Optimizing urothelial cell preparation for the human urinary micronucleus assay.

Biological monitoring of early genotoxic effects in urothelial cells using the urinary micronucleus (MNu) assay is promising for early detection of cancer, such as bladder carcinoma. But many problems are encountered, the major being the poorly differential staining of cells, particularly in women having an important amount of squamous cells. We have optimized the protocol and obtained a differential staining of the cell types present in urine on 10 subjects. Following Carnoy I fixation and Papanicolaou staining, urothelial cells were blue while most squamous cells were pink. This differential staining allowed for optimization of the MNu assay on a single urine void, for both females and males. Even if our MNu means were comparable to the literature, the great variation in reported MNu results could reside in the ability of scorers to distinguish correctly between urothelial and squamous cells. When monitoring exposed populations, this erroneous distinction could largely influence the results, even more in women's urine samples. Given a situation where exposure would not increase micronuclei frequency in vaginal squamous cells, their erroneous analysis in the MNu assay could mask an early genotoxic effect. Therefore, as transitional cell carcinoma of the bladder originates from transformed urothelial cells, restricting micronuclei analysis to urothelial cells could yield a more precise estimate of cancer risk in exposed populations. Moreover, it is hoped that the improvements proposed in this paper will allow for an easier implementation of the MNu assay in various set-ups and enhance its specificity, since MNu are considered a suitable biomarker.

Dynamic increase of a 45,X cell line in a patient with multicentric ring Y chromosomes.

Because ring Y chromosomes are unstable during cell division most reported patients are mosaics, usually including a 45,X cell line. The phenotype varies from normal males or females with streak gonads to sexual ambiguities. We present here the case of a 23-year-old man who was referred at 11 years for growth delay. The GTG-banded karyotypes of lymphocytes revealed two cell lines: 46,X,dic r(Y) seen in 76% of the metaphases analyzed and 45,X (24%). Karyotypes and FISH were performed eight years later with the following probes: DYZ3 (Y centromere), SRY (sex-region of the Y), DYZ1 (Yq heterochromatin), CEPX/Y (X centromere and Yq heterochromatin), TelVysion Xp/Yp, Xq/Yq (X and Y subtelomeres), pan-telomeric, cosmid clones LLycos130G04 and LLycos37C09 (PARII), and BAC clone RP11-5C5 (Yq11.223). The results showed an increase in the 45,X cell line (60%) and a reduction in the 46,X,dic r(Y) cell line (36.4%). The use of Yq probes showed that the ring Y chromosome was dicentric. In addition, other ring Y structures were observed. The breakpoints occurred in proximal Yp11.32 or in Yp11.31 distal to SRY and in Yq12 distal to the PARII region. Therefore, most of the Y remained intact and all genes, with the exception of those in PARI, are present in double dosage in the dic r(Y). The level of mosaicism was important in defining the phenotype.

Phenotypic variability in isodicentric Y patients: study of nine cases.

Isodicentric chromosomes are the most commonly reported aberrations of the human Y chromosome. As they are unstable during cell division and can generate various types of cell lines, most reported patients are chromosomal mosaics, generally including a 45,X cell line. Phenotypes depend on the location of the breakpoints as well as on the proportion of each cell line and vary from male to abnormal female or individual with ambiguous genitalia. Although phenotypic variability is known to also depend on the degree of mosaicism in the various tissues, gonads are rarely studied. We report nine cases of isodicentric Y chromosomes studied by conventional and molecular cytogenetic: three males, five females, and one individual with sexual ambiguity. Two males had a non-mosaic karyotype, while the third male was a mosaic with a predominant 46,XY cell line. Three of the females had a major 45,X cell line, while the last two females and the patient with ambiguous genitalia had a major 46,X,idic(Y) cell line. Analyses of gonadal tissues from the individual with sexual ambiguity and of three of the five female patients gave results concordant with their phenotype, allowing us to better understand the sexual differentiation of these patients.

Human papillomavirus (HPV) study of 691 pathological specimens from Quebec by PCR-direct sequencing approach.

Human papillomaviruses (HPV) are etiological agents of cervical cancer. In order to address clinical demand for HPV detection and sequence typing, mostly in pre-cancerous cervical lesions, we applied our two-tier PCR-direct sequencing (PCR-DS) approach based on the use of both MY09/MY11 and GP5 + /GP6 + sets of primers. We tested 691 pathological specimens, all of which were biopsies, 75% of which were diagnosed histologically as cervical intraepithelial neoplasia (CIN) grades I-III. In total, 484 samples (70%) tested HPV-positive, yielding 531 HPV sequences from 47 HPV types, including two novel types. Four most frequently found HPV types accounted for 52.9% of all isolates: HPV6, 16, 11, and 31 (21.5%, 20.0%, 7.0%, and 4.5%, respectively). Some interesting results are the following: all currently known high-risk HPV (14 types) and low-risk HPV (6 types) were detected; HPV18 was not the 1st or 2nd but rather the 4th-5th most frequent high-risk HPV type; the highest detection rate for HPV (86%) among samples suspected to be HPV-infected was found in the youngest age group (0-10 years old), including 70% (44/63) "genital" HPV types; HPV types of undetermined cervical cancer risk represented 19% and of the total HPV isolates but were strongly increased in co-infections (36.5% of all isolates). To our knowledge, this is the largest sequencing-based study of HPV. The HPV types of unknown cancer risk, representing the majority of the known HPV types, 27 of the 47 types detected in this study, are not likely to play a major role in cervical cancer because their prevalence in CIN-I, II, and III declines from 16% to 8% to 2.5%. The two-tier PCR-DS method provides greater sensitivity than cycle sequencing using only one pair of primers. It could be used in a simple laboratory setting for quick and reliable typing of known and novel HPV from clinical specimens with fine sequence precision. It could also be applied to anti-cancer vaccine development.

Pleuropulmonary blastoma: a rare pathology with an even rarer presentation.

BACKGROUND: Pleuropulmonary blastoma is among the rarest tumors of childhood. Three types have been described: cystic, solid, and mixed. To date, bilateral disease has not been documented. METHODS AND RESULTS: A 5-week-old girl presented with a history of fever. Chest x-ray showed bilateral diffuse cystic lesions. Bowel obstruction developed that required laparotomy. Multiple small bowel polyps were resected. The patient was readmitted 4 months later with deteriorating respiratory status. She underwent sequential thoracotomies for resection of multiple bullae under high-frequency oscillatory ventilation. Small bowel polypectomies were again required because of obstruction. Lung lesions were compatible with pulmonary blastoma but could not be correlated with intestinal polyposis. Bilateral cystic renal lesions were seen on ultrasound scan. Her disease progressed, despite chemotherapy, with the appearance of metastatic iris lesions. She again underwent laparotomies for multiple recurrent generalized small bowel polyps that were causing obstruction. Expanding renal cysts affected kidney function, and she died at 14 months of age. CONCLUSIONS: The rare association between pleuropulmonary blastoma and Wilms' tumor or nephroblastomatosis is known but rarely reported. Lacking pathological evidence, we can only speculate that this was the case. We have been unable to demonstrate any histological association between the renopulmonary and digestive lesions. Despite many unanswered questions, we are likely dealing with a "syndrome" of sorts with a dire outcome, despite aggressive treatments.

Short-term cyclosporine induces a remission of autoimmune hepatitis in children.

BACKGROUND/AIMS: The current immunosuppressive treatment of patients with autoimmune hepatitis consists of prednisone and azathioprine. High doses of prednisone used to obtain the remission of the disease are associated with serious adverse effects. To avoid harmful consequences of prednisone therapy, we proposed to treat patients with oral cyclosporine to obtain the remission of the inflammatory process. METHODS: This is a pilot, multinational, multicenter, clinical trial involving children with autoimmune hepatitis. Thirty-two children were recruited, who according to international criteria were considered as having definite autoimmune hepatitis. Cyclosporine alone was administered for 6 months, followed by combined low doses of prednisone and azathioprine for 1 month, after which cyclosporine was discontinued. Biochemical remission of the disease was established by the follow-up of serum transaminase activity levels. Growth parameters and adverse effects of the treatment were recorded. RESULTS: Two patients were withdrawn from the study: one for non-compliance and the other for liver failure which did not improve with cyclosporine. Of the 30 remaining patients, 25 normalized alanine aminotransferase activity levels by 6 months and all the patients by 1 year of treatment. Z-scores for height showed a trend towards improvement during treatment. Adverse effects of cyclosporine were mild and disappeared during weaning off the medication. CONCLUSIONS: Cyclosporine induced the biochemical remission of the hepatic inflammatory/necrotic process in children with autoimmune hepatitis, with few and well-tolerated adverse effects.

Autoimmune enteropathy.

Autoimmune enteropathy (AIE) is an entity reported primarily in infancy, resulting in intractable diarrhea and associated with small bowel villous atrophy and the presence of circulating anti-enterocyte (AEA) antibodies. It is a multisystem disorder with a response, in many cases, to immunosuppressive therapy.

Direct human papillomavirus (HPV) sequencing method yields a novel HPV in a human immunodeficiency virus-positive Quebec woman and distinguishes a new HPV clade.

Papillomaviruses of supergroup A exhibit genital tropism and are best known as etiologic agents for benign and malignant cervical lesions in women. A polymerase chain reaction direct sequencing approach with P-33-labeled dideoxynucleotides was used to detect and type human papillomaviruses (HPVs) in cervical biopsies. A novel sequence was found in condylomatous specimens from a human immunodeficiency virus-positive French Canadian woman. The viral gene L1 was sequenced completely, yielding a novel HPV type of supergroup A, named JC9710. This is related to a previously described HPV type from New Mexico, CP8061, and to Colobus monkey papillomavirus 1. Sequence similarity searches and phylogenetic analyses with different software packages clustered the three viral types as a new clade, for which the next available number, A15, was proposed.

A two-tier polymerase chain reaction direct sequencing method for detecting and typing human papillomaviruses in pathological specimens.

An in-house polymerase chain reaction direct sequencing (PCR-DS) approach for HPV detection and typing was developed, taking advantage of two widely used pairs of human papillomavirus (HPV)-specific PCR primers, MY09/MY11 and GP5/GP6, and 33P-labeled dideoxynucleotides. In this study, 105 pathological specimens were examined: 89% were diagnosed as cervical intraepithelial neoplasia (CIN) grade I-III, 76.2% were HPV-positive by PCR-DS. The PCR using GP5/GP6 (first tier) and MY09/MY11 primers (second tier for the GP5/GP6-negative samples) detected additional 15%-25% HPV-positive samples compared with each pair used separately. Direct sequencing was then used to type the HPV. A readout of a sequence as short as 34 nucleotides within a specific region in the L1 gene is sufficient to type known or novel sequences. Because of its high sensitivity and cost-effectiveness, the two-tier PCR-DS was adopted by the authors as the current method of choice for HPV diagnosis with ultimate sequence precision.

A case of non A, non B, non C hepatitis that relapsed into fulminant hepatic failure.

We describe a 12 year-old patient that relapsed into fulminant non A, non B, non C (NANBNC) hepatitis 10 weeks post-clinical recovery. A complete clinical and pathological evaluation, including an ultra-structural examination of a liver biopsy was consistent with the diagnosis of NANBNC hepatitis. The patient relapsed into hepatic failure and required transplantation. NANBNC hepatitis may have a relapsing form that can lead to hepatic failure requiring transplantation. Consultants in hepatology should have a high degree of clinical awareness and maintain prolonged patient follow-up.

Cervical chrondrocutaneous branchial remnants.

Similar in appearance to preauricular tags but located in the lateral neck, cervical chondrocutaneous branchial remnants are a rather less common and less well known congenital lesion. A retrospective review of admissions at Sainte-Justine Hospital between 1980 and 1993 produced 20 cases of cervical tags, of which 17 were true cervical chrondrocutaneous branchial remnants and 3 were skin tags associated with a thyroglossal duct. Of the 17 true cervical chrondrocutaneous branchial remnants, 15 were operated on in our institution. The clinical characteristics, results of investigations, surgical data, pathologic findings, and associated anomalies were documented. Several interesting facts emerged, including a male predominance (11 of 17), a scarcity of bilateral lesions (1 of 17), the presence of an elastic cartilage core in all operated specimens (15 of 15), and a high incidence of associated anomalies (13 of 17). We suggest that the second branchial arch is the most likely origin for the lesion. We propose a clear, widely acceptable name for this anomaly in order to prevent further diagnostic confusion. Most important, although simple surgical excision is all that is required for treatment, a complete physical examination of the patient and possibly an ultrasound examination of the genitourinary tract are recommended because a cervical chrondrocutaneous branchial remnant has proven in many cases to be a visible "marker" for more serious associated anomalies.

Intestinal vascular anomalies in children.

Vascular anomalies are an uncommon cause of gastrointestinal bleeding in childhood. Confusing nomenclature has made objective comparisons of published cases difficult and has interfered with an established consensus regarding diagnosis and therapeutic modalities. The purpose of this study was to clarify the situation by reviewing the records of all children who had intestinal vascular anomalies who were referred to our institution from 1975 to 1995. Thirteen lesions were identified in nine children (five boys and four girls). The median age at clinical onset was 8 years. Only two patients presented with a complex syndrome (Klippel-Trenaunay, 1; Osler-Rendu-Weber, 1). Diagnosis, location, and extension of these anomalies was only possible by angiography, which indicated that seven patients had isolated venous malformations and two had arteriovenous malformations. Because the lesions did not involve the serosa, intraoperative localization was a major problem. The main findings were a few slightly dilated mesenteric veins. Treatment was conservative in four children and surgical in five. Pathological findings on resected bowel demonstrated dilated and abnormal veins in the mucosa and submucosa. Selective angiography should not be delayed in patients with gastrointestinal bleeding if results of all other investigations are negative. Because these lesions are rarely recognizable on operative inspection, precise preoperative angiographic localization of intestinal vascular anomalies is essential to allow for a safe and limited resection of the involved bowel segment. Based on a better understanding of the natural history of these lesions, a classification of vascular anomalies of intestines in children is proposed.

Acute intrahepatic portal vein thrombosis complicating cholangitis in biliary atresia.

UNLABELLED: A 2-year-old boy with Kasai portoenterostomy had systemic features suggestive of cholangitis and acute portal vein obstruction. The rapid rise in portal pressure caused a transient, severe decompensation of hepatic function. A superior mesenteric arterial angiogram confirmed the presence of diffuse intrahepatic portal vein thrombosis. CONCLUSION: This case provides clinical and radiological evidence supporting an association between ascending cholangitis and acute intrahepatic portal vein thrombosis.

Variable expression of familial heterozygous hypobetalipoproteinemia: transient malabsorption during infancy.

Rare instances of symptomatic fat malabsorption have been reported in patients with heterozygous hypobetalipoproteinemia, but with an unclear pathogenesis. An 8-month-old boy with chronic diarrhea and failure to thrive was found to have abnormally low plasma total cholesterol (85 mg/dl), LDL-cholesterol (48 mg/dl), apoB (52 mg/dl), apoA-I (53 mg/dl), and vitamin E (0.22 mg/dl). Decreased plasma LDL-C and apoB were noted in the father (34 and 40 mg/dl, respectively), as well as several other family members. Fasting triglycerides were normal but did not increase normally in response to a fat meal test. Lipoprotein composition showed an abnormal profile of very low density (VLDL, d 1.006 g/ml), low density (LDL, d 1.063 g/ml), and high density (HDL, d 1.21 g/ml) lipoproteins. A fasting jejunal biopsy revealed lipid-laden enterocytes. Electron microscopy of the jejunal biopsy revealed the absence of lipid particles in the intercellular spaces after a fat meal. Jejunal explants cultured with [14C]palmitate and [3H]leucine showed limited synthesis of triglycerides and apolipoproteins (36 and 42% of controls, respectively), whereas the father's results were close to normal. At 1 year of age, improvement in intestinal fat absorption was accompanied by the presence of chylomicrons in the intercellular space, concomitant with the enhanced synthesis of lipids and apoB by jejunal explants. These data provide evidence that heterozygous hypobetalipoproteinemia may present early in life as transient, symptomatic lipid malabsorption. The mechanisms responsible for improved lipid transport despite persistent hypobetalipoproteinemia remain to be established.

Prolonged pH monitoring is of limited usefulness for gastroesophageal reflux.

OBJECTIVE: To assess the diagnostic value of pH monitoring. DESIGN: A prospective study. SETTING: Pediatric university hospital serving as a secondary and tertiary referral center. PARTICIPANTS: Thirty-eight infants, aged 1 to 12 months, and 26 children, aged 13 months to 18 years, admitted during a 2-year period because of clinically significant gastroesophageal reflux (GER). INTERVENTIONS: Prolonged (20-hour) pH monitoring as well as endoscopy and biopsy of the esophagus were carried out in all patients who, on the basis of clinical data, were classified as having mild, moderate, or severe GER disease. RESULTS: In the infant group, results of prolonged pH monitoring were abnormal in 34 (89.5%). In the older group, results were abnormal in less than half (11 [42.3%]) of the cases. In both groups, pH monitoring data did not correlate with the severity of GER disease or of esophagitis. Severity of GER disease was not predictive of esophagitis. CONCLUSIONS: (1) Endoscopy and biopsy of the esophagus should be the first procedures whenever there are clinical findings of moderate to severe GER disease. (2) Monitoring of pH should be restricted to those patients without a clear-cut history of GER disease.