were compared to those of 12 age- and sex-matched normal controls. The numbers of CD3+ and CD8+ T cells in the patients were not different from those in the control group, but the numbers of CD4 T cells were decreased (511 +/- 237 vs 844 +/- 247/mm3; P less than 0.01). The decrease in CD4 T cells was due to a dramatic deficiency in the CD4+ CD45RA+ subset, observed as an absolute value of blood lymphocytes (126 +/- 91 vs 384 +/- 142; P less than 0.001) and as a percentage (9.0 +/- 7.1 vs 18.8 +/- 5.0; P less than 0.01). In contrast, the CD4+ CD29+ T cell subset was not different in CVI from those in the control group. Moreover, there was a strong positive correlation between the number of percentages of CD4+ CD45RA+ blood T cells and the proliferative response of PBMC to PHA (respectively, P less than or equal to 0.02 and P = 0.05) and to Con A (P less than or equal to 0.02). The decrease of CD4+ CD45RA+ T cells could reflect an abnormality in the physiological status of T cells and could be of critical importance in the antibody deficiency.

The plasmid lipid peroxidation products: malonyldialdehyde (MDA), organic hydroperoxides (OHP) and zinc peripheral values were analysed in seven children with Kawasaki disease, in the acute phase, before and after treatment with intravenous immunoglobulins. In the acute phase, plasma levels of MDA (2.95 +/- 0.30 mumol/l, control group: 2.52 +/- 0.08 mumol/l) and OHP (235 +/- 65 mumol/l, control group: 120 +/- 10) were increased (p less than 0.05 and p less than 0.01). Moreover, plasma zinc levels were significantly decreased (10 +/- 2.15 mumol/l versus 15.6 +/- 2.5 mumol/l in control group, p less than 0.05). After treatment with immunoglobulins, MDA, OHP and plasma zinc levels returned to normal. The parallel normalisation of clinical injury draws attention to the possible role of oxygen intermediates in connective tissue degradation and in the pathogenesis of vascular abnormalities in Kawasaki disease.

Ultrasound techniques and radionuclide studies are very often used to assess vascularization of renal transplants. Although, in acute tubular necrosis or acute rejection, it is difficult to choose between these two techniques, in 3 recent cases of renal artery thrombosis, we conclude that color coded Doppler is preferable to nuclear medicine or duplex system to diagnose this form of thrombosis.

The aims of the study were the correlation between dosage and plasmatic levels of slow release theophylline and the reason for dosage adjustment. 64 pharmacokinetic studies were performed in 58 asthmatic children between 17 months and 16 years. Plasmatic levels of theophylline were performed by fluoroimmunology technique at H0 (before the dose) 2 (H2), 4 (H4), 6 (H6) and 8 (H8) hours after the dose of slow release theophylline. The best correlation between dose and plasmatic levels were observed at H4 and H6 for Armophylline and Euphylline respectively. Dosage adjustment were based both upon clinical state and plasmatic levels in 55 cases. In 9 cases the modification of dose were decided only because of plasmatic levels out the therapeutic range. The authors proposed a schema of dosage modifications based upon clinical state; plasmatic levels must be used as a guide for dose adjustment in patients clinically uncontrolled.

The progress in organ transplantation is leading an increase in the demand for organs. Any non-harvested organs from a dead child mean at least a delay or even the loss of hope for life. All brain dead children do not undergo organ harvesting because there are still obstacles. The definition of brain death must be clear and include the destruction of all the brain system. Intensive care units must be motivated by improved contacts with transplantation teams. The number of parents who refuse could decrease with better public information, good contacts with medical teams and the quality of discussion with parents when a child dies. Energy should be concentrated on improving the collective consciousness about this new aspect of medicine.

Serum IgG subclass levels were measured using an indirect competitive immunoenzymatic assay with monoclonal antibodies in 221 patients affected with definite immunodeficiency (ID) syndromes and 229 patients presenting with infection patterns suggestive of ID, but with normal immunoglobulin class levels and no clear evidence of ID. In common variable ID and IgG-IgA deficiency with normal or high IgM, subclass imbalance (mostly IgG1-IgG3 or IgG2-IgG4 deficiency) was the rule, with a higher incidence of severe infections in IgG2-IgG4 defects. One-fifth of patients with IgA deficiency, especially those with autoimmune cytopenia, had subclass deficiencies with no significant correlation with the occurrence of infections. Subclass (mostly IgG2-IgG4) deficiencies were also observed in severe combined ID, defective expression of HLA class II antigens, chronic mucocutaneous candidiasis, and IgM deficiency. Subclass levels were normal in all but one (who was IgG3 deficient) patient with the Wiskott-Aldrich syndrome and in the Buckley's syndrome, except for an unusual patient who presented with low IgG and IgA levels. Subclass (mainly IgG2) deficiency occurred in 24% of infected patients without known ID.

The communication between right pulmonary artery and left atrium is a very uncommon cause of right to left shunt. The 18th case in the world literature and the first case in France is reported here. Normal pressure in the right cavities and in pulmonary artery is evocative. Angiography assesses the diagnosis and precises the place and connections of the fistula. Surgical closing of the fistula must be promptly achieved in order to obviate embolic complications.

Improved results of organs transplantation have resulted in an increased request for organs. All brain-dead children do not undergo organ harvesting for 3 main reasons. First, the definition of brain death, which should remain strict, is not accepted by everyone, in spite of the laws on this topics. Then, organizing organs harvesting requires specific hospital structures, a co-ordinator and motivated medical staff. Finally, parent's authorization is mandatory before organs harvesting in children. Parents still often refuse. An improvement might be reached by informing the general public, a maintained trust in the medical profession and the quality of the talk with the parents when the child dies. All energies should tend to develop the collective consciousness in order to improve this new aspect of medicine.

Three children with a Bartter's syndrome have been investigated: all of them had growth retardation, hypokalemia (less than 3 mmol/l), raised plasma renin activity and urinary prostaglandins (PGE2 and PGF2 alpha) and a decreased sensibility for angiotensin. In the siblings two children had also growth retardation with mild biological signs of Bartter's syndrome, and two children had normal growth slight hypokalemia raised RPA and urinary PH, and normal sensibility for angiotensin. These data suggest mild forms of this syndrome which could be the Bartter's syndrome diagnosed in adults after laxatives or diuretics absorption. Besides these data stated a negative correlation (p less than 0,01) between plasma K+ and RPA, negative correlation (p less than 0,01) between plasma K+ and urinary PGE2 and a positive correlation (p less than 0,01) between RPA and urinary PGE2. From these observations physiopathology of Bartter's syndrome is discussed.

Autoantibodies to the cytoplasmic components of neutrophil polymorphonuclear granulocytes (ACPA) were searched for by indirect immunofluorescence on ethanol-fixed smears from normal white blood cells in 248 sera from patients with Wegener's granulomatosis, Kawasaki disease and various other vasculitides, connective tissue diseases, nephropathies and infiltrative pulmonary diseases. Apart from a weak positivity in a few Kawasaki disease sera, ACPA were found in Wegener's granulomatosis (and micropolyarteritis) only, with a correlation between ACPA titers and disease activity. These results confirm the value of ACPA in the diagnosis and evaluation of activity of Wegener's granulomatosis.

The authors describe a new observation of a congenital aerobiliar fistula in a newborn infant showing choleptysies associated with a pulmonary obstruction. The most remarkable facts are the presence of a radiological pneumogallbladder, and the existence of a real trifurcation of the trachea on the bronchography, associated with a choledocal hypoplasia. The death having occured before the operation, the autopsy allowed the confirmation of the termination on the left hepatical duct and the duality of conformation of the fistula, respiratory and biliary. On the occurence of this 9th case, a review of the literature shows the stereotyped nature of the clinical signs and the course of the fistula which seems invariable. The different aspects of the malformation are considered as well as the treatment which necessitates an exeresis by thoracic pathway.

The first patient suffered from a very severe atopic dermatitis with intense pruritus and thickened skin. He had also recurrent infections, particularly related to Staphylococcus coagulase +, and axillary and inguinal lymphodermopathy. The use of tetracosactide given intramuscularly allowed controlling the evolution of his atopic dermatitis. After several months of treatment, the skin became less infiltrated, lymphodermopathy disappeared and no severe infection had happened. The second patient had a less severe atopic dermatitis and recurrent infections without any particular severity. Topical corticosteroids allowed to control the atopic dermatitis. These two patients had high levels of circulating IgE and an important deficiency of polymorphonuclear chemotaxis which was evaluated by migration through boyden room. Study of the family showed atopic manifestations in several members, but with lower levels of IgE. The most characteristic abnormality of this syndrome is the according to considerable increase of IgE. The deficit in polymorphonuclear chemotaxis may vary according to time and even become normal. The prognosis over long periods remains to be determined.