A 2-year follow up of babies enrolled in a European multicentre trial of porcine surfactant replacement for severe neonatal respiratory distress syndrome. Collaborative European Multicentre Study Group. The postnatal growth, respiratory status and neurodevelopmental outcome of surviving babies enrolled in the first European multicentre trial of porcine surfactant (Curosurf) replacement for severe neonatal respiratory distress syndrome, were assessed at corrected ages of 1 and 2 years. Follow up rates of survivors were 93% at 1 year and 89% at 2 years. Treated and control groups were similar at both 1 and 2 years in terms of physical growth, the prevalence of persistent respiratory symptoms and the occurrence of major and minor disability. Serum antibodies recognising Curosurf and surfactant-anti-surfactant immune complexes were detected in both treated and control babies, the titres showing no difference between groups. Examination of histological lung sections from non-survivors revealed a higher incidence of severe pulmonary interstitial emphysema in control babies than in those treated with surfactant. Surfactant treatment for severe respiratory distress syndrome reduces neonatal mortality and air leaks and is not associated with an increase in disability 2 years later.


Infectious and surgical complications of childhood continuous ambulatory peritoneal dialysis. A 10-year retrospective study was performed with respect to the incidence of infectious and surgical complications in a young paediatric continuous ambulatory peritoneal dialysis population of 43 children (mean age 4.5 years [range 2.5 months-15.8 years]). The incidence of infectious complications such as peritonitis and catheter-related infections correlated well with the results of other studies. A relatively high incidence of hernias (53%) was seen in our population, probably caused by the lower mean age of the children. Obstructions were caused by omental wrapping, peritonitis, or operative procedures and did not correlate with the period of catheter function. Early leakage occurred within 5 days after catheter implantation. Later leakages were preceded by another complication, such as infection or obstruction. In early leakage, in contrast to later leakage, the catheter could be maintained.


Evidence for intact V1-vasopressin receptors in congenital nephrogenic diabetes insipidus. The binding of tritium-labelled arginine vasopressin to human platelet vasopressin receptors was investigated in patients with congenital nephrogenic diabetes insipidus. Binding characteristics, that is receptor affinity and the maximum number of binding sites, were not significantly different from those found in normal control individuals. The findings confirm the concept of intact V1 receptors in congenital nephrogenic diabetes insipidus. The defect in nephrogenic diabetes insipidus apparently only affects the cyclic adenosine monophosphate dependent V2 receptors.


Tubular function and histological findings in ifosfamide-induced renal Fanconi syndrome--a report of two cases. Two patients developed renal Fanconi syndrome (RFS) after intensive long-term chemotherapy for metastatic Ewing sarcoma and disseminated neuroblastoma. Whereas RFS was diagnosed in patient 1 before he developed osteomalacia, patient 2 experienced severe rickets and growth retardation. Renal function studies revealed slight glomerular impairment and severe tubular defects leading to increased excretion of glucose, amino acids, inorganic phosphate and low molecular weight proteins, indicating proximal tubular damage. Patient 2 additionally showed distal tubular dysfunction with acidosis and diminished concentrating capacity. Renal biopsy in patient 1 revealed marked proximal tubular defects without interstitial lymphocytic infiltration. In both patients renal damage could most likely be ascribed to previous ifosfamide (IFOS) therapy. Our patients showed no improvement in renal function after cessation of IFOS treatment, indicating a poor prognosis of once established RFS after IFOS therapy. Measurement of tubular reabsorption capacities provides exact information on the extent of tubular toxicity induced by IFOS and may be used to monitor IFOS treated patients.


Growth hormone treatment in Turner syndrome accelerates growth and skeletal maturation. Dutch Growth Hormone Working Group. Sixteen girls with Turner syndrome (TS) were treated for 4 years with biosynthetic growth hormone (GH). The dosage was 4 IU/m2 body surface s.c. per day over the first 3 years. In the 4th year the dosage was increased to 6 IU/m2 per day in the 6 girls with a poor height increment and in 1 girl oxandrolone was added. Ethinyl oestradiol was added after the age of 13. Mean (SD) growth velocities were 3.4 (0.9), 7.2 (1.7), 5.3 (1.3), 4.3 (2.0) and 3.6 (1.5) cm/year before and in the 1st, 2nd, 3rd and 4th year of treatment. Skeletal maturation advanced faster than usual in Turner patients especially in the younger children. Although the mean height prediction increased by 5.6 cm and 11 of the 16 girls have now exceeded their predicted height, the height of the 4 girls who stopped GH treatment exceeded the predicted adult height by only 0 to 3.4 cm.


Gastrointestinal endoscopy in Henoch-Schönlein purpura. Gastrointestinal (GI) endoscopy was performed in seven patients with Henoch-Schönlein purpura (HSP). In two patients there were no cutaneous lesions at the time of endoscopy, but inflammation of the duodenum, especially of the second part, led to suspicion of the disease. Upper GI endoscopy showed abnormalities in six of seven cases, and sigmoidoscopy in one of four cases. The changes were more marked in the second part of the duodenum rather than in the bulb or the stomach. The endoscopic findings included redness, swelling, petechiae or haemorrhage, erosions and ulceration of the mucosa. Histology of the mucosal biopsy specimens revealed non-specific inflammation with positive staining for IgA in the capillaries, but failed to show vasculitis. Upper GI endoscopy, including study of IgA, can be useful in the diagnosis of HSP. Colonoscopy is less helpful, especially if limited to the sigmoid colon.


Invasive aspergillosis complicating induction chemotherapy of childhood leukaemia. Two children with acute leukaemia developed histologically confirmed invasive aspergillosis within 2 weeks after onset of polychemotherapy. One child had received only prednisone and one pulse of vincristine and daunorubicin before. This child showed classical roentgenographic signs of aspergilloma following an upper pulmonary lobe infiltration. The second patient developed caecal aspergillosis obscured by clinical signs of appendicitis. He died of disseminated aspergillosis several weeks later in spite of systemic antifungal therapy. Both case reports illustrate that the possibility of invasive aspergillosis must also be expected in young patients soon after onset of induction chemotherapy.


The hepatic malignant mesenchymoma: a case report. We report a case of hepatic mesenchymoma in an 8-year-old girl who presented with abdominal pain and ultrasonographic diagnosis of hepatic echinococcosis. Due to the good general condition of the patient and the diagnostic confirmation of liver hydatid disease by the CT scan, antiparasitic therapy with albendazole was started. After 1 month of therapy the girl's general condition worsened as did the ultrasonographic picture. On laparatomy a large cystic mass was observed within the right hepatic lobe and was removed. Pathological examination of the mass excluded an echinococcal cyst and demonstrated a malignant hepatic mesenchymoma.


The inflammatory cytokines in measles: correlation between serum interferon-gamma levels and lymphocyte subpopulations. Interleukin-1 (IL-1 beta), tumour necrosis factor alpha (TNF-alpha) and interferon gamma (IFN-gamma) were measured in serum from children with measles using an immunoradiometric assay. The IFN-gamma level was increased in 52 out of 54 patients in the acute phase of measles (less than 7 days of illness), and then declined to an undetectable level in the convalescent phase. Neither IL-1 nor TNF could be detected during the course of the illness. The mean serum IFN-gamma level was at its peak on day 4 and could be detected over a 7-day period after the onset of fever, coinciding with the febrile period (6.9 +/- 1.5 days). In the acute phase, the phytohaemagglutinin responses, absolute number of platelets, total lymphocyte counts, CD3+, CD4+, CD8+ cell counts and the CD4/8 ratio were depressed, while stab cell number and lactate dehydrogenase levels were higher than those in the convalescent phase. Using Spearman rank sum test, the IFN-gamma level was correlated negatively with the peripheral lymphocyte (P less than 0.01), CD3+ (P less than 0.05), CD4+ (P less than 0.05) cell counts and the CD4/8 ratio (P less than 0.05) and correlated positively with the stab cell count (P less than 0.01) but not with any other parameter. When the acute phase findings were compared between 28 complicated and 40 uncomplicated patients, the former were younger (P less than 0.01) and had higher maximum body temperature during the illness (P less than 0.05) than the latter, there was no difference in their IFN-gamma levels. These results show that endogenous IFN-gamma appears in the circulation during the acute febrile phase of measles, but does not contribute directly to any complication of the disease.


Pseudohyperkalaemia in Kawasaki disease. Pseudohyperkalaemia was observed in 3 of 16 patients with Kawasaki disease showing remarkably increased platelet counts. Their plasma potassium concentration, which is not affected by in vitro coagulation, was in the normal range despite the increased serum level. A significant correlation was observed between the platelet count and the increase in the serum potassium level resulting from blood coagulation, which was estimated by subtracting the plasma potassium level from the serum level. This study indicates that pseudohyperkalaemia should be considered in patients with Kawasaki disease whose platelet counts are markedly increased.


Plesiomonas shigelloides sepsis and meningoencephalitis in a neonate. A newborn infant is described who presented with septicaemia and meningoencephalitis caused by Plesiomonas shigelloides, a Gram-negative rod belonging to the family Vibrionaceae. The patient in this case, the first to be documented in Europe, developed multilocular lysis of the brain despite immediate treatment with antibiotics active in vitro. A cranial CT revealed garland-like calcifications and a large amount of medullary necrosis was seen on MRI.


Dipylidium caninum in an infant.

Dipylidium caninum in an infant. The tapeworm, Dipylidium caninum, occurs worldwide in dogs and cats. It may occur rarely in children, when the infection may be unrecognised or misdiagnosed. We report such a case.


X-linked hydrocephalus: clinical heterogeneity at a single gene locus. X-linked hydrocephalus-stenosis of the aqueduct of Sylvius sequence (H-SAS, MIM number 30007) is a rare genetic disorder characterized by hydrocephalus, macrocephaly, adducted thumbs, spasticity, agenesis of corpus callosum and mental retardation. We confirm here the localisation of the mutant gene on Xq (Xq 2.8) by linkage analysis in a 5-generation pedigree (maximum lod score of Z = 4.57 at theta = 0.04 with probe St14 at locus DXS52) and emphasise the phenotypic variability of the disease. Ventricular dilatation in affected males was either severe and diagnosed antenatally or moderate and consistent with a long survival with little or no macrocephaly. Since other X-linked syndromes of mental retardation with spasticity and flexion deformities of the thumbs have previously been shown to map to the Xq 2.8 region as well (e.g. MASA syndrome and spastic paraplegia), the present results raise the question of whether H-SAS syndrome, MASA syndrome and spastic paraplegia with mental retardation might represent different phenotypic expression of various mutations at the same locus.


Aplasia of the retinal vessels combined with optic nerve hypoplasia, neonatal epileptic seizures, and lactic acidosis due to mitochondrial complex I deficiency. A newborn male with mitochondrial complex I deficiency suffered from neonatal epileptic seizures, which later developed into infantile spasms. The infant was blind due to aplasia of the retinal vessels and hypoplasia of the optic nerve. There was congenital lactic acidosis, which persisted in later life. The boy was microcephalic and retarded. Muscular hypotonia later shifted to spasticity. Succinic acid was increased in urine. We assume that the aplasia of the retinal vessels is due to damage of the retinal ganglion cells caused by the mitochondrial disease in the first 3 to 4 months of pregnancy.


Psychological and social findings in adolescents with phenylketonuria. In a retrospective study, 34 early treated, normally intelligent adolescents with phenylketonuria (PKU) and their parents were tested with several psychometric personality inventories and self-developed questionaires concerning their psychosocial situation and their disease- and diet-specific knowledge. Results show that the patients are characterized by less autonomy, a more negative evaluation of their scholastic ability, less achievement motivation, low frustration tolerance, more negative self description, less extraversion and impulsiveness, a feeling of not being quite healthy, more grave and a higher level of dependency from their families. The patients saw their whole social situation as being distinctly restricted. Their knowledge concerning disease and diet was alarmingly poor and the majority had great difficulties in managing the diet satisfactorily without parental help. Up to the age of 15 years the serum phenylalanine levels were persistently above the desired range.


Is thoraco-abdominal phase relationship an indicator of sleep state? The phase relationship between ribcage and abdominal movement in 1440 breaths from 12 infants (mean age 3.2 days) showed statistically significant differences between sleep states as judged by physiological criteria. The mean phase difference (+/- 1 SD) was 20 (+/- 16)% in active sleep, 9 (+/- 17)% in indeterminate sleep and 3 (+/- 5)% in quiet sleep (P less than 0.0005). However, the wide scatter within sleep states meant that even the mean value from 50 breaths was not specific enough to delineate sleep state.


Ventilatory requirements for respiratory distress syndrome in small-for-gestational-age infants. Neonatal ventilatory requirements and outcome were examined in 135 very preterm, small-for-gestational age (SGA) infants to determine whether fetal growth retardation protects against severe respiratory distress syndrome (RDS) in very immature infants. Their results were compared to those from gestational age- and gender-matched controls. Although there was no significant difference in the median duration of mechanical ventilation between the two groups, more SGA infants required ventilation and were ventilated because of RDS. In a subgroup also matched for mode of delivery, there was no significant difference between the proportion of SGA infants requiring mechanical ventilation for RDS compared to their matched controls. The mortality was greater in the SGA group. We conclude that fetal growth retardation does not protect against severe RDS.


Vacuum extraction, bone injury and neonatal subgaleal bleeding. In a population of 27 flemish newborns with subgaleal bleeding encountered within a period of 6 years, we studied the obstetrical, clinical and radiological data. In contrast with controversial findings from the available literature, there is little doubt that difficult, often elective vacuum extraction is the main cause of this neonatal emergency. Disturbances in haemostasis, when documented, were attributed to focal intrahaematoma consumption, except for one boy who presented with haemophilia and neonatal subgaleal bleeding. Conventional X-ray examination continues to be of importance for the documentation of suture diastasis, fissures and fractures. CT scan reveals both the amount of extra-osseous bleeding, the degree of bone displacement and injury as well as the type and extent of associated intracranial damage. Subgaleal haemorrhage rarely hides a growing synchrondrosal rupture.


Intraperitoneal administration of recombinant human erythropoietin in children on continuous ambulatory peritoneal dialysis. In 16 children treated by continuous ambulatory peritoneal dialysis (CAPD) recombinant human erythropoietin was administered intraperitoneally for the treatment of renal anaemia. The mean treatment period was 8.3 months. Mean haemoglobin values increased from 4.9 mmol/l at start of therapy to 6.2 after 6 months. While 11 out of 16 children needed a total of 22 transfusions during the 6 months prior to therapy, no transfusions were needed after initiation of therapy. Patients started with a dose of 300 units/kg per week. After 6 months of therapy, the mean dose was 370 and after 12 months 279 units/kg per week. No major side-effects were observed. The incidence of peritonitis was not increased. We conclude that intraperitoneal administration of erythropoietin is effective in the treatment of renal anaemia in children treated by CAPD.


Hypertension associated with skeletal traction in children. Since traction-associated hypertension seems to be a relatively unknown phenomenon, a survey was done of its incidence in children treated with skeletal traction for fractures and orthopaedic diseases. The correlation with hypercalcaemia, a possible aetiological factor, was also explored. Blood pressure was recorded three times a day with an automatic oscillometric unit during the stay in the hospital. Serum calcium, creatinine and total protein concentrations were measured once a week. Patients with pre-existing diseases or renal trauma were excluded. Arterial hypertension (systolic and/or diastolic) was found in 31/50 children (62%). In almost half of these the rise in systolic blood pressure was 10 mmHg or more above the 95th percentile. Hypertension occurred in most cases within the first 3 weeks of treatment; in 7 children it developed after 3 or more weeks of traction. All children became normotensive within 1 week after discontinuation of traction. Clinical symptoms were rare: two children complained of headache. In no instance had traction to be discontinued before the planned date because of hypertension. In the hypertensive group were more preschool children and more humeral fractures as compared to the normotensive group (n = 19). Hypercalcaemia occurred in 11 children and was equally distributed in hypertensive and in normotensive children. It is concluded that arterial hypertension is a frequent finding in children in traction, but its clinical relevance is uncertain. Hypercalcaemia is not a rare finding in immobilized children, but probably plays no causative role in traction-related hypertension.


Convulsions, hemiparesis and central retinal artery occlusion due to left atrial myxoma in child. Multiple embolizations were the hallmark of the disease in an 8-year-old boy with a left atrial myxoma. Embolizations occurred initially in both hands and legs, later in the brain with generalized seizures and hemiparesis, and finally in the left eye with occlusion of the central retinal artery and consecutive severe visual impairment. Echocardiography demonstrated the tumour which was removed without complications.


The role of sonography in the evaluation of gastro-oesophageal reflux--correlation to pH-metry. Sonography was compared to pH-metry and/or oesophagomanometry to evaluate the accuracy of sonography in the early diagnosis of gastro-oesophageal reflux. Thirty children with a mean age of 72 days (21-252 days) were studied. The results showed that specificity of sonographic diagnosis was 87.5% and sensitivity was 100% (with P less than 0.001). Sonography also proved helpful in providing both functional and morphological data in addition to pH-metric results. This study therefore suggests that sonography is useful as the first approach in the diagnosis of vomiting babies as it is non-invasive and provides sufficient diagnostic accuracy.


Solitary rectal ulcer: an unusual cause of rectal bleeding in children. The solitary rectal ulcer syndrome (SRUS) is a disease which is commonly diagnosed in adults but only rarely described in children. Rectal prolapse and intussusception are frequently associated with this entity. A relationship between SRUS and chronic constipation due to spastic pelvic floor syndrome (SPFS) is often observed. Thus biofeedback defaecation training is an efficient treatment of both conditions. We describe two paediatric patients suffering from SRUS associated with SPFS who showed complete recovery after biofeedback defaecation training.


Recombinant human erythropoietin in the treatment of infants with anaemia of prematurity. Recombinant human erythropoietin (rHuEPO) was administered subcutaneously three times a week to 18 infants with the anaemia of prematurity at doses of 75, 150, 300, or 600 units/kg per week for 4 weeks, starting at 3-4 weeks of postnatal age. A significant and dose-dependent increase in reticulocyte count was observed from a mean baseline value of 71 x 10(9)/l to 200 x 10(9)/l after 3 weeks of therapy, compared with a change from 69 to 97 x 10(9)/l in 66 historical controls. The haematocrit value remained unchanged during rHuEPO treatment, whereas it steadily declined until 9 weeks of postnatal age in the controls. These effects were accompanied by a marked reduction in serum iron concentration and transferrin saturation in patients receiving standard-dose iron supplements, but not in those given larger doses. Only 3 of 18 patients required a red blood cell transfusion. These infants were among the most anaemic at entry into the study and 2 of them were unable to complete rHuEPO therapy, while the third developed iron deficiency anaemia. These data indicate that rHuEPO with appropriate iron supplementation may accelerate the recovery from anaemia of prematurity. Larger scale placebo-controlled studies are now needed to confirm these findings and verify their impact on transfusion requirements of premature infants.


May-Hegglin anomaly: a rare cause of thrombocytopenia. A family with four and an unrelated family with three individuals affected by the May-Hegglin anomaly are described. Platelet counts were markedly reduced and were correctly determined only in the counting chamber. Bleeding time and platelet aggregation were always normal, but platelet nucleotide concentrations (ATP and ADP) were elevated. The platelet glycoprotein complexes Ib/IX, IIb/IIIa and Ia/IIa were quantitatively normal. Platelet-associated IgG was slightly elevated, although thrombocytopenia was presumably not caused by an immunological mechanism. Morphological investigations showed giant platelets and spindle-shaped inclusion bodies in the granulocytes, while their function (phagocytic capacity, radical production) was normal. To exclude hereditary types of thrombocytopenia, morphological and family investigations are required to avoid misdiagnosis with far-reaching diagnostic and therapeutic consequences.


Complement component deficiencies and infection: C5, C8 and C3 deficiencies in three families. Three families are described with complement component deficiencies. In one family, five children had C5 deficiency; in a second family, two children had C8 deficiency and one child in a third family had C3 deficiency. The index cases were identified during screening of patients with recurrent pyogenic infections, recurrent meningitis and meningococcaemia. Two of the five C5 deficient patients had recurrent meningitis and meningococcaemia, two had recurrent respiratory tract infections and otitis and one was healthy. One of the C8 deficient patients had meningitis, meningococcaemia and pneumonia, whereas his sibling with the same deficiency was healthy. The patient with C3 deficiency had four episodes of meningitis and recurrent otitis.


Defective B-cell and regulatory T-cell function in Wiskott-Aldrich syndrome. We report two Chinese boys with Wiskott-Aldrich syndrome presenting with gastro-intestinal bleeding, eczema and recurrent infection. They had thrombocytopenia and the mean platelet volume was small. Serum IgG and IgA were elevated and lymphocyte proliferation in response to phytohaemagglutinin, concanavalin A and pokeweed mitogen was defective. Despite documented herpes simplex virus type 1 and cytomegalovirus infection in one patient, he did not mount any humoral response. The generation of antibody-secreting cells in response to pokeweed mitogen was markedly defective in a plaque-forming cell assay. Both patients' regulatory T-cell and B-cell functions were defective in this assay. The genetic defect in Wiskott-Aldrich syndrome therefore affects T-cells, B-cells and platelets.


Influence of the development of diabetes mellitus on clinical status in patients with cystic fibrosis. The impact of pre-diabetes on clinical status was retrospectively studied in 38 cystic fibrosis (CF) patients with diabetes mellitus (DM) and 38 non-diabetic CF patients (control patients), matched in pairs for age, sex, and chronic Pseudomonas aeruginosa lung infection. Quarterly parameters of CF clinical status were collected for 6 years prior to the diagnosis of DM in the index case. Compared to the control patients, decreases in body weight, body mass index (BMI), forced expiratory volume in 1s (FEV1), and forced vital capacity (FVC) and an increase in the daily intake of pancreatic enzyme capsules were found in the pre-diabetic patients. Statistically significant differences in body weight, BMI, FEV1, FVC, and intake of pancreatic enzyme capsules between pre-diabetic and control patients emerged 4, 4, 1.25, 3 and 4.5 years prior to the diagnosis of DM, respectively. The number of lung infections did not differ between the two groups of patients. Thus, when DM develops in CF patients, an insidious decline in overall clinical status is observed for years prior to its diagnosis. Whether clinical deterioration in CF leads to DM, or pre-diabetes results in declining CF clinical status is presently unknown. Accumulating evidence suggests that the latter may be the case since insulin therapy seems to improve lung function in CF.


Complement fragment C3a in plasma of asphyxiated neonates. Recent clinical studies with adult polytrauma patients indicate that elevated plasma levels of anaphylatoxin C3a correlate with the subsequent development of the adult respiratory distress syndrome (ARDS). However, there are no parameters which allow a reliable diagnosis of ARDS in neonates. As the most predisposing condition for ARDS seems to be shock, plasma C3a was determined in 30 ventilated premature infants and neonates with respiratory distress syndrome (birth weights 660-3350 g) within the first 24 h post partum or 6-24 h after acute asphyxia or shock during the neonatal period. The range of C3a, measured by ELISA, was between 57 and 1000 ng/ml. In the asphyxia group (n = 15) peak levels of C3a in plasma (mean 388 ng/ml) were significantly higher (P less than 0.001) than in the control group (mean 153 ng/ml). In some neonates with suspected ARDS, additional samples were taken. A rise in C3a between days 2 and 8 was associated with a fatal outcome of the disease. As in adults, C3a might be a useful indicator for ARDS in neonates.


Neonatal complications of extreme prematurity in mechanically ventilated infants. Previous data have suggested that neonatal complications amongst preterm ventilated infants increase with decreasing gestational age and thus are likely to be greatest among ventilated infants of less than 28 weeks gestational age. The aim of this study was to test that hypothesis, thus we report the neonatal complications of 175 extremely preterm mechanically ventilated infants (gestational age less than or equal to 28 weeks). Of the infants 152 were ventilated because of respiratory distress syndrome (RDS) or respiratory distress of severe prematurity, 41% of these infants died. Amongst infants with RDS or respiratory distress of extreme prematurity, mortality was significantly increased in infants of gestational age less than or equal to 24 weeks and birth weight less than or equal to 1000 g. In this group 20% developed a pneumothorax, and mortality was inversely related to gestational age. In infants with RDS, 43% developed a periventricular haemorrhage and 37% were still oxygen-dependent at 28 days of age; neither of these complications was significantly related to birth weight or gestational age. Of infants with RDS 38% developed a patent ductus arteriosus and 16% developed retinopathy of prematurity. These data suggest that even amongst very immature infants there has been an impressive reduction in the neonatal complications of mechanical ventilation.


Acute deteriorations in neonatal chronic lung disease. Preterm infants with chronic lung disease (CLD) have frequent respiratory relapses. The aim of this study was to assess the aetiology of such deteriorations and in particular the proportion due to viral infections. During the study period 118 preterm infants with birth weight less than 1500 g were consecutively admitted to the neonatal intensive care unit; 22 (18.6%) developed CLD. At the onset of all respiratory deteriorations, infants were examined for the presence of patent ductus arteriosus, apnoea or aspiration; they were also carefully screened for both viral and bacterial infection. The 22 infants had a total of 74 episodes of respiratory deterioration; median 3 per baby (range 1-8). Two episodes were associated with patent ductus arteriosus, 18 with apnoea and 5 with aspiration. Infection was suspected or proven in association with all other episodes. On ten occasions the infants had positive blood cultures and on a further eight, bacteria were isolated only from the endotracheal or nasopharyngeal secretions. On the remaining 31 occasions, 27 associated with chest X-ray film abnormalities, infection was suspected, but no bacteria isolated. Viral infections were identified in association with 8 (11%) of these episodes. We conclude viral infection should be considered as a cause of otherwise unexplained respiratory deteriorations in infants with neonatal CLD.


Energy and nutrient intake of patients with mild-to-moderate chronic renal failure compared with healthy children: an Italian multicentre study. Nutritional counselling is important in the management of children with chronic renal failure (CRF). In 1988, a controlled European multicentre study was started to evaluate the effects of a low-protein diet on the progression of CRF in children. To assess the energy, macro- and micronutrient intake, 4-day weighed dietary records were obtained from 50 children with low to moderate CRF (creatinine clearance 65 to 15 ml/min per 1.73 m2) and from 93 healthy children. The mean energy intake was 90%-93% of the recommended dietary allowance for Italian children in controls and 76%-88% in CRF patients. The mean protein intake was 2.1-3.1 g/kg per day in controls and 1.6-2.7 g/kg per day in CRF patients. Overall, the energy intake was 10% and the protein intake 33% lower in CRF patients than in healthy children. Children with CRF consumed less cholesterol, calcium and phosphorus than healthy children. The lower spontaneous intake of energy, protein and other nutrients should be taken into account when planning the nutrition of children with CRF.


Intracranial blood flow velocities during seizures and generalized epileptic discharges. In 51 children with different types of epilepsy, blood flow velocities in the middle cerebral artery were recorded continuously by transcranial Doppler sonography during a standard electroencephalogram of 30 min duration. In 16 children 33 epileptic seizures were recorded. During tonic seizures, the mean flow velocity increased to a maximum of 133%-191% (median 160%) of the baseline values. Tonic-clonic seizures were also accompanied by a velocity increase. During absence seizures the mean flow velocity decreased to a minimum of 46%-82% (median 71%) of the baseline values. Changes in cerebral metabolism and arterial blood pressure in the presence of disturbed autoregulation are thought to be factors causing these alterations. No alteration of the flow velocities occurred in cases of petit-mal status, electrical status epilepticus and in 35 children with generalized epileptic discharges of up to 5 s duration without clinical manifestations.


Diagnosis of intralobar lung sequestration by colour-coded Doppler sonography. Intralobar lung sequestration in a 5-month-old dyspnoeic infant was diagnosed by colour-coded Doppler sonography (CDS). Grey scale imaging showed an echogenic mass adjacent to the right hemidiaphragm. CDS demonstrated an abnormal arterial blood supply via a vessel originating from the descending aorta at the level of the diaphragm.


Optimal BCG treatment of superficial bladder cancer as defined by American trials. Immunotherapy provides an effective alternative approach to chemotherapy in the management of superficial bladder cancer. The first widely used immunotherapy, bacillus Calmette-Guérin (BCG), eradicates residual tumour bacillus Calmette-Guérin (BCG), eradicates residual tumour in one half of patients with carcinoma in situ. Unlike chemotherapy, induction of immunity against transitional cell carcinoma has the potential of protecting patients from tumours which have not yet developed. Controlled trials suggest that BCG immunotherapy reduces disease progression, decreases the need for cystectomy and prolongs survival. While the optimal BCG treatment schedule remains unknown and may in fact vary from one patient to another, data clearly suggest that a single 6-week induction course is suboptimal. In 150 randomized patients with CIS treated with 120 mg Connaught BCG, Southwest Oncology Group (SWOG) investigators found that just 3 additional weekly treatments at week 12 increased complete response from 70 to 82% (p less than 0.05). Intravesical BCG is clearly superior to oral BCG, and controlled studies have demonstrated that percutaneous administration is not necessary. While evidence suggests that BCG is the best available treatment for superficial bladder cancer and 95% of patients have no significant toxicity, serious and even fatal toxicity can occur. Sepsis can occur with intravenous absorption and often appears to result from hypersensitivity. Limited clinical and animal model experience suggests that cycloserine improves survival over treatment with isoniazid and rifampicin, but optimal treatment of BCG sepsis is isoniazid 300 mg, rifampicin 600 mg, and prednisolone 40 mg daily.


Role of the immune response in BCG for bladder cancer. Intravesical bacillus Calmette-Guérin (BCG) has been shown in prospective, randomized clinical trials to be the treatment of choice for superficial bladder cancer. In this treatment regimen, viable bacteria are introduced into the bladder, provoking an infection which induces the immunological response that initiates antitumour activity. The role of T-lymphocytes and the T-lymphocyte subsets, helper T cells (Th) and cytotoxic T-cells (Tc) in the antitumour response has been evaluated. Depletion of total T, Th or Tc subsets in mice eliminated BCG-mediated antitumour activity. Delayed type hypersensitivity was abrogated only in Th-depleted mice, but the presence of Th-mediated delayed-type hypersensitivity (DTH) was not sufficient for expression of BCG-mediated antitumour activity. There was no evidence for the induction of protective immunity to the tumour after BCG therapy. These results show that T-lymphocytes are required for BCG-mediated antitumour activity and suggest that the antitumour activity mediated by BCG is of a nonspecific immunological type. No specific tumour cell immunity was detected.


From the 19th to the 21st centuries: BCG in the treatment of superficial bladder cancer. In superficial bladder cancer, bacillus Calmette-Guérin (BCG) is effective in the prevention of tumour recurrences, in the destruction of papillary lesions and, particularly, in the eradication of carcinoma in situ. In comparative trials BCG is superior to chemotherapeutic agents. Recent statistics indicate that though the incidence of bladder cancer continues to increase, the mortality has begun to diminish. The use of BCG has been implicated as an important contributor to this change. The exact mechanisms of action of this anticancer agent remain obscure, but clarification should lead to the development of new, safer and more effective agents. Thus, BCG, an anachronism from the early days of tumour immunology, may lead cancer immunotherapy into the next century.


Prospects for improving the efficacy of BCG. Since 1976, when Morales, Eidinger and Bruce first published on the treatment of human urothelial bladder cancer, bacillus Calmette-Guérin has gained worldwide acceptance as a therapy against bladder carcinoma recurrences. However, there are many uncertainties with regard to patient selection, treatment protocol, reduction of side effects, mechanisms of action, and thus room enough for laboratory researchers and clinicians to contribute to the improvement of BCG therapy. Initiation of mycobacterial binding to urothelial cells may be promoted by methods which increase fibronectin exposure on target cells, reduce inhibitory factors in the instillate, or by identification of more avidly binding substrains of BCG. Quantification of the ensuing immune response focuses on the local rather than the systemic immune reaction of the host and may give the opportunity to predict a favourable response, or even the endpoint of therapy. For this purpose, assessment of local cytokine production seems to be a valuable tool. Ultimately, revealing the mode of action(s) of live BCG may render the use of potentially dangerous live bacilli unnecessary and lead to the application of derivatives with better results.


BCG for carcinoma in situ.

BCG for carcinoma in situ. Bacillus Calmette-Guérin (BCG) is the most effective intravesical therapy of carcinoma in situ of the urinary bladder. Six, weekly instillations of BCG result in a complete remission in about 70-80% of patients. The optimal dose however has still to be defined, and the value of maintenance therapy is also a matter of debate. Recurrent tumours after complete remission occur mainly in the distal ureter and prostatic urethra. In these patients, cystectomy may be required. In about 60-80% of patients, local (e.g. cystitis) and/or systemic (e.g. fever, malaise) side effects are observed. The occurrence of cystitis is associated with the number of instillations, BCG dose and a positive skin test. Systemic side effects are connected with pre-existing dysuria or bacterial cystitis and with traumatic catheterization. Severe toxicity occurs in about 5% of the patients. Prognostic parameters indicating complete remission have yet to be determined, but there is evidence that cytokines detected in the urine and immune-cell infiltration into the bladder wall revealed by immunohistochemistry, can be of value in this respect.


BCG therapy in superficial bladder tumours--complications and precautions. Bacillus Calmette-Guérin (BCG) immunotherapy represents a valuable treatment in the management of superficial bladder cancer, but the therapy is not without its risks. A total of 220 patients have been included in a review of local and systemic side effects associated with BCG immunotherapy. The majority of side effects are mild and self limiting, but potentially life threatening complications can arise with BCG immunotherapy. These systemic side effects include pneumonitis/hepatitis and systemic BCG infection. Recommendations are given for the use and administration of BCG, and if these rules are correctly applied, BCG may be administered safely, and with confidence.


BCG in superficial bladder cancer: a review of phase III European trials. Shortly after Morales' original report, several phase II trials confirmed the effectiveness of intravesical bacillus Calmette-Guérin (BCG) in superficial bladder cancer therapy. Concerns have been expressed about the toxicity related to this new therapeutic modality. These phase II trial data led European urologists to try to answer some questions related to BCG therapy, such as the optimal schedule and dose, the most effective BCG strain and the value of BCG compared with current chemotherapeutic drugs. To date, phase III trials have shown that BCG is more effective than thiotepa and doxorubicin in reducing tumour recurrences and progression and that BCG seems to be as effective as mitomycin C. Toxicity is significantly higher with BCG compared to chemotherapeutic drugs; no strain of BCG seems to be superior in this respect. Further studies are required to identify the optimal schedule and dose, as well as the best therapeutic efficacy/toxicity ratio.


Development and preliminary psychometric properties of the Transition Competence Battery for Deaf Adolescents and Young Adults. This article describes the development and preliminary psychometric properties of the Transition Competence Battery for Deaf Adolescents and Young Adults (TCB). The TCB includes three subtests on employment and three subtests on independent living, in a 3-option multiple-choice format and with both written and videotaped instructions. The TCB was standardized on students from both mainstreamed and residential settings (n ranged from 180 to 230 for the different subtests). Overall, the item statistics and subtest reliabilities were adequate. An initial study of the battery's construct validity was conducted. Results demonstrated the power of all of the TCB subtests to differentiate among various groups.


Making sense of disability: low-income, Puerto Rican parents' theories of the problem. This article reports findings from an ethnographic study of the views of 12 low-income Puerto Rican parents whose children were classified as learning disabled or mildly mentally retarded. Different cultural meanings of disability and normalcy led parents to reject the notion of disability and focus on the impact of family identity, language confusion, and detrimental educational practices on children's school performance. Parents' views were in line with current arguments against labeling and English-only instruction.


Implementing a successful writing program in public schools for students who are deaf. A 2-year project to improve the writing skills of children who are deaf included instruction for teachers in the process approach to teaching writing. The project encompassed 10 public school programs for students who are deaf and included 325 students in Grades 4-10 and 52 teachers. The project included specific training goals for teachers, a self-report procedure for the teachers, and a data-collection and analysis phase to assess short-term effects on students' writing. Teacher self-reports indicated widespread involvement in the project, and pretest and posttest results showed dramatic improvement in students' writing--particularly in grammatical skills. Scoring systems for students' papers are included.


The Task Demonstration Model: a concurrent model for teaching groups of students with severe disabilities. This study investigated the use of the Task Demonstration Model (TDM) of group instruction for students with severe or moderate retardation. This model and the Standard Prompting Hierarchy (SPH) were tested against each other (and baseline) across three teachers and groups of students. Results on teacher variables showed that demands and praise were roughly equivalent for both procedures, but prompts were 12 times higher in SPH than in TDM. Data on student variables showed task engagement to be the same for SPH and TDM, percent correct to be 10% higher in TDM, but rate correct to be twice as much in TDM as in SPH.


Grading secondary vocational education students with disabilities: a national perspective. A nationwide purposive sample of secondary vocational educators completed a questionnaire that examined practices and perceptions toward grading students with disabilities. A variety of grading methods was reportedly used, but over two-thirds of respondents included a competency-based grading component. An overwhelming majority believed that student effort should be used to determine grades. Two-thirds of vocational educators had never been involved in their students' IEP development. The most common changes teachers have made over time in their grading practices included becoming more flexible and individualized in grading. Overall, teachers displayed moderately positive attitudes toward grading students with disabilities.


Teaching children with autism through task variation in physical education. The purpose of the study was to determine whether the technique of task variation (with maintenance tasks interspersed) (TV) is more effective in the acquisition of gross motor skills for students with autism than a constant task (CT) condition in a physical education setting. Subjects were 12 male students with autism, ages 11 to 15 years. The study included pretest-posttest administration of the I CAN assessment of Gross Motor Skills to assess skills such as overhand throw, kick, and vertical jump. After a 6-week treatment period, the TV condition was significantly more effective than the CT condition, at the .05 level.


Biochemical evidence of the physical association of the majority of CD3 delta chains with the accessory/co-receptor molecules CD4 and CD8 on nonactivated T lymphocytes. The association of components of the CD3 complex with the accessory molecules CD4 and CD8 was studied by immunoprecipitation experiments followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Enhanced surface iodination was achieved by a water-soluble derivative of the Bolton-Hunter reagent. Using freshly isolated nonactivated splenic T cells, we find that antibodies to CD4 and to CD8 strongly co-precipitate a 28-30-kDa band identical in mobility to the delta chain of the CD3 complex. Components corresponding in mobility to the epsilon and gamma chains of the CD3 complex are also co-precipitated but to a much lesser extent. The identity of the co-precipitated 28-30-kDa material with the CD3 delta chain was ascertained by two-dimensional nonreducing/reducing SDS-PAGE, by two-dimensional non-equilibrium pH gradient electrophoresis/SDS-PAGE and by one-dimensional peptide mapping with three different proteases. The co-precipitated 28-30-kDa material was identical to the CD3 delta chain by all these criteria. Quantitative analyses by densitometric gel tracing revealed that the amounts of CD3 delta co-precipitated with anti-CD4 and anti-CD8 add up to those in anti-V beta precipitates and to an average of 90% of those in anti-CD3 epsilon precipitates. We conclude that the majority of CD3 delta chains are associated with the accessory/co-receptor molecules CD4 or CD8 on resting T cells, and that this association is independent of antigen-specific recognition by the T cell receptor.


Improved survival from potentially lethal graft-vs.-host disease by donor pretreatment with a recipient-specific blood transfusion. I. Requirements for induction and specificity of the effect. Pretreatment of prospective donors of hemopoietic cells with a single recipient-specific blood transfusion can significantly decrease the morbidity and mortality of graft-vs.-host disease (GVHD) in lethally irradiated, allogeneically reconstituted mice. This beneficial effect of donor pretreatment could be demonstrated in donor-recipient strain combinations that were H-2 + non-H-2, H-2, or only class II-disparate, but not in the class I-disparate C57BL-B6.C-H-2bm1 strain combination. The effect was proportional to the amount of recipient-strain blood used for transfusion. Donor transfusion with a single dose of 1 ml recipient-specific whole blood resulted in minimum GVHD, lower doses being less or not effective. The interval between donor pretreatment and the use of their hemopoietic cells for reconstitution appeared to be important. The best survival was found at an interval of 4 days. Multiple transfusion was not more effective than a single one. We compared the effectiveness of whole blood and irradiated spleen cells for donor pretreatment. Both protocols have been shown previously to suppress anti-recipient delayed-type hypersensitivity. It appeared that the blood transfusion protocol was superior to the spleen cell protocol. The beneficial effect appeared to be recipient specific, since a third-party blood transfusion did not improve GVHD. We found that the beneficial effect of donor blood transfusion was due to suppression of the anti-host immune response. The donor blood transfusion was able to induce bystander suppression to alloantigens that were not used for the induction of suppression, provided they were co-expressed with the specific alloantigens by the recipients. This also indicates that, although the induction of suppression is specific, the ultimate suppressive effect is non-specific.


Inhibition of human immunodeficiency virus infection in human colon epithelial cells by recombinant interferon-gamma. Pretreatment of human colon epithelial cells HT29 by recombinant gamma interferon (IFN)-gamma was found to protect the cells from infection with various isolates of human immunodeficiency virus (HIV)-1 and HIV-2, as assessed by co-cultivation with human T lymphoblastoid cells and gene amplification by polymerase chain reaction technique. Additionally, IFN-gamma induced a dose-dependent inhibition of HIV-1 and HIV-2 production in chronically infected HT29 cells. In situ hybridization studies demonstrated that IFN-treated cells were still able to synthesize viral messenger ribonucleic acid. However, the expression of the p24 product of the gag gene was markedly decreased after IFN treatment as demonstrated by radio-immunoprecipitation assay. Taken together, these data suggested that the cytokine acted at the post-translational level by inhibiting the processing of structural viral proteins. It is concluded from this study that IFN-gamma has a potent anti-HIV effect on epithelial gastrointestinal cells.


The microbicidal activity of interferon-gamma-treated macrophages against Trypanosoma cruzi involves an L-arginine-dependent, nitrogen oxide-mediated mechanism inhibitable by interleukin-10 and transforming growth factor-beta. The present study was carried out to determine the effector mechanism of anti-Trypanosoma cruzi activity by interferon (IFN)-gamma plus lipopolysaccharide (LPS)-treated macrophages. A macrophage cell line (IC-21) that failed to mount an appreciable oxidative burst was nevertheless found able to control T. cruzi growth after exposure to IFN-gamma alone or IFN-gamma plus LPS. Moreover, microbicidal functions of both inflammatory macrophages and IC-21 against T. cruzi was found to be inhibited in the presence of NG-monomethyl-L-arginine (NGMMA), a competitive inhibitor of L-arginine. Addition of supplemental L-arginine to the culture overcame the capacity of NGMMA to block activated macrophage anti-T. cruzi functions. The ability of NGMMA to reverse both parasite growth inhibition and killing by IFN-gamma plus LPS-activated macrophages was found to correlate with the suppression of nitrite accumulation in the culture supernatants. Together, these results implicate the L-arginine-dependent production of nitric oxide in T. cruzi killing by activated macrophages. We also tested the ability of interleukin(IL)-10 and transforming growth factor (TGF)-beta, to block regulation of T. cruzi growth in this system. Both IL-10 and TGF-beta inhibited anti-parasite function by IFN-gamma-activated macrophages, with an optimal dose of 100 units/ml and 0.5 ng/ml, respectively. Moreover, when used in combination, suboptimal doses of IL-10 and TGF-beta were found to produce a synergistic inhibitory effect in the regulation of T. cruzi growth. The ability of IL-10 and TGF-beta to suppress microbicidal function was also positively correlated with inhibition of nitrite generation in macrophage culture supernatants. These results predict an in vivo role for IL-10 and TGF-beta in promoting parasite survival in the face of the host cell-mediated immune response.


Membrane region of surface IgM is not sufficient for transducing growth inhibitory signals in an immature B cell line WEHI-231. The murine B lymphoma line WEHI-231 is representative of immature B cells. Like normal immature B cells, WEHI-231 is susceptible to growth arrest following cross-linking of surface IgM (sIgM). Previously, we have shown using a WEHI-231 immunoglobulin (Ig) delta-transfectant that sIgD cross-linking failed to initiate growth arrest, in contrast to sIgM. In this report, we extend our research to investigate the structural requirement of Ig mu chain for regulating growth inhibition. Recombinant, chimeric Ig molecules delta/mu m and mu/delta m consisting of exons encoding extracellular delta and mu domains and membrane regions of different isotypes were constructed and introduced into WEHI-231 cells. A similar approach was used for sIgG2b-expressing transfectants. Our findings indicate that the mu m region is not sufficient for regulation of growth inhibition in WEHI-231 cells and suggest that additional extracellular region(s) of mu chain may be required for this response.


Modulation of restricted class II T cell responses by peptides derived from self class II molecule. We have explored the possibility of using peptides derived from a major histocompatibility complex (MHC) class II (I-Ab) molecule to modulate I-Ab-restricted T cell responses. Six peptides spanning the polymorphic regions of I-Ab were analyzed for competitive binding to the I-Ab molecule, and for efficacies in blocking I-Ab-specific T cell response. Only PB1 (residues 75-91 of beta chain) bound the I-Ab molecule with high affinity. When these MHC-derived peptides were administered simultaneously with antigen, PB1 effectively inhibited I-Ab-restricted T cell responses as well as another peptide PB2 (residues 59-78 of beta chain). PB2 inhibited specific T cell response only when it was administered simultaneously with antigen. Since PB2 is a weak binder of I-Ab, an additional mechanism must account for its inhibitory activity. Both PB1 and PB2 peptides elicited specific T cell responses, indicating that these peptides were not tolerogenic in syngeneic mice. However, the induction of T cells in response to PB1 and PB2 did not increase reactivity to I-Ab. MHC class II-derived peptides thus can be used to regulate T cell responses without the risk of autoreactivity.


Effects of the thymic microenvironment on the response of thymocytes to stimulation. We show that, in vitro, the response of thymocytes to certain stimuli, and their survival largely depend on the nature of the culture environment, i.e. whether thymocytes are stimulated within intact thymus lobes or in cell suspension. Exposure of isolated thymocytes to 12-O-tetra-decanoylphorbol 13-acetate (TPA)+ionomycin rapidly abolishes the expression of recombination-activating gene-1 (RAG-1) mRNA (3 h), down-regulates CD4 surface antigen expression (3 h), and enhances apoptosis (24 h). On the other hand, when thymocytes are cultured in intact lobes, TPA plus ionomycin down-regulate rather than abolish RAG-1 mRNA expression (3 h), have little effect on CD4 expression even following 24-h exposure, and only marginally induce apoptosis (24 h). Differences between the culture systems are less pronounced in response to anti-CD3 antibodies. Therefore, it appears that removing thymocytes from their thymic microenvironment makes the cells more susceptible to certain stimuli, possibly by altering their physiological status. In addition, it has been suggested that termination of RAG-1 expression can be linked to thymocyte selection processes. We found that the down-regulation of RAG-1 expression was not dependent on the induction of apoptosis, supporting a proposed link with positive selection.


Neurons and neuroblastoma as a source of macrophage colony-stimulating factor. Accumulation of macrophages in brain tissue as observed in nervous system injury may be due to local production of hematopoietic colony-stimulating factors (CSF). The present work shows human neuroblastoma cells and murine neurons, namely granule cells of the cerebellum, to produce macrophage (M)-CSF which guides expansion and differentiation of macrophage lineage cells. The mRNA-encoding M-CSF but not the respective protein is present in mouse brain including cerebellum. Neither granulocyte M-CSF nor IL-3 is produced by cerebellar neurons or neuroblastoma. By their production of M-CSF, neurons may regulate the macrophage response and lead to local expansion and enhanced function of macrophages in inflammatory diseases of the central nervous system.


A trypsin-like serine protease activity on activated human B cells and various B cell lines. We have studied the trypsin-like serine protease activity of human tonsillar B lymphocytes. The lysate of the low-density, in vivo activated B cells as well as the lysate of cells stimulated with anti-human IgM F(ab')2 show elevated trypsin-like serine protease activity compared to the resting subset as monitored by the cleavage of Tos-Gly-Pro-Arg-pNA. The cleavage is sensitive to N-tosyl-L-lysyl-chloromethyl ketone and benzamidine but not to iodoacetamide. Experiments with intact cells give similar results. The finding that the intact cells hydrolyze the substrate, while their supernatant does not, suggests that the protease activity is cell membrane associated. It is possible that C3 is a substrate of the enzyme since the activated B cells cleave C3, whereas the resting B cells do not, and also C3 inhibits the enzyme-substrate reaction. In addition to the ex vivo B cells, we studied the serine protease activity of certain well-characterized B cell lines. The results show a correlation between the phenotype and the enzyme expression of the cell lines. BL41, an Epstein-Barr virus (EBV)-negative Burkitt lymphoma line, with a resting phenotype, has low activity, while its EBV genome-carrying convertants E95-A-BL41, E95-C-BL41, EHR-A-BL41 and BL41/95 that have the phenotype of activated B cells, have high proteolytic activity. The lymphoblastoid cell line WW-1-LCL which has the phenotype of an immunoblast, has the highest serine protease activity. On the basis of the above data, we suggest that a rather tight correlation exists between the degree of activation and the appearance of serine protease(s) on the surface of human B cells.


Genetics of nonspecific immunity: I. Bidirectional selective breeding of lines of mice endowed with maximal or minimal inflammatory responsiveness. The genetic regulation of acute inflammatory reaction (AIR) was studied by the method of bidirectional selective breeding, used to produce a line of mice giving the maximal and a line of mice giving the minimal inflammatory reaction (AIR max and AIR min, respectively). The AIR was triggered by subcutaneous injection of a neutral substrate (suspension of polyacrylamide microbeads), and measured by the leukocyte and serum protein accumulation in the exudate. The two parameters are positively correlated and present a normal frequency distribution. The highly genetically heterogeneous foundation population was produced by the equipoised intercrossing of eight inbred strains of mice, and selective breeding carried out by assortative matings of extreme phenotypes. The response to selection in 11 consecutive generations was highly asymmetrical: a marked AIR increase in the AIR max and no change in the AIR min line occurred. The mean value of realized heritability in the AIR max line was 0.26 and 0.18 for cell and protein concentrations, respectively. The response to selection must have resulted from the interaction of seven to nine independent gene loci endowed with additive effects. The lack of response to selection of the AIR min line is discussed. The large inter-line difference opens new possibilities for studying the biochemistry and molecular genetics of inflammation, and also for investigating the beneficial or detrimental effect of inflammatory responses.


Tyrosine kinase activation in thymic epithelial cells: necessity of thymocyte contact through the gp23/45/90 adhesion complex. Interactions between thymocytes and thymic stromal cells are necessary for T cell differentiation, maturation and proliferation. The signals required for these events to occur often necessitate close contact, and indeed adhesion, between the cell types involved. While the transmission of signals from stromal cells to thymocytes has been well documented, there is little evidence that binding of thymocytes to stromal cells can result in stromal cell activation. We have recently identified a novel thymic epithelial adhesion complex composed of three non-covalently associated glycoproteins (gp23, gp45 and gp90). While gp23 and gp45 are jointly required for adhesion to thymocytes, the function of gp90 is unknown. In the present work, we show that gp23/45-mediated contact with thymocytes induces de novo tyrosine phosphorylation of gp90. Furthermore, the protein tyrosine kinase responsible for gp90 neophosphorylation is itself an integral part of the adhesion complex.


Evidence for oligoclonality of T cell receptor delta chain transcripts expressed in rheumatoid arthritis patients. The inflamed synovium of rheumatoid arthritis (RA) patients contains gamma/delta T cells which express predominantly T cell receptor (TcR) variable (V) delta 1 and V delta 2 chains. Such T cells may contribute to the pathogenesis of RA. To assess the extent of clonality among these T cell populations we sequenced the junctional regions of rearranged TcR V delta 1-C delta and V delta 2-C delta chain cDNA, after using the polymerase chain reaction (PCR) to amplify TcR delta chain transcripts isolated from synovial membrane mononuclear cells (SMC) of five RA patients. The sequences of these delta chain transcripts were compared with those found in peripheral blood mononuclear cells (PBMC) of the same patients and in PBMC of four healthy controls. In contrast to control PBMC, V delta 1 chain cDNA derived from PBMC of three patients showed a strong bias towards usage of the same V-joining (J) combination and junctional region sequences, although the specific sequences were unique in each patient. However, oligoclonality of the V delta 1 chain was less marked in SMC of two of these patients and absent in SMC of the other patients. For V delta 2, oligoclonality was detected in PBMC of two patients. In SMC of a single patient, a dominant V delta 2 transcript was detected that utilized the J delta 2 segment, which was rarely expressed in the normal TcR repertoire. These results indicate in vivo clonal expansion of V delta 1- and V delta 2-expressing gamma/delta T cells in the peripheral blood of RA patients and a synovial T cell infiltrate which consists largely of polyclonally expanded gamma/delta T cells, but shows clonal dominance in some patients. Our data strongly support a role for V delta 1+ and V delta 2+ gamma/delta T cells in the pathogenesis of RA, and, although the nature of the antigen(s) recognized by these cells remains elusive, this report suggests the potential involvement of antigen(s) specific for the V region and V-J junction.


Analysis of human/mouse interleukin-6 hybrid proteins: both amino and carboxy termini of human interleukin-6 are required for in vitro receptor binding. The multifunctional cytokine interleukin-6 (IL-6) is a single polypeptide chain consisting of 184 amino acids in man and 187 amino acids in mouse. Despite the relatively high degree of sequence similarity of these two molecules (about 57%), the biological activity in mouse and human IL-6 shows species specificity. Starting with this observation, we constructed interspecies hybrids with the goal of defining which segments of the human IL-6 molecule are involved in human receptor binding. In this manner we generated multiple amino acid substitution mutants which do not contain insertions or deletions as compared with the parental proteins, and which, therefore, should not show dramatic changes in folding. Using two biological assays on cells of human and mouse origin and a recently developed in vitro binding assay to recombinant soluble human IL-6 receptor, we obtained results which indicate that both the amino and carboxy termini are necessary and sufficient for efficient binding, but that the carboxy terminus plays the dominant role in receptor recognition.


Lipopolysaccharide induces human interleukin-1 receptor antagonist and interleukin-1 production in the same cell. A new member of the interleukin-1 (IL-1) family has recently been described. Human IL-1 receptor antagonist (IL-1ra) is structurally related to IL-1 alpha and IL-1 beta but binds to IL-1 receptors on various target cells without demonstrable agonist activity. Understanding the mechanisms of regulation of IL-1ra production may clarify the biology of this unique cytokine as well as elucidate its possible role as a natural anti-inflammatory protein. The effects of lipopolysaccharide (LPS) on IL-1 alpha, IL-1 beta and IL-1ra production was studied at a single-cell level by use of cytokine-specific antibodies and indirect immunofluorescence technique. The peak synthesis of IL-1ra and IL-1 alpha/beta occurred in peripheral blood monocytes obtained from healthy blood donors within 4 and 6 h of cell stimulation, respectively. By double-staining procedure all IL-1ra-positive cells were also IL-1 alpha and/or beta positive. Thus, endotoxin induced simultaneous synthesis of the IL-1 gene family in the same cells. Only monocytes contributed to the production of IL-1 alpha, beta and IL-1ra during the 96 h of cell culture. The maximum number of IL-1ra-producing monocytes was 48 +/- 16% as compared to peak production of IL-1 alpha and beta which occurred in 75 +/- 9% and 80 +/- 12% (p < 0.001), respectively, of all peripheral blood monocytes. The incidence of IL-1 alpha- and beta-containing cells was not only significantly higher but also occurred for a longer time period, 72 h as compared to 24 h for IL-1ra localized in the Golgi organelle. However, IL-1ra-containing cells with a diffuse cytoplasmic appearance were also evident (20%-30%) at a later stage, 12 to 72 h after stimulation. Blocking IL-1 surface receptors by addition of exogenous recombinant IL-1 beta before stimulation could not inhibit the diffuse cytosolic localization. This indicates that the "late" staining pattern did not reflect IL-1ra being secreted and internalized after binding to extracellular receptors. Thus, perhaps IL-1ra modulates IL-1 effector mechanisms by receptor interactions both inside and outside the cell.


T cell repertoire in tuberculosis: selective anergy to an immunodominant epitope of the 38-kDa antigen in patients with active disease. It is generally accepted that both host protection and pathogenic reactions in tuberculosis are mediated by T lymphocytes. However, little is known about the structures and discreet functions of epitopes stimulating the immune response. In this study, proliferative responses of blood T lymphocytes to synthetic peptides derived from the sequence of the 38-kDa antigen from Mycobacterium tuberculosis have been investigated in 41 healthy individuals and in 36 patients with active tuberculosis. Of the healthy purified protein derivative (PPD)-positive donors, 90% responded to a permissively recognized peptide, 38.G (residues 350-359), located at the carboxy terminus of the molecule. Four other permissively recognized epitopes of this molecule (38.A, 38.I, 38.E, 38.K) were stimulatory for more than 50% of healthy PPD-positive individuals. Patients with lymphatic tuberculosis responded to these peptides in a similar manner. In contrast, we observed a selective anergy to stimulation with peptide 38.G in the majority of patients with pulmonary (11% responders) and nonlymphatic extrapulmonary tuberculosis (25% responders). The lack of responsiveness to 38.G was epitope specific since the degree of responsiveness to the other four permissively recognized peptide epitopes was similar for patients and PPD-positive controls. Using the PEPSCAN technology and truncated peptides, the core epitope of 38.G was localized to a peptide 10 amino acids long (HFQPLPPAVV). This minimal structure was capable of inducing a proliferative response in all healthy 38.G responders tested. The mechanisms influencing this epitope-specific anergy in patients could give new insights into the immunopathogenesis of tuberculosis.


Pore-forming protein in individual cytotoxic T lymphocytes: the effect of senescence provides a probe for understanding the lytic mechanism. The senescent decline of cytolytic T lymphocyte (CTL) activity was examined (a) to learn more about the effect of aging on the immune system, and (b) to probe the mechanism of cell-mediated cytolysis. The effect of age on the generation of pore-forming protein (Pfp) was examined at the cellular level in a murine model using CTL stimulated in allogeneic mixed lymphocyte culture (MLC). Pfp expression was analyzed by immunocytochemistry and enzyme-linked immunosorbent assay (ELISA). Immunocytochemical analyses of Pfp in MLC-stimulated splenic T cells from a large number of mice revealed that although stimulated cells from aged mice exhibited fewer Pfp-producing cells than those from young, the diminution in the proportion of Pfp+ cells was small compared to the age-related decrease in lytic activities (approximately 2-fold vs. approximately 7.4-fold, respectively). Time-course analysis disclosed similar kinetics for the generation of Pfp+ cells among responding cells from young and aged mice. No significant age-related difference in the proportion of Pfp+ cells was observed in MLC-stimulated lymph node cells despite a large and significant difference in lytic activity (approximately 6.5-fold). Purified CD8+ T cells demonstrated a large age-related difference in CTL activity (approximately 3-11-fold) and accounted for virtually all the Pfp. Although little difference in the proportion of Pfp+ CD8+ T cells could be detected between age groups, stimulated CD8+ cells or whole splenic T cells from old mice consistently exhibited a striking reduction in both the intensity of Pfp staining and the apparent numbers of granules per cell. This difference in Pfp was examined by ELISA and total Pfp levels were found to be approximately 12-fold greater in CTL generated from splenic T cells of young compared to aged mice. The results demonstrate that Pfp levels are reduced in CTL from aged compared to young mice at the level of the individual cells and suggest the possibility that a threshold level of Pfp may be required for potency of effector cell function.


Expression of major histocompatibility complex class I antigens at low levels in the thymus induces T cell tolerance via a non-deletional mechanism. Transgenic CBA (H-2k haplotype) mice expressing the H-2 Kb major histocompatibility complex (MHC) class I gene under control of transcriptional promoter elements from a milk protein gene display high-level H-2 Kb transcription in lactating mammary glands and low-level transcription in skin and thymus of male and virgin female transgenic mice. However, H-2 Kb antigen could be detected only in lactating mammary gland epithelial cells by immunohistological methods. All transgenic mice are tolerant of H-2 Kb since they fail to reject skin grafts from mice expressing H-2 Kb molecules. Furthermore, anti-H-2 Kb cytotoxic responses could not be generated using responder T cells from transgenic mice but T cells from the same mice proliferated, in the presence of interleukin-2, in response to stimulator cells expressing H-2 Kb. Tolerance to H-2 Kb is induced in the thymus since CBA mice grafted with thymus tissue from transgenic mice fail to reject H-2 Kb disparate skin grafts. However, experiments with double-transgenic mice also expressing a T cell receptor with anti-H-2 Kb specificity reveal that tolerance induction is not brought about by elimination of thymocytes bearing H-2 Kb-reactive receptors. Instead, a non-deletional mechanism which results in down-modulation of both CD8 and T cell receptor expression in peripheral T cells correlates with the induction of tolerance in these mice. These data reveal that extremely low levels of self-antigen expression in the thymus are sufficient to induce tolerance via non-deletional mechanisms.


Dissection of major histocompatibility complex (MHC) and T cell receptor contact residues in a Kb-restricted ovalbumin peptide and an assessment of the predictive power of MHC-binding motifs. The effect of alanine substitution on the major histocompatibility complex (MHC) binding and T cell receptor recognition of the Kb-restricted ovalbumin 257-264 peptide was investigated. Positions 3, 5 and 8 of the octamer were important for Kb binding, as predicted from the motifs found in Kb-associated peptides, while mutations at positions 4, 6 and 7 affected cytotoxic T lymphocyte recognition. Substitutions at positions 1 and 2 had very minor effects on T cell recognition. In addition, we tested the capacity of sequence motifs to predict MHC binding by analysis of a series of peptides which all bear the minimal Kb motif. We found that possession of good motifs was not always sufficient to give strong MHC binding, indicating secondary effects of the residues flanking the "MHC anchor" positions.


The CD39 molecule defines distinct cytotoxic subsets within alloactivated human CD8-positive cells. Lymphocyte activation induces or increases the expression of several surface structures, none of which is characteristic of an activated cell subset. In particular, structures such as CD45RO, CD25, CD26, CD49b, CD54, CD71 are expressed by the vast majority of lymphocytes at various times following in vitro activation. CD39 molecules were originally identified on activated B lymphocytes and have recently been described on activated T cell clones. In the present report, we have characterized phenotypically and functionally defined cell subsets generated during an in vitro allostimulation. Results indicated that the percentage of CD39+ cells reached a maximum at day 6 and remained stable thereafter. We demonstrate that CD39 expression allows the identification within the allosensitized CD8+ cytotoxic cells of distinct subsets of cells mediating allo cytotoxic T lymphocyte or natural killer (NK)-like reactivity. More precisely, CD8+CD39+ alloactivated cells mainly mediate specific killer activity, whereas CD8+CD39- alloactivated cells predominantly exhibit NK-like reactivity. Further, we show a high functional correlation associated with the lack of CD39 expression on NK-like alloactivated CD8+ cells, while there is no association with CD56 or CD57 NK-associated structures.


Alpha 1-acid glycoprotein potentiates lipopolysaccharide-induced secretion of interleukin-1 beta, interleukin-6 and tumor necrosis factor-alpha by human monocytes and alveolar and peritoneal macrophages. Although the physiological role of alpha 1-acid glycoprotein (AGP), an acute-phase protein, is poorly understood, several lines of evidence support a modulatory action on the immune response. In this study, we investigated the effect of AGP on the production of interleukin (IL)-1 beta, IL-6 and tumor necrosis factor (TNF)-alpha by human monocytes, macrophages and the monocytic THP-1 cell line. AGP significantly enhanced (2- to 7-fold) the production of these cytokines in monocytes induced by suboptimal concentrations of lipopolysaccharide [E. coli lipopolysaccharide (LPS): 100 ng/ml] in serum-free conditions, whereas it had little or no effect in the absence of LPS. The potentiating effect of AGP was inhibited by specific antibodies. It was concentration dependent and the greatest enhancement was observed with 250-500 micrograms/ml. Moreover, AGP only potentiated the effect of suboptimal concentrations of LPS. AGP did not alter the time course of LPS-induced IL-1 beta, IL-6 or TNF-alpha secretion. AGP acts as a co-inducer and could also potentiate cytokine secretion triggered by Neisseria meningitidis LPS and muramyl dipeptide. The glycan moiety of AGP did not seem to be involved in its potentiating effect, since both its major glycoforms and asialo-AGP potentiated the effect of LPS to the same extent as native AGP. Possible differences in the effect of AGP according to cell maturation were investigated using isolated human macrophages: AGP potentiated LPS-induced cytokine production by both peritoneal and alveolar macrophages. These data suggest that AGP can modulate monocyte/macrophage functions, thereby contributing to the amplification and regulation of immune and inflammatory responses.


Self tolerance to human A and B histo-blood group antigens exists at the B cell level and cannot be broken by potent polyclonal B cell activation in vitro. It is generally considered that tolerance to self antigens is less complete in B than in T lymphocytes. However, B cell tolerance through either functional inactivation (anergy) or clonal deletion has been demonstrated in transgenic mice. In the present study, we investigated whether B cells specific for self A/B histo-blood group antigens can be detected in normal humans. It is a key feature of the ABO system that all normal individuals make natural antibodies against those A or B carbohydrates which are not present in their organism. To detect B cells by the limiting dilution approach we used a specific enzyme-linked immunosorbent assay for the quantitation of anti-A/B antibodies, and a culture system in which polyclonal B cell activation occurs through cell contact with EL4 thymoma cells. As was reported for other B cell studies, we frequently detected "polyreactive" immunoglobulin (Ig)M (but not IgG) with apparent autoreactivity but of uncertain significance regarding physiologic conditions. However, A- or B-specific B cell responses occurred with selective patterns in agreement with classical blood group serology in 14 individuals with A, B, AB or 0 blood group phenotypes: 1/11,600 B cells made anti-allo A/B IgM and 1/26,500 B cells such as IgG, while only 1/104,000 B cells apparently made anti-self A/B IgM and 1/350,000 B cells such as IgG. This shows self tolerance at the B cell level. Since anergy of B cells can frequently be broken by polyclonal B cell activation in vitro, and EL4 cells are potent B cell stimulators, the present results argue for either a highly resistant anergic state or for clonal deletion of self-A/B histo-blood group-specific human B cells.


Effects of HIV-1 Tat protein on human T cell proliferation. In inducing immunodeficiency in human immunodeficiency virus (HIV)-1 infection, a role for the HIV-1 Tat protein has been suggested. In addition to effects on viral transcription, the protein has been ascribed immunosuppressive functions, which were investigated in this study. Proliferation of purified T cells to CD3 monoclonal antibodies immobilized to plastic plates was inhibited up to 70% by addition of 5 micrograms/ml of the Tat protein. This inhibitory effect, however, was not observed in the presence of accessory cells. Furthermore, no effect of Tat protein could be observed on T cell proliferative responses to recall antigen which most likely is related to the presence of accessory cells in the cultures. Taken together, our results do not imply an important role for immunosuppressive effects of Tat in induction of immunodeficiency as observed in HIV infection in vivo.


c-myc-induced natural killer cell sensitivity of human melanoma cells is reversed by HLA-B27 transfection. In human melanoma, activation of the c-myc oncogene results in locus-specific down-modulation of the HLA-B antigen expression. Moreover, overexpression of c-myc induces an increase of natural killer (NK) sensitivity of the tumor cells. To show that this effect on susceptibility to NK cells is mediated by the down-modulation of the HLA-B expression rather than by the activation of the oncogene, we supertransfected a c-myc transfectant with the gene encoding the HLA-B27 protein. The resulting supertransfectants with HLA-B27 surface expression were all less sensitive to NK cells than their parental cell line and showed a level of resistance equal to the original melanoma cell line with low c-myc expression. This indicates that the induction of NK sensitivity by c-myc activation in human melanoma cells is mediated through down-modulation of the HLA-B expression. These data also imply differential effects of HLA-A and HLA-B molecules on lysis by NK cells, because the level of NK susceptibility can apparently be defined by the level of HLA-B, irrespective of a substantial level of HLA-A expression present in the tumor cells.


Nitric oxide mediates suppression of T cell responses in murine Trypanosoma brucei infection. African trypanosomes induce a generalized state of immunosuppression in their mammalian hosts. One characteristic of this is a suppression of lymphocyte responses to mitogen, which is mediated by suppressor macrophages. We investigated the involvement of nitric oxide in this phenomenon. Both peritoneal and splenic cell cultures from infected mice released nitrite and this was inhibitable by NG-monomethyl L-arginine (L-NMMA). The release of nitrite correlated with suppressed splenic T cell proliferative responses to concanavalin A. It was shown that adherent spleen cells from infected mice mediate suppression, which could be abrogated by L-NMMA. These results suggest that in T. brucei infection, the activation of macrophages to produce nitric oxide leads to impaired lymphocyte responses and immunosuppression.


A versatile method to produce antibodies to human T cell receptor V beta segments: frequency determination of human V beta 2+ T cells that react with toxic-shock syndrome toxin-1. Human V beta (hV beta) regions have been expressed in the context of mouse T cell receptor (TcR)-CD3 complexes, and subsequently used to raise hV beta-specific monoclonal antibodies (mAb). The method of expression of hV beta outlined in this report contrasts in its versatility with the one reported by Choi et al. (Proc. Natl. Acad. Sci. USA 1991. 88: 8357). For instance, we have applied it successfully to the construction of mouse T cell transfectants expressing hV beta 1, hV beta 2, hV beta 3, hV beta 8, hV beta 9, hV beta 13.5, hV beta 19, hV beta 21, and hV beta 22 gene segments. mAb against the hV beta 2 and hV beta 19 regions have been raised by using these transfectants as immunogens in mice. Here, we illustrate the application of the anti-hV beta 2 mAb to the measurement of human T cells that react with the staphylococcal toxic-shock syndrome toxin-1.


The B29 and mb-1 polypeptides are differentially expressed during human B cell differentiation. Surface immunoglobulin on mouse B cells is associated with a heterodimer comprising the products of the mb-1 and B29 genes. Here we report that antibodies raised against a peptide sequence from the intracytoplasmic C terminus of the B29 murine gene product detect the 37-kDa component of the human heterodimer, indicating that this component in man is also encoded by the B29 gene. The immunocytochemical reactivity of these anti-B29 antibodies was compared with those of antibodies to the mb-1 protein. Of 25 cases of acute lymphoblastic leukaemia (ALL), 24 were positive for mb-1 whereas B29 was expressed in only 13 cases. Most of these B29-positive ALL expressed immunoglobulin mu heavy chain in their cytoplasm (pre-B ALL). In lymphoid tissue sections, anti-B29-labeled B cell follicles in a similar fashion to anti-mb-1, with the striking exception that plasma cells were unreactive for B29, but positive for mb-1. These results suggest that the synthesis of B29 begins later in precursor B cells than that of mb-1, and ceases before the terminal plasmacyte phase.


Quantitative analysis of the response of human T cell receptor V gamma 9+ cells to Plasmodium falciparum. Plasmodium falciparum stimulates peripheral blood gamma delta + T cells from unexposed donors. The responding cells bear V gamma 9+ chains of the T cell receptor, the majority of which, but not all, are associated with V delta 2 chains. We have analyzed whether the precursor frequency of these V gamma 9+ cells approaches that expected of superantigens or mitogens and whether, like a superantigen, the response is major histocompatibility complex (MHC) unrestricted. Limiting dilution analyses of the response of enriched gamma delta + T cells to P.falciparum using autologous and heterologous antigen-presenting cells suggest that the response is more characteristic of an antigen-specific MHC-restricted response. The frequencies are lower than would be expected if all V gamma 9+ cells respond, and there is a dramatically reduced response when allogeneic antigen-presenting cells are used.


High frequency of somatically mutated IgM molecules in the human adult blood B cell repertoire. Nucleotide sequence analysis of cDNA encoded by the single member of the human immunoglobulin VH6 gene family show that blood B cells in adults, but not in neonates, frequently express somatically mutated IgM molecules. The number of mutations in VH6-encoded cDNA from adult blood ranged from 2 to 19 mutations/VH gene (average 10.1/VH gene). The distribution of silent and replacement mutations suggests that at least some of the VH6 genes were derived from B cells that were activated and selected by antigen. We conclude that the blood B cell repertoire in adult humans, in contrast to its much-studied murine splenic counterpart, is a rich source of highly mutated IgM molecules.


Electropharmacology of dofetilide, a new class III agent, in anaesthetised dogs. In open chest anaesthetised dogs, dofetilide increased the ventricular effective refractory period over the dose range 1-100 micrograms/kg i.v. and the ventricular fibrillation threshold at doses between 3-100 micrograms/kg and was 80-1000 times more potent than sematilide, racemic sotalol, d-sotalol or quinidine. The maximal increases in ventricular fibrillation threshold induced by sematilide and quinidine were less than that induced by the other drugs. A change in the character of the induced arrhythmia from true ventricular fibrillation to a rapid ventricular flutter, with frequent spontaneous conversion, was observed following all drugs. No adverse haemodynamic effects of dofetilide, sematilide or d-sotalol were observed, but quinidine induced marked cardiac depression and racemic sotalol also impaired left ventricular contractility. All drugs reduced heart rate, though the effect of racemic sotalol was clearly greater than that of the other agents. Dofetilide is a potent class III antiarrhythmic agent with antifibrillatory properties and a favourable haemodynamic profile.


Effects of drugs on behavior before and during chronic diazepam administration. The effects of diazepam, pentobarbital, and phencyclidine (PCP) were studied on punished and unpunished responding maintained by fixed-interval schedules before and during chronic administration of diazepam. Before chronic diazepam administration, increasing doses of diazepam and PCP increased and then decreased rates of both punished and unpunished responding. Increases in punished responding were larger than increases in unpunished responding. Pentobarbital only increased punished responding, while higher doses decreased both rates of punished and unpunished responding. During chronic diazepam administration, rates of punished and unpunished responding showed further increases with all three drugs and the dose-effect curves also shifted to the right. Analysis of local rates of responding within fixed-interval components suggested that increases in low rates early in the interval were responsible for the rate increases produced by these drugs before chronic diazepam administration. During chronic diazepam administration low rates early in the interval showed greater increases after all three drugs and their ability to produce rate-increasing effects extended further into the interval. The similar effects of these drugs before and during chronic diazepam administration suggests a similar mechanism despite the widely recognized differences in their interaction with receptors. This common mechanism may relate to rate-increasing effects more than to specific effects on punished responding.


Chronic estrogen alters contractile responsiveness to angiotensin II and norepinephrine in female rat aorta. Sex hormones have been postulated to play an important role in the modulation of vascular responsiveness to endogenous vasoactive substances. This study was designed to establish the effects of chronic treatment with estrogen in vivo on subsequent contractile responsiveness of aortic rings to angiotensin II or norepinephrine in vitro. Concentration-response curves for angiotensin II were compared in aortic rings with or without endothelium isolated from ovariectomized rats (275-299 g) pretreated with 17 beta-estradiol (approximately 1 mg/kg per day) or placebo for 14 days and from normal prepubertal female rats (125-149 g) pretreated with 17 beta-estradiol (approximately 5 mg/kg per day) or placebo for 14 days. Whether or not endothelium was present, angiotensin II-induced contraction was significantly depressed in rings from 17 beta-estradiol-treated ovariectomized or prepubertal rats when compared to controls, and the pattern of the effects of 17 beta-estradiol-treatment on the concentration-response curves for angiotensin II was similar in the two models. In contrast to angiotensin II-induced contraction, norepinephrine-induced contraction was significantly enhanced in aortic rings with or without endothelium from ovariectomized rats pretreated with 17 beta-estradiol (approximately 1 mg/kg per day, 14 days) and from normal prepubertal female rats pretreated with 17 beta-estradiol (approximately 5 mg/kg per day, 14 days) when compared to controls. The results demonstrate that chronic treatment with 17 beta-estradiol selectively reduced and enhanced contractile responsiveness of aortic rings to angiotensin II and norepinephrine, respectively. Further, the results indicate that the normal prepubertal female rat is an efficient model to investigate modulation by estrogen of aortic responsiveness to vasoactive agents in vitro.


Effects of K+ channel blockers on the relaxant action of dihydralazine, cromakalim and nitroprusside in isolated rabbit femoral arteries. The relaxant responses to dihydralazine and the influence of different K+ channel blockers were studied in isolated rabbit femoral arteries. The prototype K+ channel opener, cromakalim, and nitroprusside, which does not produce relaxation by K+ channel activation were used for comparison. Dihydralazine was most effective on contractions induced by noradrenaline (EC50 = 1.1 microM; Emax = 95%) and relaxed the contractions elicited by 20 mM K+ (EC50 = 2.0 microM; Emax = 81% in preference to 124 mM K(+)-induced contractions (EC50 = 30.1 microM; Emax = 54%). Cromakalim, but not nitroprusside, also selectively relaxed 20 mM K(+)-induced contractions. In noradrenaline-contracted arteries, glibenclamide (10 microM) completely suppressed the relaxant response to cromakalim but did not influence the vasorelaxation produced by dihydralazine or nitroprusside. Tetraethylammonium (8 mM) and Cs+ (4 mM) shifted the concentration-relaxation curve for dihydralazine 2-fold to the right, whereas Ba2+ (0.1 mM), 4-aminopyridine (5 mM) and procaine (0.1 mM) failed to influence dihydralazine-induced responses. Tetraethylammonium (8 mM) shifted the concentration-relaxation curve for cromakalim and nitroprusside 6-fold to the right and suppressed the maximal relaxant effects by about 30%. It is concluded that dihydralazine produces vascular smooth muscle relaxation by a mechanism different from the opening of glibenclamide- and ATP-sensitive K+ channels.


Inhibition of [3H]paroxetine binding by various serotonin uptake inhibitors: structure-activity relationships. Fifty-four compounds structurally related to zimeldine or alaproclate and eight reference substances were examined as inhibitors of the high affinity binding of [3H]paroxetine to rat cerebral cortical membranes as a measure of the affinity of the 5-hydroxytryptamine (5-HT) transporter. None of the compounds had an affinity as high as paroxetine (KD = 0.026 nM). The most potent compound, 3-(4-methoxyphenyl)-1-methyl-3-phenylpropylamine (2) had a 5 times lower affinity than paroxetine. Some other diphenyl-1-methyl-propylamines displayed high affinity, e.g. the 4-bromo (4) and 2-bromo (7) derivatives. The primary amine analogue of zimeldine substituted with an alpha-methyl group (19) had an affinity only slightly less than that of norzimeldine (11) but an almost 100 times higher affinity than that of the unsubstituted primary zimeldine analogue (57). These observations indicate that a methyl group on the alpha-carbon and on the nitrogen both increase the affinity for the [3H]paroxetine binding site. The structure activity relationship for the compounds to inhibit [3H]paroxetine binding was highly significantly correlated to the inhibition of 5-HT uptake in mouse brain slices (P less than 0.01) and to the inhibition of noradrenaline uptake in the same slices (P less than 0.05). QSAR analysis of the zimeldine series of compounds indicates that substitution of halogens of the 2-position of the phenyl ring is unfavourable. The cis configuration promotes higher activity than the trans configuration.


Anticonvulsant activity of competitive antagonists of NMDA receptor in genetically epilepsy-prone rats. The potency of some competitive antagonists of N-methyl-D-aspartate (NMDA) to antagonize audiogenic seizures was evaluated in genetically epilepsy-prone rats following intraperitoneal or oral administration. The anticonvulsant effects were evaluated on seizures evoked by auditory stimulation (109 dB, 12-16 kHz) in animals placed singly under a perspex dome. Sixty and hundred-twenty minutes after intraperitoneal or 120 min after oral administration all compounds showed antiseizure activity with ED50 against clonus ranging from 11.6 to 384 mumol/kg after intraperitoneal and higher doses after oral administration. DL-(E)-2-Amino-4-methyl-5-phosphono-3-pentenoic acid (CGP 37849) and its carboxyethylester (CGP 39551) and 3-((+/-)-2-carboxypiperazin-4-yl)-1-propenyl-1-phosphonic acid (CPPene), were the most potent compounds when administered intraperitoneally and orally. 3-((+/-)-2-Carboxypiperazin-4-yl)-propyl-1-phosphonic acid ((-)-CPP) and (+)-CPP showed minor potency as anticonvulsants and 2-amino-7-phosphonoheptanoic acid (2AP7) was the least potent. Following oral treatment, the duration of action of CPPene was about 8 h, while CGP 39551 still protected against audiogenic seizures after 16 h. All compounds were anticonvulsant at doses below those at which overt behavioural side-effects were apparent. CPPene and (+/-)-CPP showed the best therapeutic index among the NMDA receptor antagonists studied. Since some of these compounds showed anticonvulsant properties after oral administration, potential use of these or other selective NMDA antagonists for antiepileptic therapy in man is suggested.


Effects of imidazoline-related compounds on the mechanical response to nicorandil in the rat portal vein. The purpose of this study was to investigate the interactions of compounds structurally related to imidazoline at K+ channels located in the rat portal vein. Nicorandil, a K+ channel activator, dose dependently inhibited spontaneous contractions of the isolated rat portal vein. Glibenclamide (0.1-1 microM), an ATP-sensitive K+ channel blocker, competitively antagonized the response to nicorandil, whereas methylene blue (10 microM), a guanylate cyclase inhibitor, did not. Phentolamine, antazoline, tolazoline, and midaglizole also shifted the dose-response curve for nicorandil to the right in the dose range of 1-100 microM. The rank order of potency was glibenclamide much greater than phentolamine = antazoline = midaglizole greater than tolazoline. In contrast, clonidine, idazoxan, imidazole, 1-benzylimidazole, and yohimbine were ineffective. In addition, cromakalim (1-100 nM), a selective K+ channel activator, also inhibited spontaneous contractions of the rat portal vein, and this effect was antagonized by phentolamine in a similar way to that found with nicorandil. These results suggest that some 2-substituted imidazolines, including phentolamine, possibly act as K+ channel blockers, like glibenclamide, in vascular smooth muscle.


Possible implication of peptidase activity in different potency of angiotensins II and III for displacing [125I]angiotensin II binding in pig aorta. A single class of [125I]angiotensin II ([125I]AII) binding sites was found in porcine aortic smooth muscle membranes. Des-Asp1-AII (AIII) and des-Asp1-[Ile8]AII were 20 times less potent than AII or [Sar1,Ile8]AII to displace [125I]AII binding. In contrast, AII and AIII equipotently induced an increase in cytosolic free Ca2+ concentration in cultured porcine aortic smooth muscle cells. Des-Asp1-[Ile8]AII and [Sar1,Ile8]AII equipotently inhibited the increase induced by either AII or AIII. In order to explain this discrepancy, we studied [125I]AIII binding. In the presence of amastatin or a potent inhibitor of aminopeptidase M, [125I]AII binding remained stable for 90 min, but [125I]AIII binding decreased gradually after the peak at 30 min. The decrease was completely blocked by the presence of amastatin and phenylmethylsulfonylfluoride. Consequently, AIII was as potent as AII to displace [125I]AII binding in the presence of these two protease inhibitors. These results suggest that the lower potency of AIII to displace [125I]AII binding is related primarily to AIII-specific degradation by proteases.