Nephrogenesis and the development of renal disease. Over the last few years there has been considerable progress in analysing the cellular basis of nephrogenesis and a start has been made towards elucidating the underlying molecular controls of this process. In this review we begin by describing how the kidney forms and then consider recent work on the mechanisms underlying these events. We review evidence implicating a neural basis for kidney induction and go on to show how the induced metanephric mesenchyme aggregates and forms condensations which themselves polarize to form epithelia and filtering nephrons. We then discuss how changes in the extracellular matrix are implicated in these processes and how the expression of nuclear transcription factors may regulate the final phenotype of the kidney. Finally, we show how the study of nephrogenesis is beginning to shed light on the aetiology of a range of disorders that include renal malformations, renal tumours, and inherited glomerular and polycystic kidney diseases.


Rehabilitation of young adults during renal replacement therapy in Europe. 1. The presence of disabilities. The aim of this study was to analyse rehabilitation during RRT in 617 young adults from different European countries who started dialysis or transplantation before the age of 15 years. The data were derived from the EDTA Registry patient data files and a special questionnaire that was sent to centres reporting to the EDTA Registry. The duration of RRT was more than 10 years in 63% of patients. Fifty-four percent were living with a functioning graft and 46% were on dialysis. The prevalence and severity of motor, hearing, sight, and mental disabilities were analysed retrospectively. They were found to vary according to primary renal disease and method of treatment. One-third of patients had one or more disabilities at the start of RRT. Although disability status had changed in many patients by 31 December 1986, some disability remained in one-third of the patients available for study. Disabilities were recorded as mild in the majority of patients. Both improvement and worsening of motor and mental disability occurred more often than changes of hearing capacity and sight. It is concluded that prevention and treatment of disabilities need special attention in children and young adults on RRT in order to improve rehabilitation.


Rehabilitation of young adults during renal replacement therapy in Europe. 2. Schooling, employment, and social situation. The educational status, employment rate and social situation were studied in 617 patients between 21 and 35 years of age who started renal replacement therapy (RRT) as children. The data were derived from a special questionnaire concerning disability and rehabilitation sent to dialysis and transplant centres reporting to the EDTA Registry. Fifty-six percent of patients completed secondary school and one in three went on to vocational training. Eleven percent of patients attended university, and 16% were reported to have gone to a special school for the handicapped. Up to one-third of patients who attended different school types failed to complete their education. There were notable geographical differences in schooling and in employment. Fifty-six percent of all patients were employed. Lack of schooling was considered to be a major reason for unemployment. Sixty-one percent of patients with disabilities and 34% without disabilities were receiving invalidity payments. The place of residence of these patients aged 21-35 was usually the parental home. Compared to the general population of similar age, only a few patients were married (13.5% of the total study group) and 8% had children. In summary, the present report shows that the major factors influencing rehabilitation on RRT are the presence of disabilities, the method of treatment, geographical factors, duration of RRT, and the underlying primary renal disease.


Familial occurrence of primary glomerulonephritis: evidence for a role of genetic factors. Chronic glomerulonephritis was diagnosed in 23 patients born in a small valley in Northern Italy and in five additional patients with one parent or a more remote ancestor born in the same area, all belonging to three potentially related families. Eighteen patients had biopsy-proven glomerulonephritis: 11 IgA nephropathy, 3 IgM nephropathy, 2 membranoproliferative type I, and 2 mesangial proliferative glomerulonephritis with isolated C3 deposits. Ten had clinical glomerulonephritis. A community screening programme discovered six additional related patients. Two underwent renal biopsy (1 IgA nephropathy; 1 focal glomerulosclerosis); four were diagnosed as having clinical glomerulonephritis. Genealogical investigation identified five deceased family members with diagnoses of chronic nephritis recorded on their death certificates. No environmental nephrotoxic factors were identified. Restriction fragment length polymorphism (RFLP) analysis of HLA-DR beta, HLA-DQ alpha and beta genes, and complement typing for C4A, C4B, and Bf polymorphisms were carried out for 29 patients, 168 healthy relatives, and 24 local controls. The frequency of HLA-Dw8.1 specificity, related to DR beta 8, DQ beta 3b, and DQ alpha 1a RFLPs, was significantly increased more in the affected and unaffected pedigree members than in Italian controls.


Lipoprotein(a) in patients with proteinuria. Lipoprotein(a) (Lp(a)) has recently been recognized to be a risk factor for coronary heart disease. Lp(a) median values in the absence of renal disease are around 10 mg/dl. Higher levels (greater than or equal to 30 mg/dl) correlate with the occurrence of coronary heart disease, particularly in the presence of elevated cholesterol. We have studied Lp(a) in 76 adults with proteinuria. Fifty had glomerular diseases and 26 non-glomerular diseases, with renal function varying from normal to advanced chronic renal failure. Lp(a) values were shifted to the right, with a median of 21.0 mg/dl, and 25% of patients had values of 30 mg/dl or more. Lp(a) did not correlate with cholesterol, age, lipoprotein subclasses, apoproteins A-I or B-100, albumin, creatinine, or creatinine clearance. Median Lp(a) values did not differ significantly comparing men versus women, or glomerular versus non-glomerular disease. Lp(a) may inhibit fibrinolysis, and is deposited in atherosclerotic lesions. Although the cause of these elevated Lp(a) levels is uncertain, we propose that they contribute to the increased risk of coronary heart disease in the nephrotic syndrome, and may play a role in progressive renal disease.


Acute renal vascular response to ACE inhibition in two rat strains with different susceptibility to the development of glomerulosclerosis. Normotensive male Wistar rats are susceptible to the development of glomerulosclerosis, a process which can be accelerated by partial renal ablation, while normotensive male Wistar Kyoto (WKy) rats are resistant to glomerulosclerosis. Long-term treatment with angiotensin-I-converting enzyme inhibitors prevents the development of glomerulosclerosis. We studied the acute renal vascular response to bolus injections of captopril (1, 3, and 10 mg/kg). Mean arterial blood pressure (MAP) was significantly less in Wistar rats compared to WKy and not influenced by unilateral nephrectomy. Renal vascular resistance (RVR) and the filtration fraction (FF) were significantly greater in sham and unilateral nephrectomy Wistar rats as compared to WKy. Captopril significantly reduced the RVR in all four groups, but at comparable doses, RVR remained greater in the Wistar sham and Wistar rats following unilateral nephrectomy compared to sham and uninephrectomized WKy respectively. Captopril reduced FF in Wistar rats only. These data indicate an enhanced level of activity of the renal renin-angiotensin system in glomerulosclerosis-susceptible Wistar rats compared to glomerulosclerosis-resistant WKy rats, suggesting that vascular reactivity involving the renin-angiotensin system is an important determinant of the genetically determined differences in susceptibility to glomerulosclerosis in these two rats strains. The strain-dependent difference in RVR at the highest dose of captopril indicates that additional, possibly structural features may play a role in the difference in susceptibility to glomerulosclerosis.


Role of kidney and liver in the renotropic activity generated in rats after uninephrectomy. Following uninephrectomy the remnant kidney undergoes a compensatory growth apparently regulated by a humoral renotropic factor(s). Our studies were carried out to assess the role of renal and hepatic tissue in the generation of such a renotropic factor(s). The renotropic activity in extracts of rat kidney and liver obtained at different times after uninephrectomy was assayed in cortical tubules from rat kidney removed 24 h after uninephrectomy. We found that kidney extracts from 6 h and 24 h uninephrectomized rats increased [3H]thymidine incorporation into tubular cell DNA, dose-dependently, compared to those from sham-operated rats. Maximal stimulation was observed at 10(4)-fold (6 h) or 10(2)-fold (24 h) extract dilution. This activity was undetected in perfused kidney extracts, or in extracts of kidneys from simultaneously uninephrectomized and partially hepatectomized rats. However, only 10-fold dilution of unperfused liver extract from 6 h or 24 h uninephrectomized rats showed significant activity. Kidney or liver extracts from 30 min uninephrectomized animals failed to display activity. Our results point to an extrarenal origin for the humoral renotropic activity generated after uninephrectomy, although the kidney seems to modulate this activity.


Effect of treatment mode on the natural history of acquired cystic disease of the kidney in patients on renal replacement therapy. The natural history of acquired cystic disease of the kidney has been investigated in a long-term follow-up study of patients on renal replacement therapy. A cohort of 145 end-stage renal failure patients was initially investigated with ultrasonography to determine the degree of cystic change. Seventy-three patients were available for follow up a minimum of 3 years later. The grade of cystic disease progressed in dialysis patients and progression was more marked in haemodialysis patients than patients maintained on CAPD. Patients with functioning renal transplants did not show progression of cystic change and in two patients regression was seen. Nine patients maintained on chronic dialysis at the time of initial ultrasound subsequently received renal grafts, and three of these patients had evidence of regression of cystic change on follow-up scanning. After 3 years follow-up a single haemodialysis patient had evidence of a solid lesion in a cystic kidney and this has not progressed during a further 12 months of follow-up. Acquired cystic disease of the kidney is a progressive disease in chronic dialysis patients. However, over a follow-up period of 3 years, patients with functioning renal grafts do not show similar progression. The incidence of solid renal tumours has been shown to be low.


Does haemodialysis impair macrophage Fc receptor function? Patients treated with chronic haemodialysis are at risk of infections, possibly because of impaired function of macrophage Fc receptors. Using [123I]-labelled aggregates of human IgG ([123I]-AIgG) as a probe of Fc-receptor-mediated function, we examined eight patients treated with chronic intermittent haemodialysis (HD), eight patients treated with CAPD, eight patients with preterminal renal failure who had not yet received renal replacement therapy, and eight healthy controls. In all three patient groups the first elimination half-life of [123I]-AIgG was decreased, suggesting accelerated binding of the probe. In the HD group overall clearance of [123I]-AIgG was similar to the value found in healthy controls. In the CAPD and preterminal renal failure group clearance was decreased as compared with the HD patients. Uptake of [123I]-AIgG by liver and spleen was quantitatively similar in patients and controls, but hepatic uptake of [123I]-AIgG reached its maximum earlier in the patients treated with HD. These results suggest that Fc receptor function is not impaired in patients who undergo chronic haemodialysis.


Effect of bezafibrate on lipoprotein (a) and triglyceride-rich lipoproteins, including intermediate-density lipoproteins, in patients with chronic renal failure receiving haemodialysis. The effect of bezafibrate, at doses of 200 mg three times weekly throughout a period of 10 weeks, on lipoprotein (a) and triglyceride-rich lipoproteins including intermediate-density lipoproteins (IDL) has been studied in 12 patients with chronic renal failure receiving haemodialysis. No side-effects were observed, and serum creatine phosphate kinase values remained within normal limits throughout the duration of the study. Serum cholesterol, triglycerides and apoprotein (apo) B significantly decreased after bezafibrate, whereas apo A-I increased significantly. Serum lipoprotein (a) decreased after bezafibrate, although differences reached no statistical significance. The very-low-density lipoproteins (VLDL) cholesterol and the VLDL triglycerides decreased from 0.93 +/- 0.45 mmol/l (Mean +/- SD) to 0.54 +/- 0.27 mmol/l (P less than 0.05) and from 1.50 +/- 0.58 mmol/l to 0.91 +/- 0.35 mmol/l (P less than 0.01) respectively. The IDL cholesterol and IDL triglycerides decreased from 0.44 +/- 0.35 mmol/l to 0.20 +/- 0.18 mmol/l (P = NS) and from 0.46 +/- 0.35 mmol/l to 0.17 +/- 0.12 mmol/l (P less than 0.05) respectively. These data, with a substantial improvement of the lipoprotein profile in patients with chronic renal failure on haemodialysis after treatment with bezafibrate, suggest a decrease in the coronary heart disease risk.


Peritoneal drainage: an important element in host defence against staphylococcal peritonitis in patients on CAPD. The growth of Staphylococcus aureus and coagulase-negative staphylococci were studied in fresh and effluent peritoneal dialysate from patients on continuous ambulatory peritoneal dialysis (CAPD). Peritoneal drainage during CAPD removes bacterial contaminants from the peritoneal cavity with an efficiency that depends upon the volume of peritoneal fluid remaining after drainage (residual volume). Combination of our data on the growth of coagulase-negative staphylococci in dialysate with a mathematical model of peritoneal drainage during CAPD shows that a residual volume of less than 800 ml (normal = approximately 400 ml) will prevent survival in the peritoneal fluid. A residual volume of less than 200 ml is required to eliminate S. aureus because of its faster rate of growth in dialysate. Previous work has shown that numbers of macrophages are too few to influence bacterial growth in the peritoneal dialysate. Coagulase-negative staphylococci adhere poorly to mesothelial cells in culture. Survival within the peritoneal cavity during CAPD probably depends on colonization of the PD catheter. Coagulase-negative staphylococcal peritonitis is likely to be localized to areas of the peritoneal membrane in close contact with the PD catheter. S. aureus is able to multiply in the peritoneal dialysate during CAPD and thereby causes generalized peritonitis.


Audit of the use of calcium carbonate as a phosphate binder in 100 patients treated with continuous ambulatory peritoneal dialysis. We studied the effect of converting 100 established CAPD patients from aluminium- to calcium-based phosphate binders. After a follow-up of 1 year only 60% of patients remained on calcium carbonate. Hypercalcaemia was the major problem, with more than 40% of patients having a serum calcium in excess of 3.0 mmol/l. Several patients required hospitalization for symptomatic hypercalcaemia. Hypercalcaemia was more common in patients with normal serum parathyroid hormone concentrations (65 versus 25%, P less than 0.01). Serum phosphate control was better prior to commencing calcium carbonate when patients were treated with aluminium phosphate binders mean 1.71 +/- 0.15 mmol/l (SEM) than at the time of maximum serum calcium concentration, 1.81 +/- 0.25, P less than 0.05. This study does not confirm the findings of others, which have suggested that calcium carbonate is a safe and effective phosphate binder for patients with end-stage renal failure.


Diabetic glomerulosclerosis without diabetes mellitus--two case reports and a review of the literature. The duration of diabetes mellitus and presence of hyperglycaemia appear to be important in the development of diabetic nephropathy. The presence of nodular glomerulosclerosis is thought to be pathognomonic of the condition. We report two patients with histological features of diabetic glomerulosclerosis who did not have diabetes mellitus. The discussion reviews the literature and concludes that diabetic glomerulosclerosis with normal glucose tolerance is very rare and that most cases are due to overt diabetes mellitus or a degree of glucose intolerance. However, cases with only minimal glucose intolerance suggest that factor(s) other than hyperglycaemia are responsible for diabetic renal damage.


Autosomal dominant polycystic kidney disease with primary hyperaldosteronism. We report three cases of primary aldosteronism associated with autosomal dominant polycystic kidney disease. The diagnosis of primary hyperaldosteronism was based on the presence of hypokalaemia with excessive urinary potassium excretion and/or the characteristic hormonal changes. Renal function impairment due to autosomal dominant polycystic kidney disease could mask hypokalaemia. The interpretation of adrenal imagery may be hindered by adjacent renal cysts. In one case an adrenal adenoma was detected and surgically removed, with only partial correction of the blood pressure. This could be explained by the persisting underlying autosomal dominant polycystic kidney disease. We conclude that in a hypertensive patient with polycystic kidney disease, extrarenal causes of hypertension may be present.


The use of vaginal pessaries for uterine prolapse. Vaginal pessaries are useful for treating uterine prolapse when surgical intervention is not a safe option. This article covers recommendations for selection, insertion and care of the appliance. Careful follow-up is necessary to minimize possible complications, which can include infection, ulceration of the mucosa, incarceration of the pessary, and an increased incidence of vaginal and cervical malignancy. Disagreement within the literature about the proper schedule for follow-up, and inadequate documentation of the risks of pessary usage underscore the need for clinical research in this area.


Kidney depth measured by using 99Tcm-DMSA. Comparison with CAT scan: review of 50 measurements. Measurement of kidney depths using computed axial tomographic (CAT) scans, considered as the gold standard, has been compared in 25 patients with depth measured using an isotopic method (99Tcm-DMSA). 99Tcm-DMSA underestimates renal depth in 78% of the cases (mean underestimation: 0.78 cm). The correlation coefficient between the two methods is 0.82. As a comparison, kidney depths calculated using Tonnesen's formula are also given and related to CAT scan results. The presence of renal morphological abnormalities in 18% of the studied kidneys allowed the authors to study their potential influence on 99TcmDMSA measurements. They do not seem to be very different from those obtained in morphologically normal kidneys. The different methods for kidney depth measurement described in the literature are briefly reviewed.


Replacing 99Tcm-DMSA for renal imaging?

Replacing 99Tcm-DMSA for renal imaging? We have tried to evaluate the usefulness of an early 99Tcm-MAG3 image, obtained during the second minute after injection of the tracer, in predicting the renal alterations seen on a 99Tcm-DMSA scintigraph, in children clinically suspected of pyelonephritis. It appears that the accuracy of the MAG3 image is population dependent: when, in most of the patients of a study group, DMSA scintigraphy is either normal or very abnormal, the MAG3 image will correctly reflect the DMSA scintigraph. However, when the DMSA alterations are less pronounced, the early MAG3 image will miss the lesions in about 50% of the cases.


An evaluation of human papillomavirus testing as part of referral to colposcopy clinics. To determine the usefulness of human papillomavirus (HPV) testing as a triage method for predicting which women referred to a colposcopy clinic were most likely to have histologically confirmed cervical intraepithelial neoplasia (CIN). Papanicolaou tests, ViraPap tests for HPV infection, and colposcopically directed biopsies were performed concurrently on 482 women referred to a student health colposcopy clinic. The results demonstrated that HPV positivity was associated with a greatly increased likelihood of histologic confirmation of CIN, especially among women with concurrent cytologic findings that were negative or showed only atypical squamous cells of undetermined significance. Testing for HPV appears to have a role in the triage of students now being referred to our colposcopy clinic. A combination of HPV testing and repeated cytologic screening would provide reasonably sensitive screening for cervical neoplasia while limiting the use of colposcopic services, which are currently overburdened. The eventual usefulness of HPV testing will depend on the cost and availability of colposcopy services, the cost of Papanicolaou tests, the cost and accuracy of HPV tests, and the predictive value of HPV detection in the population being screened.


Fetal abdominal echogenic mass: an early sign of intrauterine cytomegalovirus infection. Recent advances in ultrasound technology have made it possible to diagnose fetal diseases much earlier in pregnancy. Reports have indicated that intrauterine infections, specifically cytomegalovirus, can be diagnosed prenatally by ultrasound. We encountered two cases in which cytomegalovirus infection was associated with hyperechoic areas in the fetal abdomen. Hyperechoic areas in the fetal abdomen may be an early sign of fetal cytomegalovirus infection. Persistence of this sign may indicate a mild form of infection. Disappearance of these hyperechoic areas in subsequent ultrasonographic examinations and development of other ultrasound signs may indicate severe infection and poor prognosis.


Percutaneous transcatheter embolization for control of life-threatening pelvic hemorrhage from gestational trophoblastic disease. Pelvic hemorrhage from gestational trophoblastic disease remains a common and vexing problem. Traditional surgical therapy, including hysterectomy and hypogastric artery ligation, may be technically difficult as well as hazardous to debilitated patients. In contrast, percutaneous transcatheter embolization specifically occludes the vessels that directly contribute to bleeding. Other potential advantages include avoidance of general anesthesia and major surgery, a rapid recovery period, and preservation of fertility. Various embolic materials allow one to tailor the duration of occlusion to the underlying disorder. Reported complications are rare and generally involve aberrant emboli or inadequate collateral circulation leading to ischemic injury. We believe that transcatheter embolization should be considered an alternative to operative intervention for control of pelvic hemorrhage from gestational trophoblastic disease.


Attenuation by the GABA receptor agonist baclofen of experimental carcinogenesis in rat colon by azoxymethane. The effects of the gamma-amino-n-butyric acid (GABA)A receptor agonist muscimol, and the GABAB receptor agonist baclofen, on colon carcinogenesis induced by azoxymethane in Wistar rats were tested. Administration of either muscimol or baclofen had no influence on the incidence of colon tumors at week 30. However, almost all the colon tumors that developed in rats treated with baclofen were adenomas, whereas in the control group the tumors were mainly adenocarcinomas. In contrast, administration of muscimol did not affect the histological types of the colon tumors. These findings indicate that baclofen inhibits the growth of colon tumors via the GABAB receptor.


Incidence and significance of congenital hypertrophy of the retinal pigment epithelium (CHRPE) in familial adenomatous polyposis coli (FAPC). As part of a population based study of familial colorectal cancer 33 affected patients with familial adenomatous polyposis coli (FAPC) and 33 relatives, at 50% risk of inheriting FAPC, from 24 kindreds, were identified and examined. Fourteen of the affected patients had extracolonic manifestations of the FAPC gene. Twenty-five of the 33 affected patients had one or more areas of congenital hypertrophy of the retinal pigment epithelium (CHRPE) and 20 had more than three CHRPEs, all having bilateral lesions. There were significant interfamilial differences in the ocular findings. Because of this interfamilial difference in the predisposition to develop CHRPEs it is important to establish the CHRPE status of individual FAPC families before the results of ophthalmoscopy can be used to predict the carrier status of at risk relatives.


[Management of migraine].

[Management of migraine]. When considering treatment for migraine patient, the physician should bear in mind how little is known about this remarkably complex and diverse condition with no miracle cure or single uniform method of treatment. Experience suggests that adherence to a number of principles based on a combination of common sense, modesty, and open-mindedness is essential to effective management of migraine. The patient needs to be both reassured and taken seriously, and should be told that the cause of migraine is unknown and that performing multiple investigations would be unhelpful. The difference between acute treatments and maintenance prophylactic treatments should be clearly explained. The patient should participate in therapeutic decisions such as whether or not to use prophylaxis and whether pharmacologic or nonpharmacologic treatment is most appropriate. Finally, and most importantly, both the physician and patient must be aware that much patience is needed but that this patience will usually be rewarded, not by complete resolution of the disease, but by an often dramatic improvement in the migraine patient's quality of life.


[Extracellular and intracellular activity of sparfloxacin against Mycobacterium avium complex and Mycobacterium xenopi]. Activity of the new fluoroquinolone sparfloxacin against 30 strains of M. avium complex and 25 strains of M. xenopi was tested in vitro. Sparfloxacin was used alone (determination of MICs and MBCs) and in combination with ethambutol and rifabutin. Synergy studies with determination of the FIC and FBC indices showed that the sparfloxacin-ethambutol combination was synergistic against 10 M. avium complex strains and 12 M. xenopi strains. With the three-drug combination (sparfloxacin-ethambutol-rifabutin), synergy was found against 12 M. avium and 14 M. xenopi strains. Studies of intracellular bacteria showed that the decrease in viable bacteria with the three-drug combination was 1 log for M. avium and 2 log for M. xenopi.


[In vitro activity of sparfloxacin against mycoplasmas]. The in vitro activity of the new difluorinated quinolone sparfloxacin against mycoplasmas was studied comparatively with ofloxacin, doxycycline, and erythromycin. Minimal inhibitory concentrations (MICs) were determined by agar dilution for 21 strains of Mycoplasma pneumoniae (Mp), 20 strains of M. hominis (Mh), 7 strains of M. genitalium (Mg), and 3 strains of M. fermentans (Mf), and by broth dilution for 49 strains of Ureaplasma urealyticum (Uu). Sparfloxacin was very active against all tested mycoplasmas, with the following MICs (microgram/ml): 0.1 for Mp, 0.05-0.1 for Mg, less than or equal to 0.01 for Mh, less than or equal to 0.01-0.05 for Mf, and 0.1-0.5 for Uu. Minimal bactericidal concentration was 1 microgram/ml for Uu. Sparfloxacin was more active than ofloxacin against all the mycoplasmas tested and was the most active compound against Mh and Mf. Erythromycin had the lowest MICs against Mp and Mg. Sparfloxacin exhibited comparable effectiveness against doxycycline-susceptible and doxycycline-resistant strains. Sparfloxacin appears to be one of the most active agents in vitro against mycoplasmas.


Clinical characteristics of parainfluenza virus infection in hospitalized children. Severity of illness and clinical characteristics of parainfluenza virus (PIV) infection were evaluated in 81 hospitalized children over a 4 year period. Fifty three patients were previously healthy and 28 had preexisting pulmonary abnormalities associated with bronchopulmonary dysplasia (BPD), congenital heart disease (CHD), asthma, or prematurity. When compared with formerly healthy children, the patients with preexisting pulmonary abnormalities were more likely to develop lower than upper respiratory tract illness (P less than 0.0001). In the lower respiratory tract infection group, patients with preexisting pulmonary abnormalities were sicker (P = 0.047), were hospitalized longer (P = 0.016), required more supplemental oxygen (P = 0.004), and were older (8.8 vs. 5.1 months) than previously healthy patients. Nosocomial infection occurred only in BPD patients. All these patients developed pneumonia. They were sicker (P = 0.0018), requiring more therapy (P = 0.0038) than other patients with preexisting pulmonary abnormalities and lower respiratory tract disease. Patients with BPD should be placed in protective isolation during PIV epidemics.


Hepatoblastoma: a clinico-pathologic review. Hepatoblastoma, the most common and peculiar tumor of the liver in infancy and childhood, has recently been the object of an international protocol of the European (SIOP) Histopathological Study Group in order to establish common anatomicoclinical criteria provided with therapeutic and prognostic significance. A general review of hepatoblastoma concerning its possible association with congenital anomalies, endocrine and metabolic disorders, as well as physical and laboratory data, macroscopic and histological patterns, is reported. The different histotypes of hepatoblastoma, represented by anaplastic, embryonal, fetal, mixed (epithelial and mesenchymal) and teratoid, are described, and hypothesis of the pathogenesis of this tumor is discussed, based upon immunohistochemical investigations.


Significance of binding to Na,K-ATPase in the tissue distribution of ouabain in guinea pigs. Ouabain binds specifically to Na,K-ATPase on the plasma membrane and therefore serves to measure the tissue concentration of Na,K-ATPase. We examined the role of ouabain binding to Na,K-ATPase in its overall tissue distribution. The tissue-to-plasma concentration ratio (Kp,vivo) was defined in each tissue after intravenous administration of 3H-ouabain in guinea pigs, and specific binding of ouabain to Na,K-ATPase was measured in tissue homogenate to obtain the dissociation constant and binding capacity in each tissue. A predicted tissue-to-plasma concentration ratio (Kp,vitro) was calculated using the obtained binding parameters and the volume of extracellular space in each tissue. The absolute values of Kp,vitro were comparable to those of Kp,vivo, except in brain. Regression analysis showed that the specific binding capacity of Na,K-ATPase in each tissue is the main factor in the tissue variation of Kp,vivo. Therefore, the binding of ouabain to Na,K-ATPase plays a significant role in the tissue distribution of ouabain.


[Value of the in situ hybridization technique in the diagnosis of human papilloma virus infections of the uterine cervix. Correlations between the human papilloma virus type and the morphological features]. Clinico-pathologic, epidemiologic and molecular analytic studies have shown that: i) the squamous cancer of the uterine cervix derives from intraepithelial lesions with different degrees of atypia; ii) human papillomavirus (HPV) can play a fundamental role in the progression of these lesions. We have examined 219 uterine cervix biopsies by means of the in situ hybridization technique (ISH) using biotinylated probes for 6/11, 16/18 and 31/35/51 HPV strains. The histologic lesions were as follows: 104 (49.7%) focal koilocytotic lesions 26 (11.8%) low grade intraepithelial lesions (SIL) 45 (20.5%) high grade intraepithelial lesions (SIL) 18 (8.2%) condylomata acuminata 21 (9.8%) invasive cancers. The percentage of the positivity found in the whole material examined is 14.1%. In particular, none of the 109 cases of focal koilocytotic lesions was positive, whereas the 28.1% of the remaining 110 biopsies turned out to contain HPV. We hypothesize that our small percentage of positivity in preinvasive and invasive lesions of the uterine cervix can be due to the fact that the HPV is not the only cancerogenic factor involved, although the possibility of a low sensitivity of the technique cannot completely be excluded.


Low, intermediate and high grade breast carcinomas as determined by histotyping, immunohistochemical prognosticators and histological grading. Low grade breast cancers i.e. mucinous (17 cases--3.2%), tubular (7 cases--1.3%) and invasive cribriform carcinomas (3 cases--0.5%) have been identified within a series of 524 breast cancers only by histotyping in hematoxylin-eosin stained sections: the reactivities of immunohistochemical prognosticators as estrogen/progesterone receptors (ER, PgR), growth fraction (GF: Ki67), p53 and c-erbB-2 oncoproteins are in agreement with clinical behaviours. Invasive papillary carcinomas (9 cases--1.6%) are not to be considered low grade carcinomas. Intermediate grade cancers are also determined by histotyping. Medullary carcinoma (13 cases--3.4%) has a paradoxical behaviour displaying a favourable clinical prognosis together with high grading and GF, absence of ER, PgR, high p53 and c-erbB-2 values, as compared with invasive ductal carcinomas: an extensive tissue immune response as suggested by a heavy lymphocyte infiltration may explain this behaviour. Invasive lobular carcinoma (62--11.6%) shows an intermediate immunohistochemical pattern, paralleling an intermediate prognosis, when compared with low and high grade carcinomas: ER, PgR and GF positivities are nearly the same as in ductal carcinomas whereas grading, p53 and c-erbB-2 are less expressed. These data are confirmed both for lobular carcinomas as a whole and for all variants of this kind of tumors. Invasive ductal carcinomas (413 cases--79%) may be stratified on three prognostic classes corresponding to histological grading (G1, G2, G3). Significant relationships of grading with all the immunohistochemical prognosticators studied has been observed. It may be concluded that grading is a parameter of paramount importance in this group of tumors.


Absorptive-mediated endocytosis of an adrenocorticotropic hormone (ACTH) analogue, ebiratide, into the blood-brain barrier: studies with monolayers of primary cultured bovine brain capillary endothelial cells. The internalization of a neuromodulatory adrenocorticotropic hormone (ACTH) analogue, [125I]ebiratide (H-Met(O2)-Glu[125I]His-Phe-D-Lys-Phe-NH(CH2)2NH2), was examined in cultured monolayers of bovine brain capillary endothelial cells (BCEC). HPLC analysis of the incubation solution showed that [125I]ebiratide was not metabolized during the incubation with BCEC. The acid-resistant binding of [125I]ebiratide to BCEC increased with time for 120 min and showed a significant dependence on temperature and medium osmolarity. Pretreatment of BCEC with dansylcadaverine or phenylarsine oxide, endocytosis inhibitors, and 2,4-dinitrophenol, a metabolic inhibitor, decreased significantly the acid-resistant binding of [125I]ebiratide. The acid-resistant binding of [125I]ebiratide was saturable in the presence of unlabeled ebiratide (100 nM-1 mM). The maximal internalization capacity (Bmax) at 30 min was 7.96 +/- 3.27 pmol/mg of protein with a half-saturation constant (Kd) of 15.9 +/- 6.4 microM. The acid-resistant binding was inhibited by basic peptides such as poly-L-lysine, protamine, histone, and ACTH but was not inhibited by poly-L-glutamic acid, insulin, or transferrin. These results confirmed that ebiratide is transported through the blood-brain barrier via an absorptive-mediated endocytosis.


Cystosarcoma phyllodes of the breast: histologic features, flow cytometric analysis, and clinical correlations. The histologic features of 187 cases of cystosarcoma phyllodes of the breast were reviewed. The tumors were divided into histologically benign, borderline, and malignant categories. Correlation with clinical outcome was available in 100 cases. Overall rate of local recurrence was 28% (benign, 27%; borderline, 32%; malignant, 26%). Metastases occurred in eight of 100 cases (two borderline and six malignant). Although no histologic features were predictive of local recurrence, stromal overgrowth, mitotic rate greater than 15 per 50 high-power fields, and cytologically atypical stromal cells characterized seven of the eight tumors that metastasized. These features were not evident in the eighth case. Flow cytometric analysis of eight tumors (four benign, two borderline, and two malignant) showed discordance between histology and DNA content in three cases. There was slightly better correlation of histology and S-phase fractions. Based on these results demonstrating the difficulty in predicting clinical outcome, wide local excision remains an appropriate initial method of treatment. Simple mastectomy may be necessary for very large tumors and should be considered in histologically malignant tumors and cases with multiple recurrences, since some recurrent tumors in this series showed increasingly unfavorable histologic features.


Multiple HPV infection: microanatomy by in situ hybridization and immunohistochemistry. Specific human papillomavirus (HPV) types have been shown to be associated with proliferative epithelial lesions with variable biological consequences in infected patients. Simultaneous infection by more than one HPV type has been infrequently reported, and its clinical significance is unknown. We have examined four biopsies of cervical and vulvar tissue, each with evidence of infection by two different HPVs. Using both in situ hybridization and immunohistochemical techniques, we determined the cellular distribution of the viral infections. Using biotinylated type-specific probes and stringent conditions we were able to demonstrate that in each case the two HPVs occupied distinct, non-overlapping foci within the lesions. The condylomatous tissues contained DNA from HPV types that are associated with high-grade neoplasia and invasive cancer (16 and 18), as well as types commonly associated with benign proliferative lesions. Immunohistochemical analysis of the lesions with antibody to bovine papillomavirus capsid antigen failed to detect HPV in regions shown by in situ hybridization to contain HPV 16 and 18 DNA, whereas type 6 and 11 infected areas were readily identified. These results provide indirect evidence of viral interference between HPV types and indicate that interference may limit the number of HPV types that produce active infections within a single cell.


Is cytomegalovirus associated with renal disease in AIDS patients? Although it has been suggested that cytomegalovirus (CMV) infection of the kidney might facilitate the development of human immunodeficiency virus-associated nephropathy (HIVAN) or other morphologic renal changes in patients with AIDS, no systematic study has been performed on kidneys from AIDS patients. We examined 75 autopsy kidneys, two renal biopsy specimens, and a nephrectomy specimen from 78 HIV-infected patients (five with HIVAN) for the presence of CMV. Immunocytochemistry (ICC) utilizing a monoclonal antibody against the late antigen of CMV and in situ hybridization (ISH) with a biotinylated DNA probe for CMV sequences were used. The detection system for both ICC and ISH was streptavidin-conjugated alkaline phosphatase with Fast Red TR chromogen. CMV was detected in only 10 of the 78 kidneys examined (12.8%): eight by both methods, one by ISH only, and another by ICC only. All 10 positive kidneys were obtained from autopsies of patients with AIDS. The average number of positive cells (in approximately 15 x 10 mm sections) was 22 with ICC and 10 with ISH. Glomerular intracapillary cells (possibly endothelial cells) were the most commonly stained, followed by positive cells in the interstitium and peritubular capillaries. Relatively few tubular epithelial cells were stained. The majority of positive cells by either ICC or ISH did not show nuclear or cytoplasmic inclusions; however, only two of the 10 positive kidneys did not contain cells with typical Cowdry type-A intranuclear CMV inclusions. The most frequent pathologic finding in the kidneys positive for CMV by either ICC or ISH was acute tubular necrosis (in six of 10, 60%).(ABSTRACT TRUNCATED AT 250 WORDS)


In situ hybridization for Epstein-Barr virus NotI repeats in posttransplant lymphoproliferative disorder. Epstein-Barr Virus-associated posttransplant lymphoproliferative disorder (PTLD) is a serious complication of solid organ transplantation. In PTLD, B-cell expansions range from reactive hyperplasias to large cell lymphomas and are often associated with active Epstein-Barr virus (EBV) infection. The lymphoproliferations may infiltrate transplant allografts and therefore may need to be distinguished from acute cellular rejection (ACR). A total of 36 tissue specimens from 11 transplant patients (six kidney, two heart, three liver) with PTLD were studied for EBV content by automated in situ hybridization (ISH) on formalin-fixed or Bouin's-fixed, paraffin-embedded tissue using a synthetic 3' terminally biotin-labeled oligonucleotide DNA probe from the EBV NotI tandem repeat region. The NotI repeat is abundantly transcribed during productive EBV infection and may encode an EBV early antigen. EBV serologies from the 11 patients showed seven primary acute infections and one acute reactivation. Two serologic studies indicated infection of indeterminant onset, and serology was not performed on one patient. Histologically, seven patients presented with polymorphous infiltrates in transplant allograft biopsies, three of which progressed to disseminated monomorphous cell populations and death within 3 to 6 wk. Tissues examined by ISH from all 11 patients showed nuclear staining for EBV in the atypical lymphoid infiltrates (34/36 specimens). The nuclear signal ranged from a stippled pattern of positivity to homogeneous nuclear staining and was localized predominantly in follicular center cells and immunoblasts, although some smaller lymphocytes also contained the viral genome. ISHs performed on 31 allograft biopsies with ACR from 24 transplant patients (six kidney, five heart, 13 liver) without clinical evidence of PTLD and with serological evidence of past EBV infection were negative for the virus. Cell lines containing EBV in the productive state (EB3 and P3HR1) were positive with ISH for NotI, while a latently infected cell line (Raji) was negative. These data indicate that ISH with the NotI probe identifies amplified genome in EBV infections and is useful in discriminating the atypical infiltrate of EBV-associated PTLD from that seen in ACR.


In situ hybridization with nonisotopic probes using different detection systems. In order to evaluate a sensitive nonisotopic in situ hybridization method for routine work in pathology laboratories, we compared seven different detection systems, using digoxigenin- and biotin-labeled probes. The sensitivity of these methods was tested on four cases of cervical condyloma all known to be positive for HPV 6. Four of these methods gave satisfactory results without any background staining. The single biotin method and the single digoxigenin method were equally sensitive, while the two triple biotin methods, using mouse anti-biotin/anti-mouse IgG/alkaline phosphatase mouse anti-alkaline phosphatase or mouse anti-biotin/alkaline phosphatase anti-mouse IgG/alkaline phosphatase mouse anti-alkaline phosphatase as the detection systems, tremendously improved the sensitivity. The enhanced sensitivity of the nonisotopic in situ hybridization method make it useful in investigation of pathologic tissues.


Multiplex polymerase chain reaction.

Multiplex polymerase chain reaction. The polymerase chain reaction (PCR) is a widely utilized assay for specifically amplifying small fragments of DNA. Multiplex PCR is the amplification of more than one DNA fragment per reaction and has many potential uses. When more than one primer set per reaction tube is utilized, the total number of tubes in any one experiment may be reduced, conserving expensive reagents and decreasing possible contamination. Multiplex PCR allows for an assay of the gene of interest and assures that the amplification process proceeds as expected with the use of a companion control genome primer set. Multiplex PCR is useful in assaying DNA extracted from samples of immunocompromised patients in which more than one infectious agent may be suspected such as simultaneous EBV and CMV detection. Multiplex PCR offers many advantages over single reaction PCR and has been found to be an useful adjunct in our laboratory.


Sinonasal Schneiderian papillomas: human papillomavirus typing by polymerase chain reaction. A series of 35 formalin-fixed, paraffin-embedded Schneiderian papillomas (24 inverted, nine cylindrical cell type, and two fungiform) of the nasal cavity were evaluated for the presence of human papillomavirus (HPV) types 6b/11, 16, and 18 DNA sequences using both a highly sensitive and specific modification of the polymerase chain reaction technique and conventional in situ hybridization. The HPV gene sequences (E6-E7 portions) were not detected in any of the 24 inverted or nine cylindrical cell papillomas. One of the fungiform papillomas was positive for HPV 6b/11. We conclude: (a) the origin of most Schneiderian sinonasal papillomas is not associated with HPV infection of these common types, and (b) fungiform papilloma is a distinctive clinicopathologic subtype of Schneiderian papilloma that may be HPV-related.


In situ localization of Epstein-Barr virus in thymic carcinoma. Epstein-Barr virus (EBV) genome is associated with a variety of lymphoid and epithelial malignancies. EBV DNA has been detected in some cases of thymic carcinoma, but the cellular locus of the virus has never been defined. Detection of EBV has also been reported in normal thymus, thymic lymphoid hyperplasia, and thymoma by some investigators but not by others. In order to better define the association of the virus with benign and malignant thymic tissues and to characterize its cellular locus, we applied a recently developed in situ hybridization technique using a very abundant EBV transcript (EBER1) as target to a variety of thymic tissues. We detected expression of this transcript only in the malignant epithelial cells in one case of thymic lymphoepithelioma-like carcinoma. EBV expression was not detected in six other cases of thymic carcinoma, nor in tissue from 16 normal thymuses, 14 thymomas, and 10 thymic lymphoid hyperplasias.


Papillary immature metaplasia of the cervix: a distinct subset of exophytic cervical condyloma associated with HPV-6/11 nucleic acids. A subset of exophytic cervical precursor lesions are composed of immature metaplastic cells that differ from conventional condylomata by the virtual absence of koilocytotic atypia and the presence of slender filiform papillae. We evaluated a series of exophytic cervical lesions containing this morphology for HPV nucleic acids and compared the associated HPV types with conventional exophytic condylomata of the cervix. Six of six exophytic condylomata and five of six papillary immature metaplasias (PIM), respectively, contained HPV type 6/11 by in situ hybridization. Subtyping of three PIM by polymerase chain reaction combined with direct sequencing revealed nucleic acid sequences consistent with HPV 6/11. PIM were distinguished from high-grade squamous intraepithelial lesions by the rarity of mitoses and by the uniformity of nuclear size and staining intensity with multiple chromocenters. However, these lesions tended to involve the more cephalad region of the cervical transformation zone, and three cases extended deeply into the endocervix with two requiring conization for a definitive diagnosis. Although their bland morphology and association with HPV 6/11 nucleic acids suggest a benign process, their location within the endocervical canal implies that these variants of condyloma may differ biologically from conventional exophytic condylomas of the cervix. The differential diagnosis of PIM and potential explanations for their distinctive morphology, are discussed.


Prevalence of human papillomavirus in sinonasal papillomas: a study using polymerase chain reaction and in situ hybridization. Inverted and fungiform papillomas of the sinonasal cavity share a common origin from the Schneiderian membrane, but they differ widely in their rates of recurrence and progression to carcinoma. To determine the role of human papillomavirus in the etiology of these lesions, 15 inverted papillomas, five fungiform papillomas, and two squamous cell carcinomas associated with inverted papilloma were examined for the presence of HPV by in situ hybridization (ISH) and polymerase chain reaction (PCR). ISH was carried out on formalin-fixed, paraffin-embedded material using HPV types 6/11, 16/18, and 31/33/35 DNA probes. Tissue DNA was amplified by PCR with HPV L1 consensus primers, and the product was detected by gel electrophoresis, Southern blotting, and hybridization with type specific probes (HPV types 6/11, 16, 18). Three of 15 inverted papillomas and two of five fungiform papillomas were positive for HPV 6/11 by ISH, whereas PCR detected HPV 6/11 sequences in two of 15 inverted and three of five fungiform papillomas. Biopsies from two patients who had serial resections contained HPV 6/11 in the original lesions and all recurrences. No HPV was detected in the carcinomas by ISH, whereas PCR detected HPV 16 in one carcinoma. These findings confirm the presence of HPV DNA sequences in both inverted and fungiform sinonasal papillomas as well as in an associated squamous carcinoma. This would suggest a role for HPV in the pathogenesis of Schneiderian membrane lesions. Furthermore, our data indicate that ISH and PCR are equally sensitive in detecting HPV in sinonasal papillomas.


Expression of carcinoembryonic or related cross-reacting antigens in primary pediatric neuroectodermal brain tumors. Various amounts of carcinoembryonic antigen (CEA) or cross-reacting antigen (CRA) were expressed in eight childhood primitive neuroectodermal tumors (PNETs) and in three of 12 astrocytomas (ASTR) using frozen section immunocytology. The antigen detection was performed using two recently developed anti-CEA monoclonal antibodies (MoABs), B.18.7.7 and D.14, reacting with a CEA specific antigenic epitope. Unfortunately, the two new MoABs also demonstrated cross-reactivity with nonspecific cross-reacting antigens (i.e., NCA-55) specific for the intratumoral granulocytes and cytotoxic mononuclear effector cell population. Three of eight PNETs contained 1 to 10% of CEA- or CRA-positive brain tumor cells. In two of eight, the antigenic epitopes were found in 10 to 50% of tumor cells, and in three of eight a large population (50 to 90%) of such cells was detected. Three pilocytic ASTRs contained CEA or CRA in 1 to 10% of cells. CEA or CRA positivity was located both on the cell surface and intracellularly in both tumor types. CEA or CRA expression in both tumor types was detectable only in fresh frozen sections and D.14 was preferable to B.18.7.7. The three pilocytic ASTRs lost CEA or CRA reactivity after formalin fixation and paraffin embedding. CEA-related cross-reacting antigen (NCA-55) was detected on the cell surfaces of tumor-infiltrating mononuclear cells, macrophages, neutrophilic granulocytes, and probably in some brain tumor cells also.


Detection of amplified HPV 6 and 11 DNA in vulvar lesions by hot start PCR in situ hybridization. We analyzed the distribution pattern of human papillomavirus (HPV) 6 and 11 DNA in vulvar lesions by in situ hybridization after amplification by the "hot start" polymerase chain reaction (PCR). HPV DNA was routinely detected in granular layer cells showing perinuclear halos and nuclear atypia by in situ hybridization with or without PCR. Cells that lack these changes rarely exhibited HPV DNA with standard in situ hybridization. After amplification, in situ analysis showed that many of the cells that lacked halos and atypia did contain HPV DNA and that the hybridization signal often localized to areas where there was a thickened granular layer. HPV DNA was not noted in the basal cells. The one copy of HPV 16 in SiHa cells was detectable after PCR with a single primer pair by in situ analysis only if the hot start modification was employed. Prior reports describing the PCR in situ methodology noted the need for from five to seven primer pairs. The hot start technique, which may be done by withholding the DNA polymerase until the temperature is sufficiently high to disfavor nontarget specific pathways, allowed the use of a single primer pair and showed that the degree of target-specific amplification, and not the size of the amplified product, determines the success of the PCR in situ technique.


A simplified method for detecting cytomegalovirus by polymerase chain reaction from histologic sections of small biopsies. Using a simplified procedure, we have extracted DNA from unstained paraffin sections of needle biopsies of kidney and liver transplants and identified the presence of CMV using the polymerase chain reaction. This method utilizes oligonucleotide primers for two genes shown to be specific for cytomegalovirus (CMV) as well as an internal control gene (hemoglobin) in a single reaction. Utilizing nested PCR amplification with agarose gel electrophoresis, CMV can be detected without radioisotopes to a level of sensitivity equivalent to one one-hundredth of a cytomegalic virocyte per cm2 of a 3-microM paraffin section. This method is applicable to situations where only scarce paraffin-embedded tissue is available.


A tobacco-specific N-nitrosamine or cigarette smoke condensate causes neoplastic transformation of xenotransplanted human bronchial epithelial cells. Using a xenotransplantation system in which immortalized nontumorigenic human bronchial epithelial cells (BEAS-2B cells) are grown in deepithelialized rat tracheas that are subcutaneously transplanted into athymic nude mice, we exposed BEAS-2B cells either to cigarette smoke condensate or to the tobacco-specific N-nitrosamine 4-(methylnitrosamine)-1-(3-pyridyl)-1- butanone. After 6 mo the carcinogen-exposed BEAS-2B cells were neoplastically transformed to invasive adenocarcinomas. Cell lines obtained from xenografts exposed in vivo to chemicals exhibited several features typical of malignant lung cancer cells, such as increased in vivo invasiveness that correlated well with enhanced type IV collagenolytic activity, resistance to serum-induced growth inhibition, and increased expression of transforming growth factor alpha and its cellular-membrane receptor. Invasiveness, similar to that seen after exposure to phorbol esters, was also detected after in vitro exposure of BEAS-2B cells to cigarette smoke condensate. Collectively, these data indicate that cigarette smoke condensate and N-nitrosamine 4-(methylnitrosamine)-1-(3-pyridyl)-1-butanone induce in vivo phenotypic changes in BEAS-2B cells similar to the progressive changes that occur during human lung carcinogenesis.


Five subtypes of type A gamma-aminobutyric acid receptors identified in neurons by double and triple immunofluorescence staining with subunit-specific antibodies. The extraordinary structural diversity of subunits forming type A gamma-aminobutyric acid (GABAA) receptors in the brain is expected to give rise to different modes of GABAergic synaptic inhibition and different profiles of modulatory drugs effective in anxiolytic, hypnotic, and antiepileptic therapy. To identify receptor subtypes in situ, the most prevalent subunits were visualized by double and triple immunofluorescence staining in rat brain, using polyclonal antibodies to the alpha 1, alpha 3, and gamma 2 subunits and a monoclonal antibody to locate both the beta 2 and the beta 3 subunit. At both cellular and subcellular levels five distinct patterns of subunit colocalization were identified: I, alpha 1 beta 2,3 gamma 2; II, alpha 3 beta 2,3 gamma 2; III, alpha 1 alpha 3 beta 2,3 gamma 2; IV, alpha 3 gamma 2; and V, alpha 1 alpha 3 gamma 2. As analyzed by confocal laser microscopy, different subunits displayed the same local variations of staining intensity ("hot spots") along the plasma membrane. The covisualized subunits appear therefore to be coassembled in receptor subtypes. Most neurons expressed only a single major receptor subtype with no apparent distinction between synaptic and extrasynaptic sites. However, in some neurons, most notably in Purkinje cells, the subunit composition varied between the soma and the dendrites, pointing to the existence of receptor heterogeneity within single neurons. Furthermore, different populations of neurons may be characterized by particular receptor subtypes. Cells displaying alpha 1-subunit immunoreactivity were mostly identified as GABAergic, whereas monoaminergic neurons displayed intense alpha 3-subunit immunoreactivity but virtually no alpha 1-subunit immunoreactivity. The allocation of defined GABAA receptor subtypes to identified neurons opens the way for a functional analysis of receptor heterogeneity.


Stable association between the bovine papillomavirus E5 transforming protein and activated platelet-derived growth factor receptor in transformed mouse cells. The 44-amino acid E5 transforming protein of bovine papillomavirus is the shortest protein known to induce tumorigenic transformation of fibroblasts. We showed previously that expression of the E5 protein activates the cellular beta receptor for platelet-derived growth factor (PDGF) and proposed that the activated receptor transmits the transforming signal to the cell. Here we use coimmunoprecipitation analysis to show that the E5 protein and the activated PDGF receptor exist in a stable complex in transformed mouse C127 cells. These results suggest a distinct mechanism of growth factor receptor activation and provide further evidence that the PDGF receptor is an important target of the E5 protein.


Requirement for a conserved serine in both processing and joining activities of retroviral integrase. Retroviruses encode a protein, the integrase (IN), that is required for insertion of the viral DNA into the host cell chromosome. IN alone can carry out the integration reaction in vitro. The reaction involves endonucleolytic cleavage near the 3' ends of both viral DNA strands (the processing step), followed by joining of these new viral DNA ends to host DNA (the joining step). Based on their evolutionary conservation, we have previously identified at least 11 amino acid residues of IN that may be essential for the reaction. Here we report that even conservative replacements of one of these residues, an invariant serine, produce severe reductions in both the processing and joining activities of Rous sarcoma virus IN in vitro. Replacement of the analogous serine of the type 1 human immunodeficiency virus IN had similar effects on processing activity. These results suggest that this single conserved serine is a component of the active site and that one active site is used for both processing and joining. Replacement of this serine with certain amino acids resulted in a loss or reduction in DNA binding activities, while other replacements at this position appeared to affect later steps in catalysis. All of the defective Rous sarcoma virus INs were able to compete with the wild-type protein, which supports a model in which IN functions in a multimeric complex.


Mouse macrophage receptor for acetylated low density lipoprotein: demonstration of a fully functional subunit in the membrane and with purified receptor. The functional molecular mass of the macrophage receptor for acetylated low density lipoprotein (Ac-LDL) was determined in membranes by radiation inactivation analysis. Membranes from tumors induced by the mouse macrophage cell line P388D1 were frozen and irradiated with high-energy electrons. Residual binding activity indicated a minimum functional molecular mass of 35,000 Da, considerably smaller than the active 260,000 M(r) protein seen on ligand blots under nonreducing conditions. Scatchard analysis of receptor binding gave no evidence of partially inactivated molecules. The receptor protein, purified by affinity chromatography and preparative gel electrophoresis, was incubated with dithiothreitol (0.1-100 mM) and retested for binding activity. Active subunits of 158,000 and 80,000 M(r) could be demonstrated by ligand blotting, with quantitative conversion of binding activity to the 80,000 M(r) species at 10 mM dithiothreitol. At 100 mM dithiothreitol, all binding activity was lost. Further size reduction was not detected by silver staining. These data suggest that the isolated mouse macrophage Ac-LDL receptor is a trimer with one class of SH groups involved in trimerization and another in the actual binding site. The monomeric species is fully active in vitro under mild reducing conditions. The radiation inactivation data also suggest that each monomeric unit is fully active and capable of functioning independently in the binding of ligands in the membrane.


A single-stranded DNA-binding protein from Crithidia fasciculata recognizes the nucleotide sequence at the origin of replication of kinetoplast DNA minicircles. A sequence-specific single-stranded DNA-binding protein from the trypanosomatid protozoan Crithidia fasciculata binds to a sequence of 12 nucleotides located at the origin of replication of kinetoplast DNA minicircles. This sequence, termed the universal minicircle sequence (UMS), is conserved in the kinetoplast DNA minicircles among species of the family Trypanosomatidae. The purified protein binds specifically to the heavy strand of the DNA at this site, which consists of the sequence 5'-GGGGTTGGTGTA-3'. Binding analyses using mutated UMS dodecamers have revealed the significant contribution of each of the individual residues at the binding site, with the exception of the 3'-terminal adenine residue, to the generation of specific protein-DNA complexes. The possible role of this sequence-specific single-stranded DNA-binding protein in replication of kinetoplast DNA minicircles and the relation of the UMS to chromosomal telomeric sequences are discussed.


Intracellular neutralization of virus by immunoglobulin A antibodies. IgA is thought to neutralize viruses at the epithelial surface of mucous membranes by preventing their attachment. Since IgA, a polymeric immunoglobulin, is transported through the lining of epithelial cells by the polymeric-immunoglobulin receptor and since viruses are obligate intracellular parasites, we hypothesized that IgA antibodies may also interfere with viral replication by binding to newly synthesized viral proteins within infected cells. Polarized monolayers of Madin-Darby canine kidney epithelial cells expressing the polymeric-immunoglobulin receptor were infected on the apical surface with Sendai virus. Anti-Sendai virus IgA monoclonal antibody delivered from the basolateral surface colocalized with viral protein within the cell, as documented by immunofluorescence. More importantly, anti-viral IgA reduced virus titers greater than 1000-fold (P less than 0.0001) in apical supernatants and greater than 10-fold (P less than 0.0001) in cell lysates from monolayers treated with anti-viral IgA compared with those treated with either anti-viral IgG or an irrelevant IgA monoclonal antibody. We believe that the differences in viral titers between cell layers treated with specific IgA, which enters the epithelial cell by binding to the polymeric-immunoglobulin receptor, and those treated with specific IgG, which does not enter the cells, or irrelevant IgA indicate that specific intracellular IgA antibodies can inhibit viral replication. Thus, in addition to the classical role of humoral antibodies in extracellular defense, IgA antibody may be able to neutralize microbial pathogens intracellularly, giving IgA a role in host defense that has traditionally been reserved for cell-mediated immunity.


New transients in the electron-transfer dynamics of photolyzed mixed-valence CO-cytochrome c oxidase. Electron transfer following photolysis of CO from mixed-valence (cytochrome a3+ Cu2+A cytochrome a2+3-CO Cu+B) cytochrome oxidase (ferrocytochrome-c; oxygen oxidoreductase, EC 1.9.3.1) was studied on time scales of nanoseconds to milliseconds by multichannel time-resolved optical absorption spectroscopy. In this method, the optical absorption was measured at many wavelengths simultaneously by using an optical spectrometric multichannel analyzer system. The high-quality time-resolved difference spectra showed a large increase on a microsecond time scale in the visible region centered at approximately 520 nm and in the UV region centered at approximately 390 nm. These absorbance changes were not observed after photodissociation of CO from the fully reduced enzyme and therefore are attributed to intramolecular electron transfer. Simultaneously, there was a blue shift and a small increase in the alpha band, which is attributed to the reduction of cytochrome alpha. Approximately one-third of the absorbance change at 520 nm can be attributed to reduction of cytochrome a. The absorbance changes associated with the 520- and the 390-nm bands are on the same time scale (t1/2 approximately 2 microseconds) as the dissociation of CO from Cu+B reported previously by time-resolved infrared spectroscopy. The position and shape of these bands are reasonable for charge-transfer transitions involving copper(II). We suggest that the absorbance increase at 520 nm, which cannot be attributed to a reduction of cytochrome a, may represent a charge transfer involving Cu2+B accompanying the oxidation of Cu+B to Cu2+B. The absorbance increase at 390 nm is also partially attributed to this transition. These results suggest that Cu2+B may be observed spectrophotometrically in the electron-transfer dynamics of cytochrome oxidase.


Saccharomyces cerevisiae sec59 cells are deficient in dolichol kinase activity. The temperature-sensitive Saccharomyces cerevisiae mutant sec59 accumulates inactive and incompletely glycosylated protein precursors in its endoplasmic reticulum at the restrictive temperature. O-mannosylation and glycosyl phosphatidylinositol membrane anchoring of protein are also abolished, consistent with a deficiency in dolichyl phosphate mannose. Membranes prepared from sec59 cells that had been shifted to the restrictive temperature, however, made normal amounts of dolichyl phosphate mannose when exogenous dolichyl phosphate was supplied, but dolichyl phosphate mannose synthesis was severely depressed in the absence of exogenous dolichyl phosphate. Quantitative measurements of dolichyl phosphate in sec59 cells showed that the levels were decreased to 48% of wild type at the permissive temperature and to less than 10% at the restrictive temperature. Assays of enzymes from the dolichyl phosphate synthetic pathway, cis-prenyltransferase and dolichyl pyrophosphate phosphatase, gave wild-type levels. However, dolichol kinase activity was greatly decreased. When sec59 cells were transformed with a plasmid that overexpresses the wild-type gene, dolichol kinase activity increased 10-fold over wild-type levels. These results strongly suggest that the sec59 gene encodes dolichol kinase.


Expression of platelet-derived growth factor and its receptor in AIDS-related Kaposi sarcoma in vivo suggests paracrine and autocrine mechanisms of tumor maintenance. As previously described, proliferation of Kaposi sarcoma (KS)-derived cells in vitro is dependent on the presence of platelet-derived growth factor (PDGF). To test the hypothesis that PDGF may also be a major growth factor for KS cells in vivo, we performed in situ hybridization and immunohistochemical staining for PDGF and PDGF receptors in tissue sections of AIDS-related KS. The data suggest that KS consists of two types of tumor cells. (i) The main population are spindle-shaped cells with elongated nuclei (KS-s cells). They reveal a strong expression of PDGF beta receptors but do not express the PDGF-A and PDGF-B isoforms. (ii) A minor population of KS cells express PDGF beta receptor as well as PDGF-A and PDGF-B (KS-p cells). These cells are often grouped in whorls and surrounding vascular slits. They reveal spherical nuclei with evenly distributed chromatin and inconspicuous nucleoli. PDGF alpha receptor is not expressed in either form of KS cells. The results suggest that the isoforms of PDGF and the PDGF beta receptor are differentially expressed in two different cell types in KS and that PDGF isoforms may contribute to the pathogenesis of KS.


Brief dendritic calcium signals initiate long-lasting synaptic depression in cerebellar Purkinje cells. We have performed experiments designed to test the hypothesis that long-term depression (LTD) of excitatory synaptic transmission in the cerebellar cortex is caused by a rise in postsynaptic Ca concentration. These experiments combined measurements of synaptic efficacy, performed with the thin slice patch clamp technique, with fura-2 measurements of intracellular Ca concentration ([Ca]i) in single cerebellar Purkinje cells. Simultaneous activation of the climbing fiber and parallel fibers innervating single Purkinje cells caused a LTD of transmission of the parallel fiber-Purkinje cell excitatory synapse. This LTD was associated with large and transient rises in [Ca]i in the Purkinje cell and apparently was due to Ca entry through voltage-gated Ca channels in the Purkinje cell dendrites. The rise in [Ca]i produced by climbing fiber activity was necessary for LTD, because addition of the Ca chelator bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate (BAPTA) to the interior of the Purkinje cell blocked LTD. Further, elevation of [Ca]i, produced by depolarizing pulses delivered in conjunction with parallel fiber activation, induced a depression of synaptic activity that closely resembled LTD in both time course and magnitude. Thus, a rise in [Ca]i appears to be sufficient to initiate LTD. From these results, we conclude that LTD of the parallel fiber-Purkinje cell synapse is initiated by a brief, climbing fiber-mediated rise in postsynaptic [Ca]i and that LTD is maintained by other, longer-lived processes that are triggered by the rise in postsynaptic [Ca]i.


Evidence for two promoters upstream of the pts operon: regulation by the cAMP receptor protein regulatory complex. Several potential target sites for multiple regulatory mechanisms were previously identified in the 5' flanking region of the pts operon. We have investigated the in vitro interactions of the cAMP receptor protein (CRP).cAMP regulatory complex with two DNA binding sites, by gel mobility-shift assays, and report the analysis of the functional role of each of the binding sites in vivo. Promoter-reporter gene fusion studies identified two CRP.cAMP-dependent promoters (the previously identified P1 and another promoter, P0) upstream of ptsH. The crr promoters (P2) within ptsI may be negatively regulated by CRP.cAMP.


A retinoic acid receptor alpha antagonist selectively counteracts retinoic acid effects. Retinoic acid (RA) exerts its pleiotropic effects on cell growth and differentiation through the activation of a family of transcription factors-the RA receptors (RARs). Three subtypes of these receptors exist, RAR alpha, RAR beta, and RAR gamma. The receptors are differentially expressed in different cell types and stages of development, suggesting that they may regulate different sets of genes. We have identified a synthetic retinoid with the characteristics of a selective RAR alpha antagonist. This antagonist counteracts RA effects on HL-60 cell differentiation and on B-lymphocyte polyclonal activation. Beyond its potential practical relevance, this and other specific antagonists will be useful to dissect the RAR system and to assign to one given receptor each of the many RA-regulated functions.


At least five related, but distinct, hepatitis C viral genotypes exist. Hepatitis C virus, the major causative agent of blood-borne non-A, non-B hepatitis in the world, has been the subject of considerable nucleic acid sequence analysis. Although all reported hepatitis C sequences from the United States have been represented by the prototype hepatitis C virus type 1 sequence, two groups of variant sequences have been reported in Japan. However, we have noted five distinct, but related, genotypes (I-V) throughout the world, based on detailed sequence determination and analysis of the first 1700 nucleotides and part of the nonstructural region 5 at the C terminus of the open reading frame. The nucleotide sequence for a large number of hepatitis C virus isolates spanning six continents was obtained by direct sequence analysis of PCR products after reverse transcription. Genotype was classified by using several distinct sequence motifs. We observed that most genotypes coexist in several geographic regions, including the United States, Japan, Germany, and Italy. So far, genotype V has been found only in South Africa. Interestingly, each distinct genotype seems to be maintained throughout the genome in the segments studied. These genotype distinctions should be considered when designing specific diagnostic tests, developing potential vaccines, and studying viral transmission.


Elimination of smooth muscle cells in experimental restenosis: targeting of fibroblast growth factor receptors. Factors in plasma and platelets do not fully account for the proliferation of smooth muscle cells in vascular injury, implying that additional factors are involved. Recently, we and others have observed that vascular injury regulates basic fibroblast growth factor, suggesting a further role for this pleiotropic factor. We report here that injury of rat arteries leads to an increase in fibroblast growth factor receptors in vascular smooth muscle cells. This up-regulation makes smooth muscle cells susceptible, in vitro and in vivo, to the lethal effects of a conjugate of basic fibroblast growth factor with the ribosome inactivator saporin. Saporin alone has no effect, whereas the conjugate kills proliferating, but not quiescent, smooth muscle cells in vitro. In vivo, one to three doses inhibit neointimal proliferation but have no apparent effect on the uninjured artery. Thus, the up-regulation of fibroblast growth factor receptors in vascular injury suggests new therapeutic possibilities for such refractory conditions as restenosis following balloon angioplasty.


Antibodies to a peptide from the maize auxin-binding protein have auxin agonist activity. The major auxin-binding protein in maize membranes is thought to function as a physiological receptor. From earlier information, including the use of site-directed irreversible inhibitors, several of the amino acids likely to form part of the active auxin-binding site were provisionally assigned. Inspection of the amino acid sequence of the auxin-binding protein showed a short region containing all but one of these amino acids. We find that antisera raised against a synthetic peptide encompassing this region recognize all isoforms of the maize auxin-binding protein together with homologous polypeptides in other species. We further find that the antibodies hyperpolarize protoplast transmembrane potential in an auxin-like manner. We conclude that these antibodies display auxin agonist activity and that we have identified an essential portion of the auxin-binding site.


Regulated high level expression of a human gamma-globin gene introduced into erythroid cells by an adeno-associated virus vector. Gene therapy of severe hemoglobinopathies will require high-level expression of a transferred globin gene in erythroid cells. Distant regulatory elements flanking the beta-globin gene cluster, the locus control region, are needed for appropriate expression. We have explored the use of a human parvovirus, the adeno-associated virus (AAV), for globin gene transfer. The human A gamma-globin gene, linked to hypersensitivity site 2 from the locus control region of the beta-globin gene cluster, was subcloned into a plasmid (psub201) containing the AAV inverted terminal repeats. This construct was cotransfected with a helper plasmid containing trans-acting AAV genes into human 293 cells that had been infected with adenovirus. The recombinant AAV vector containing hypersensitivity site 2 stably introduced on average one or two unrearranged proviral copies into human K562 erythroleukemia cells. The transferred globin gene exhibited normal regulation upon hemin induction of erythroid maturation and was expressed at a level equivalent to a native chromosomal A gamma-globin gene.


Quality control of radiotherapeutic treatment of medulloblastoma in a multicentric study: the contribution of radiotherapy technique to tumour relapse. The French Medulloblastoma Group. Between 06.86 and 11.89, 88 medulloblastoma or primitive neuroectodermic tumour (PNET) localised in the posterior fossa have been included in the M7 multicentric protocol, 82 received the totality of the radiotherapy treatment and were evaluable for this study. Twenty-two of these 82 patients relapsed: their radiotherapy treatment is analysed in the present study. In 10 cases out of the 22 relapses treatment failure was probably due to a radiotherapeutic imperfection. This study confirms the necessity of a strict radiotherapy control, particularly in multicentric study.


[When the leg is the victim of the foot]. Man (homo-erectus, plantigrade) is in constant contact with the ground via his feet which are platforms on which the legs and, above all, the entire body rest. When the body moves, the only segment which remains stable and fixed is the foot, which forms the point of departure of all the muscular actions of this moving body. The slightest deformity of this platform, this foot, will lead to skeletal, musculotendinous and neurovascular problems in the legs, and to pain. Pain due to skeletal problems may be related to fatigue fractures (tibia, fibula) and to posterior tibial periostitis. Pain may also be due to musculotendinous problems which are characterised by a muscle bed syndrome (anterolateral, posterior) or tensosynovitis. Neurovascular problems are typified by a tarsal sinus syndrome, a musculocutaneous nerve syndrome and dysfunction of Lejears' sole during filling in flat feet and during emptying in hollow feet.


Pathogenesis of Sjögren's syndrome.

Pathogenesis of Sjögren's syndrome. Sjögren's syndrome is a systemic autoimmune disease characterized by lymphocytic infiltrations of lacrimal and salivary glands. SS patients produce a variety of autoantibodies, including RF and ANA. Genetic factors, including HLA-DR3, predispose to primary SS. In contrast to normal SGs, the SS SG epithelial cells express high levels of HLA-DR antigens. This class II gene expression on the target organ may represent the structural basis for HLA-associated disease susceptibility. The glands are infiltrated with CD4+ T cells that can produce cytokines, including IL-2 and interferon-gamma. B cells within the SG produce autoantibodies, including RF. These SG B cells frequently use the VKIIIb subgroup of kappa light chain, a feature that SS patients share with Waldenstrom's macroglobulinemia patients. B cells undergo small clonal expansions that can be detected on Southern blot as immunoglobulin gene rearrangements, and SS patients have a markedly increased risk of developing non-Hodgkin's B-cell lymphoma involving the SGs and cervical lymph nodes. Due to accessibility of the SG for biopsy and the characteristic patterns of autoantibody production, SS provides an opportunity to study the target organ for autoimmune destruction and the transition from autoimmunity to lymphoma.


[Leg pain in neurology].

[Leg pain in neurology]. A frequent presenting symptom encountered by both neurologists and phlebologists, pain in the lower limbs often results in clinical practice in the suggestion of a neurological disorder. Only pain relating to neurological conditions will be dealt with here. A review of the anatomical pathways of different types of sensation is followed by a semiological review of descriptions of pain and the underlying mechanisms. Mention is then made "from below upwards" of a number of common conditions in which pain is a frequent presenting symptom: firts peripheral: polyneuropathy (alcoholic and diabetic), polyradiculoneuropathy, mononeuropathy, root pain (sciatica, cruralgia), narrow lumbar canal; then central: spinal cord pain, thalamic pain, so-called projected encephalic pain. This merely provides a differential diagnostic approach in relation to phlebological pain of the lower limbs.


[Vaccination against hepatitis A].

[Vaccination against hepatitis A]. Hepatitis A is a worldwide disease transmitted by oral-fecal route, the endemicity level is high in developing countries and in the Far East. In northern Europe the endemicity level is low, but it is intermediate in the European Mediterranean area. Seroepidemiological surveys show that in Italy, as in many other European countries, hepatitis type A endemicity is declining. Therefore, immunity to infection shifts towards the older generations, leaving an increasing number of teenagers and young adults susceptible to the infection. Since in adults the infection is associated with higher levels of morbidity and mortality than in children, hepatitis A remains a public health problem. Moreover, travellers visiting an endemic area are at risk of acquiring the disease. Short-term, passive immunization with immune serum globulins is not a satisfactory method of controlling hepatitis A. The best way of controlling HAV endemicity is vaccination and a satisfactory vaccine will be available by 1993. Soon after HAV propagation in cell culture was achieved, three different approaches to HAV vaccine production were investigated: recombinant vaccines, live attenuated vaccines, inactivated vaccines. The production of a recombinant vaccine is, as yet, unavailable, because two HAV surface proteins linked together in a definite three dimensional configuration are needed to stimulate an effective immune response. A satisfactory live attenuated vaccine has not yet been obtained because of difficulties in maintaining a stable level of attenuation. Consequently, attention has shifted towards inactivated vaccines, and the HM 175 strain inactivated vaccine has been produced and widely studied in over 25,000 human volunteers.(ABSTRACT TRUNCATED AT 250 WORDS)


[Appendicitis: microbial interactions and new pathogens]. The Authors present an exhaustive review on microbial agents of appendicitis by means of literature and personal research data. Thus, a detailed analysis is made on common autochthonous agents and their pathogenetic interactions and on less common exogenous bacterial, viral, mycotic, protozoan and helminthic agents with emphasis to the role of Yersinia enterocolitica. In fact this bacterium seems responsible for 3% to 8% of cases in accordance with literature and personal research data (more detailed, Y. enterocolitica has been isolated in 3.8% of 208 inflamed appendices from both pediatric and adults surgical florentine patients). At the end, the pathogenetic role of "new" other bacteria, like Buttiauxella agrestis, Aeromonas hydrophila, Arizona, Streptococcus lactis, is debated on the basis of a personal study.


Functional modulation of GABAA receptors by cAMP-dependent protein phosphorylation. gamma-Aminobutyric acidA (GABAA) receptors are ligand-gated ion channels that mediate inhibitory synaptic transmission in the central nervous system. The role of protein phosphorylation in the modulation of GABAA receptor function was examined with cells transiently transfected with GABAA receptor subunits. GABAA receptors consisting of the alpha 1 and beta 1 or the alpha 1, beta 1, and gamma 2 subunits were directly phosphorylated on the beta 1 subunit by adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase (PKA). The phosphorylation decreased the amplitude of the GABA response of both receptor types and the extent of rapid desensitization of the GABAA receptor that consisted of the alpha 1 and beta 1 subunits. Site-specific mutagenesis of the serine residue phosphorylated by PKA completely eliminated the PKA phosphorylation and modulation of the GABAA receptor. In primary embryonic rat neuronal cell cultures, a similar regulation of GABAA receptors by PKA was observed. These results demonstrate that the GABAA receptor is directly modulated by protein phosphorylation and suggest that neurotransmitters or neuropeptides that regulate intracellular cAMP levels may modulate the responses of neurons to GABA and consequently have profound effects on synaptic excitability.


Repression of the insulin-like growth factor II gene by the Wilms tumor suppressor WT1. The Wilms tumor suppressor gene wt1 encodes a zinc finger DNA binding protein, WT1, that functions as a transcriptional repressor. The fetal mitogen insulin-like growth factor II (IGF-II) is overexpressed in Wilms tumors and may have autocrine effects in tumor progression. The major fetal IGF-II promoter was defined in transient transfection assays as a region spanning from nucleotides -295 to +135, relative to the transcription start site. WT1 bound to multiple sites in this region and functioned as a potent repressor of IGF-II transcription in vivo. Maximal repression was dependent on the presence of WT1 binding sites on each side of the transcriptional initiation site. These findings provide a molecular basis for overexpression of IGF-II in Wilms tumors and suggest that WT1 negatively regulates blastemal cell proliferation by limiting the production of a fetal growth factor in the developing vertebrate kidney.


Immunoglobulins and complement components levels in patients with lung cancer. Sera from 96 patients with lung cancer were assayed in order to evaluate the concentrations of IgG, IgA, IgM, C3c and C4. Histologically, 49 of the patients had squamous cell carcinoma, 11--adenocarcinoma, 20--small cell carcinoma and 16--not identified lung cancer. No statistically significant differences were found between the concentrations of IgG and IgM in patients with carcinoma of the lung versus subjects in the control group. Both serum IgA and complement components (C30 and C4) were significantly elevated in almost all patients from the tumor group as compared with the levels in the control group.


Long-term survival of xenogeneic pancreatic islet grafts induced by CTLA4lg. Antigen-specific T cell activation depends on T cell receptor-ligand interaction and costimulatory signals generated when accessory molecules bind to their ligands, such as CD28 to the B7 (also called BB1) molecule. A soluble fusion protein of human CTLA-4 (a protein homologous to CD28) and the immunoglobulin (lg) G1 Fc region (CTLA4lg) binds to human and murine B7 with high avidity and blocks T cell activation in vitro. CTLA4lg therapy blocked human pancreatic islet rejection in mice by directly affecting T cell recognition of B7+ antigen-presenting cells. In addition, CTLA4lg induced long-term, donor-specific tolerance, which may have applications to human organ transplantation.


Activation-induced ubiquitination of the T cell antigen receptor. The zeta subunit of the T cell antigen receptor (TCR) exists primarily as a disulfide-linked homodimer. This receptor subunit is important in TCR-mediated signal transduction and is a substrate for a TCR-activated protein tyrosine kinase. The zeta chain was found to undergo ubiquitination in response to receptor engagement. This posttranslational modification occurred in normal T cells and tumor lines. Both nonphosphorylated and phosphorylated zeta molecules were modified, and at least one other TCR subunit, CD3 delta, was also ubiquitinated after activation of the receptor. These findings suggest an expanded role for ubiquitination in transmembrane receptor function.


Selective role of N-type calcium channels in neuronal migration. Analysis of neuronal migration in mouse cerebellar slice preparations by a laser scanning confocal microscope revealed that postmitotic granule cells initiate their migration only after the expression of N-type calcium channels on their plasmalemmal surface. Furthermore, selective blockade of these channels by addition of omega-conotoxin to the incubation medium curtailed cell movement. In contrast, inhibitors of L- and T-type calcium channels, as well as those of sodium and potassium channels, had no effect on the rate of granule cell migration. These results suggest that N-type calcium channels, which have been predominantly associated with neurotransmitter release in adult brain, also play a transient but specific developmental role in directed migration of immature neurons before the establishment of their synaptic circuits.


IP3 receptor: localization to plasma membrane of T cells and cocapping with the T cell receptor. Immune responses in lymphocytes require cellular accumulation of large amounts of calcium (Ca2+) from extracellular sources. In the T cell tumor line Jurkat, receptors for the Ca(2+)-releasing messenger inositol 1,4,5-trisphosphate (IP3) were localized to the plasma membrane (PM). Capping of the T cell receptor-CD3 complex, which is associated with signal transduction, was accompanied by capping of IP3 receptors. The IP3 receptor on T cells appears to be responsible for the entry of Ca2+ that initiates proliferative responses.


Evidence that free radicals are involved in graft failure following orthotopic liver transplantation in the rat--an electron paramagnetic resonance spin trapping study. The purpose of these studies was to determine whether free radicals were formed as a consequence of reperfusion during orthotopic liver transplantation and whether their formation was related to graft failure. Grafts were stored for 18 hr in Euro-Collins solution or for 48 hr in University of Wisconsin solution (nonsurvival conditions) and reperfused with blood containing the spin trap alpha-phenyl N-tert-butylnitrone (PBN). Venous blood samples (4-5 ml) were collected, and serum was extracted with chloroform and methanol (2:1) and analyzed for radical adducts by electron paramagnetic resonance (EPR) spectroscopy. In samples from livers stored under nonsurvival conditions, EPR spectra were detected indicating the presence of PBN radical adducts. In contrast, radical adduct formation was 3- to 4-fold lower in similar experiments performed with untransplanted livers or with livers stored under survival conditions (1 hr in Ringer's solution or 24 hr in UW solution). Oxygen radicals are more likely involved in the production of radical adducts because formation was nearly completely prevented by superoxide dismutase plus catalase or Carolina rinse, which contains glutathione, desferrioxamine mesylate, and allopurinol. Radical adduct formation was much greater in a blood-free perfusion system where oxygen delivery was high, suggesting that blood elements are not necessary for radical adduct formation. An inverse correlation between survival of livers stored in UW solution and radical adduct signal was observed in this study. Thus, it is concluded that free radicals formed during reperfusion are involved in the mechanism of graft failure following liver transplantation in the rat.


Treatment of central nervous system B lymphoproliferative syndrome by local infusion of a B cell-specific monoclonal antibody. A 9-month-old infant developed Epstein-Barr virus-induced lymphoproliferative syndrome with mediastinal and central nervous system localizations, associated with mediastinal tuberculosis, 5 months after heart transplantation. As a combination of anti-B cell antibodies (CD21- and CD24-specific) and recombinant interferon alpha 2b, given intravenously, was not effective on the central nervous system disease, the anti-CD21 antibody was infused intrathecally via an Ommaya reservoir. High local concentrations of monoclonal antibodies were achieved, with no adverse effects. A dramatic clinical response was obtained, with clearance of abnormal cells from the cerebrospinal fluid and a clear reduction in the abnormalities on the brain images. The patient is well 7 months later. This observation indicates that treatment of B lymphoproliferative syndrome with central nervous system localization is feasible using a nontoxic, local B cell-specific approach.


A recent decrease in the time to development of monomorphous and polymorphous posttransplant lymphoproliferative disorder. We have noted a decrease in the time to development of posttransplant lymphoproliferative disorder (PTLD) over the last two and one-half years in our multiorgan transplant program. From February 1965 until December 1990, 1622 transplants were performed including 1489 kidneys (KTxp), 87 livers (LTxp), and 46 pancreata. Between February 1965 and July 1988 (group 1), there were 1260 transplants performed and nine cases of either monomorphous PTLD (M-PTLD, n = 8) or polymorphous PTLD (P-PTLD, n = 1) were diagnosed. The mean time to development of PTLD was 163 +/- 128 weeks, all after KTxp. Five of these nine patients received haploidentical living-related grafts. All patients had presented with advanced disease, none had transplant nephrectomy, and all died of their disease. Between July 1988 and December 1990 (group 2), 362 transplants were performed, and four cases of M-PTLD and three cases of P-PTLD were recognized. Of the seven cases of PTLD in group 2, six developed within 90 days posttransplant (early PTLD). The mean time to development of PTLD was 11 +/- 16 weeks. This was significantly earlier than group 1 (P less than .01). Four of the five cases after KTxp had a 1 or 2 DR-matched donor. Five of these seven patients had serological evidence of recent Epstein-Barr Virus infection, and four of these five had received OKT3 and then developed early PTLD. In group 2, three patients are alive 7-15 months after KTxp nephrectomy, the remaining four have died. We hypothesize that risk factors for the development of PTLD may include heavy immunosuppression, including the use of OKT3, good DR matching, and active EBV infection. Treatment should include graft removal, if applicable, and reduction or cessation of immuno-suppression.


Inflammatory mediator release from human monocytes via immobilized Fc receptors. Its potential role in adverse reactions to systemic monoclonal antibody therapy. Human monocytes released superoxide anion, IL-1, and TNF subsequent to binding of their Fc receptor I to murine IgG2a or rabbit IgG. Fc receptor II binding to murine IgG2b or IgG1 had similar consequences. Immobilized murine monoclonal antibodies, IgG2a anti-CD3 (OKT3) or IgG1 anti-CD44 also induced superoxide anion and monokine production. Monocytes bound OKT3 via FcRI and responded to immobilized OKT3 by inflammatory mediator release in the absence of T cells. These results suggest that direct interaction of immunoglobulins with monocytes via FcR may represent an important phase of the pathophysiology of adverse reactions to systemic monoclonal antibodies.


Observations on the effect of verapamil with sodium stibogluconate in kala azar. 40 parasitologically confirmed cases of kala azar, were randomly allocated into four treatment groups to assess the effect of verapamil on fresh and antimony resistant cases of kala azar. Untreated patients received sodium stibogluconate only or in combination with oral verapamil. Antimony-resistant patients were treated with sodium stibogluconate combined with oral verapamil or pentamidine. The patients were followed up for six months. Verapamil neither shortened the duration of treatment nor increased parasitological cure rate nor improved the ultimate cure. In antimony unresponsive patients it did not reverse unresponsiveness.


The problem of follow-up of renal transplant patients in the tropics. A 40-year-old male Nigerian with end-stage renal disease (ESRD) received a renal allograft from a live related donor, in the United Kingdom in 1986. Subsequently, in Nigeria, he developed recurrent episodes suggestive of chronic allograft rejection or allograft nephritis which necessitated hospitalisation in our establishment. He subsequently died during one such episode. The peculiar management problems and challenges posed by the patient during his recent admission in our clinical setting are discussed.


Hepatoma in pregnancy.

Hepatoma in pregnancy. We present the third case of hepatoma in pregnancy to be reported in an African. Despite the high incidence of hepatoma in the male population, hepatoma complicating pregnancy is very rare in this environment. Death usually occurs within one year of onset of symptoms. The clinical features during pregnancy do not differ appreciably from its presentation in the non-pregnant state. However, after pregnancy, as demonstrated in our case, the disease appears to pursue a more rapid course. These observations lend more weight to suggestions of a hormonal dependence of hepatoma.


Normal cortisol secretion in sickle cell anemia. In the present work, 14 sickle cell anemia patients and 16 normal controls were submitted to rapid ACTH stimulation test for basal cortisol determination. No significant differences were observed between the two groups for the basal and stimulated cortisol levels or for the increment at 30, 50 and 120 min. after infusion. The results obtained for the basal cortisol were in disaccordance with a previous report; however, we used a sensitive test to detect precocious involvement of the adrenal glands. This suggests that in sickle cell anemia there is not hypocortisolism. The adrenal gland is vascularized by several arteries and so the sickling phenomenon may be unusual in the adrenal cortex.


Causes of mortality and parasites and incidental lesions in harbour porpoises (Phocoena phocoena) from British waters. Detailed post mortem examinations were carried out on 41 harbour porpoises (Phocoena phocoena) found dead on the coast of the United Kingdom. The commonest causes of death were entanglement in fishing gear, and parasitic and bacterial pneumonia. Among the non-fatal conditions parasitoses of various organs were common and there was a very wide variety of other conditions. In total 295 diseases and other lesions were found, an average of 7.2 per animal.