Exaggerated vasomotor response to ANG II in rats with fetal programming of hypertension associated with exposure to a low-protein diet during gestation.

The renin-angiotensin system plays a key role in the initiation and maintenance of elevated blood pressure associated with altered intrauterine milieu. The current studies were undertaken to verify whether vascular response to ANG II is increased in adult offspring of low-protein fed dams (LP) compared with control (CTRL) and if so, to examine underlying mechanism(s). ANG II-induced contraction of carotid rings was increased in LP (E(max), the maximum asymptote of the curve, relative to maximal response to KCl 80 mM: 230 +/- 3% LP vs. 201 +/- 2% CTRL, P < 0.05). In both groups, contraction to ANG II was mediated solely by AT1R. Responses to thromboxane A2 analog U-46619 and to KCl 80 mM under step increases in tension were similar between groups. Endothelium depletion enhanced contraction to ANG II in both groups, more so in LP. Blockade of endothelin formation had no effect on response to ANG II, and ANG-(1-7) did not elicit vasomotor response in either group. Superoxide dismutase (SOD) analog Tempol normalized LP without modifying CTRL response to ANG II. Basal levels of superoxide (aortic segments, lucigenin-enhanced chemiluminescence and fluorescent dye hydroethidine) were higher in LP. ANG II further increased superoxide production in LP only, and this was inhibited by coincubation with diphenylene iodonium or apocynin (inhibitor of NADPH oxidase complex). AT1R expression in carotid arteries was increased in LP, whereas SOD expression was unchanged. In conclusion, vasoconstriction to ANG II is exaggerated in this model of developmental programming of hypertension, secondary to enhanced vascular production of superoxide anion by NADPH oxidase with concomitant increase of AT1R expression.

Human molecular embryogenesis: an overview.

Molecular embryology is a rapidly evolving field of great complexity. This overview is primarily for the newcomer to this field, in an attempt to demystify the processes by which a human single-celled zygote eventually forms an embryo. Although all embryonic cells share the same genetic information, they differentiate according to the basic plan dictated not only by multiple families of transcription factors to silence some genes and activate others but also through DNA methylation, histone acetylation, and heterochromatinization. Regional expression of various transcription factors causes embryos to establish primary embryonic axes. Once the basic body plan is established, the region-specific diversity becomes progressively finer, and each cell eventually develops a "molecular address" characterized by the expression of specific genes. The overview is divided into two main parts: embryonic cell growth and morphogenesis. At the present time, more is known about the details of molecular regulation of the embryonic growth than about morphogenesis.

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.

Molecular characterization of a pediatric pheochromocytoma with suspected bilateral disease.

An 11-year-old boy with hypertension was suspected of having bilateral adrenal pheochromocytomas and hyperplasia. Molecular analysis of specific tumor suppressor genes and oncogenes excluded the familial syndromes, von Hippel-Lindau (VHL) disease and multiple endocrine neoplasia (MEN) type 2A. Further evaluation identified a unilateral adrenal pheochromocytoma with a VHL heterozygous somatic mutation (G695A) and loss of the maternal allele at 11p15.5-11p14 exclusively in the tumor tissue. Both reverse-transcriptase polymerase chain reaction and immunohistochemistry confirmed increased expression of IGF2 within the tumoral tissue, relative to a normal control adrenal gland. These results ruled out familial syndromes and suggested that the VHL mutation and the loss of maternal allele on chromosome 11 could have contributed to tumor development.

Hyperfunctioning malignant thyroid nodule in an 11-year-old girl: pathologic and molecular studies.

We identified a papillary carcinoma in an 11-year-old girl with a hyperfunctioning thyroid nodule. A met453thr mutation in TSHR was found in the nodule but not in normal thyroid tissue or in leukocytes. This case documents that this activating mutation is associated with neoplasia.

Pediatric adrenocortical tumors: molecular events leading to insulin-like growth factor II gene overexpression.

It has been previously shown that adrenocortical tumors (ACT) in adults exhibit structural abnormalities in tumor DNA in approximately 30% of cases. These abnormalities involve chromosome 11p15 and include loss of heterozygosity, paternal isodisomy, and overexpression of the gene for insulin-like growth factor II (IGF2), correlating with DNA demethylation at this locus. It has been hypothesized that these events occur late in the tumorigenic process in adults and seem to correlate with a worse prognosis. We present 4 pediatric cases of ACT diagnosed at 2.5 yr, 10 months, 12 yr, and 2.2 yr. All 4 patients presented with virilization, and 1 patient also showed signs and symptoms of glucocorticoid excess. The youngest patient's maternal aunt had surgical excision of a more than 15-cm ACT 18 yr previously, but the aunt is doing well at age 23 yr. They all had surgical removal of their tumors. The 2.5-yr-old child also received chemotherapy and radiotherapy because of capsular rupture and, after 3 local recurrences, died 3.3 yr after initial presentation. We investigated all 4 tumors for chromosome 11 structural abnormalities (11p15.5 to 11q23), IGF2 and H19 expression by competitive RT-PCR analysis, and IGF2 methylation patterns by Southern analysis. All 4 tumors (100%) showed a combination of structural abnormalities at the 11p15 locus with mosaic loss of heterozygosity involving 11p. All tumors also had significantly increased IGF2 messenger ribonucleic acid levels relative to normal adrenal (up to 36-fold) and significant IGF2 demethylation (mean, 87%). H19 messenger ribonucleic acid levels were undetectable in 3 of 4 tumors, explained in part by mosaic loss of the actively expressed maternal allele for this imprinted gene. By immunohistochemistry we were able to confirm increased IGF-II peptide levels within the tumor tissue in 10 pediatric patients, including the 4 patients described above. Concomitantly, we also observed nuclear accumulation of p53, suggesting somatic mutations. For the 10-month-old patient, sequencing revealed a p53 germline mutation. We therefore conclude that in pediatric ACT, structural abnormalities of tumor DNA and IGF2 overexpression as well as p53 mutations are very common and are therefore less useful for prognosis than in adults. Our findings support the theory that pediatric ACT, whose IGF2 expression and steroidogenesis evoke the phenotype of the fetal adrenal cortex, may arise because of defective apoptosis.

Searching for an intrinsic neuroendocrine cell in the kidney. An immunohistochemical study of the fetal, infantile and adult kidney.

The pathogenesis of primary renal carcinoid tumor is unknown. One hypothesis has implied derivation from a yet unrecognized intrinsic neuroendocrine cell in the renal parenchyma/hilum either as a minute endocrineparacrine constituent or resulting from entrapped/misplaced progenitor cells of the so-called dispersed neuroendocrine system during organogenesis. Immunohistochemical staining for chromogranin and serotonin was systematically performed on a whole-mount and geographically mapped normal adult kidney, kidneys from 15 fetuses (age range: 15 to 38 weeks), and renal specimens from 18 infants/children (age range: 7 days to 123 months). Minute paraganglion nests (composed of chromogranin positive/serotonin negative chief cells and S-100 protein positive dendritic cells) were incidentally detected within the renal hilum primitive stroma (unilaterally) of two fetuses at 22 and 26 weeks. Sequestration and persistence of such paraganglion nests during renal growth and maturation would offer a basis for the rare occurrence of extra-adrenal paraganglioma involving the renal hilum/pedicle. Otherwise, no neuroendocrine cell was detected within the renal parenchyma or hilum, therefore not validating/sustaining the aforementioned hypothesis in the pathogenesis of renal carcinoid tumor.

New method for automatic identification identification and typing of single and multiple superimposed human papillomavirus sequences.

This study of specimens of human papillomaviruses (HPV) through HPV-specific polymerase chain reaction (PCR), followed by direct sequencing, resulted in 11% (38/354) superimposed HPV sequences, signifying coinfection with more than one HPV type. To address the diagnostic problem that these superimposed ("degenerated," overlapping) sequences pose, the authors created a papillomavirus database in Microsoft Excel (Microsoft Corporation, Redmond, WA, U.S.A.) and Corel Quattro Pro 9 (Corel Corporation, Ottawa, Ontario, Canada) formats, retrievable from http://www2.crosswinds.net/ -crosswindswatson/index.html. This sequence database is simultaneously a search and comparison tool for quick (several seconds) typing of HPV from regular and "degenerated" sequencing results. Some of the advantages of the method are as follows: (1) superimposed HPV sequences that differ in length could be readily identified from a single input; (2) the search is restricted to the currently known 127 PV types, which speeds up the typing; (3) the most common HPV sequencing artifacts are included for quick detection; (4) there is no proprietary code and the database could be easily improved; (5) HPV sequence identification does not require internet connection; and (6) new HPV types could be easily detected. This method allowed resolution of all but 1 of 354 HPV-positive specimens. From 38 superimposed HPV sequences, this method identified one known HPV type (3 specimens), two HPV types (30 specimens) and three HPV types (4 specimens).

3-Hydroxy-3-methylglutaryl-CoA lyase (HL): gene targeting causes prenatal lethality in HL-deficient mice.

3-Hydroxy-3-methylglutaryl-CoA lyase (HL, EC 4.1.3.4) catalyses the last step of ketogenesis from leucine and fatty acids. HL deficiency in humans is one of the many inborn errors of CoA ester metabolism. By gene targeting, we created a strain of HL-deficient mice. Heterozygous HL-deficient mice are clinically normal and fibroblasts from homozygous HL-deficient embryos grow normally despite absence of HL activity. In contrast, homozygous HL-deficient embryos die at approximately 11.5 days post-coitum. Histologically, HL-deficient embryos show marked vacuolization, particularly in liver. Ultrastructural studies of hepatocytes obtained before death from HL-deficient embryos reveal abnormal dilated mitochondria. HL-deficient mice are the first mammalian example of a disease primarily affecting CoA ester metabolism with abnormal prenatal development.

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.

JC9813-A putative novel human papillomavirus identified by PCR-DS.

Papillomaviruses consist of more than 130 viral types described so far. Most of them are human papillomaviruses (HPV) of supergroup A, demonstrating ano-genital tropism and characterized as etiological agents for benign and malignant cervical lesions in women. A PCR-direct sequencing (PCR-DS) approach with P-33 labeled dideoxynucleotides was used to detect and type human papillomaviruses in cervical biopsies. One novel sequence was identified in a LSIL (low-grade squamous intraepithelial lesions) specimen from an HIV-positive English Canadian patient. The structure of the viral gene L1 was determined, yielding a putative novel HPV type of supergroup A (clade A8) named JC9813.

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.

Childhood adrenocortical tumors: case series and reevaluation of prognosis--a 24-year experience.

Adrenocortical neoplasms are rare in childhood and adolescence. The prognostic significance of tumor size, weight, and histological grade are still very much unclear. Eleven patients, (3 boys, 8 girls), with a median presentation age of 7 years (range, 0.8 to 16 years) were identified. Six presented with virilizing symptoms, two with cushingoid symptoms, one with both, and two others had nonspecific symptoms. The interval between onset of symptoms and diagnosis was an average of 18 months (median, 8 months). Hormonal profile correlated well with clinical presentation in nine patients. Two patients with nonspecific symptoms had an aldosterone-producing lesion and an androgen-secreting tumor. Ten patients underwent complete surgical excision, with one intraoperative spillage. Median tumor weight was 94.5 g (range, 4 to 750 g). Three lesions were less than 5 cm in maximal width, six were between 5 and 10 cm, and two were greater than 10 cm. Two tumors had capsular or vascular invasion. Three patients received chemotherapy: one who had inoperable metastatic disease, and two based on clinical and histopathologic findings. Ten patients are doing well, without evidence of recurrent disease with a median follow-up of 3 years (range, 9 months to 15 years), eight patients have been followed up for more than 2 years. The medically treated patient who had metastatic disease died 3 years after diagnosis. A review of the pediatric literature, in some cases, indicates that larger tumors have a worse prognosis, while other investigators claim histological grade is more important. The authors' results do not support these conclusions, but rather suggest that in the pediatric population, when excision is complete, guarded optimism is warranted even with tumors larger than 5 cm. Addendum: Since submission of the manuscript, patient 4 has been operated on twice for local recurrences 13 and 16 months after the initial surgery. She was the only patient in the series to have an intraoperative capsular tear. All other surgical patients remain free of disease.

Echogenic dilated bowel loops before 21 weeks' gestation: a new entity.

OBJECTIVE: The purpose of this study was to determine the outcome at birth for fetuses in which echogenic dilated bowel loops (EDBL) are detected before 21 weeks of gestation. MATERIALS AND METHODS: A retrospective study was undertaken of 45 patients with EDBL. Sonographic criteria included dilated bowel with an echogenic wall. EDBL was divided into two categories: the isolated and the complex (associated with other abnormalities). When the EDBL affected only one quadrant of the fetal abdomen, it was referred to as the local form, and when it was observed in more than one quadrant, it was termed the diffuse form. RESULTS: Intestinal dilatation of 2-8 mm was revealed on sonography in 21 fetuses with the isolated form of EDBL. Follow-up sonography showed resolution of EDBL in 20 cases. The outcome at birth was normal in 19 (90%) cases. The 20th fetus was born with jejunal atresia, and the remaining fetus died after cordocentesis. Twenty-four fetuses with the complex form of EDBL also had intestinal dilatation of 2-8 mm shown on sonography. Nine (38%) of these fetuses were born alive: six had gastroschisis; two had meconium peritonitis; and one had vertebral defects, imperforate anus, tracheoesophageal fistula, and radial and renal dysplasia (VATER). In these cases, resolution of EDBL was noticed at sonographic follow-up. Fourteen (58%) of the 24 fetuses were aborted. The remaining fetus died after amniocentesis. The incidence of normalcy is comparable in both local and diffuse dilatation. Amniocentesis was performed in 15 cases. Disaccharidase activity was measured in 13 patients. Low disaccharidase activity was detected in 10 (77%) of 13 cases. We saw cystic fibrosis in none of the 45 fetuses. Infection was detected before and at birth in five (11%) cases. CONCLUSION: EDBL is a new entity that is most likely related to temporary obstruction. A fetus with the isolated form has a good prognosis, whereas a fetus with the complex form has an outcome directly related to the severity of associated abnormalities.

Hepatic sinusoidal ectasia.

Oral contraceptive-associated sinusoidal ectasia is a rare lesion of unknown pathogenesis. We describe the case of a 31-year-old woman who had used oral contraceptives for 10 years and was found to have abnormal liver function tests on "routine" examination. A single 9 x 5 x 4 cm hypervascular lesion was demonstrated radiographically. The hepatic immuno-diacetic acid and liver-spleen scans were normal. One subcapsular lesion was identified in the resected right hepatic lobe; it was characterized by focal marked dilatation of the sinusoidal spaces associated with rare hepatocyte necrosis and early intrasinusoidal fibrosis. The subcapsular location and the vascular nature of this wedge-shaped lesion suggest it may represent the telangiectatic precursor of a centrally scarred focal nodular hyperplasia.

PIP: A 31-year old mother of a 3-year old child visited a rheumatologist complaining of pain in both wrists, nose bleeding, and headaches. Since her delivery her weight dropped 12 kg and she has taken Synthroid daily. Before her pregnancy, she took the combined oral contraceptive (OC) Norinyl for 7 years. After childbirth, she took the OC Ortho Novum 1/80. She did not smoke or abuse drugs or alcohol. She did not have a history of hepatic or gastrointestinal diseases. Her alkaline phosphatase and glutamyl transferase levels were higher than normal (632 IU/L and 142 IU/L vs. =or 110 IU/L and =or 55 IU/L, respectively). Other liver function tests, her electrolytes, and her complete blood cell count were not unusual. She underwent an abdominal computed tomography scan and ultrasonography which indicated a single right hepatic lobe lesion (9 cm in diameter). It was well vascularized and circumscribed (12 x 10.5 cm) with big arteries leading to the immediate subcapsular region. The veins and arteries of the other lobe and the spleen appeared normal. Based on these tests, surgeons removed a segment of the right liver. She recovered well. 18 months after surgery, she was in good health. Pathologic examination of the 748 gm segment revealed that endothelial cells lined the sinusoidal spaces indicating true sinusoidal ectasia. The segment also exhibited individual hepatocyte atrophy and necrosis and inflammatory reaction and early fibrosis. Bile ducts had proliferated many portal spaces. Hepatic sinusoidal ectasia may be a forerunner of focal nodular hyperplasia.

Spontaneous diabetes mellitus syndrome in the rat. III. Pancreatic alterations in aglycosuric and untreated diabetic BB Wistar-derived rats.

The pancreatic alterations in aglycosuric and untreated diabetic BB Wistar-derived rats are described. A common finding, often seen in young aglycosuric rats, is that of discrete foci of periductular and/or acinar aggregates of lymphocytes and macrophages. Sites of periductular mononuclear cell infiltrates usually lack endocrine cells. In contrast, foci of acinar infiltrates, although distinct from the predominant endocrine cell mass in the islets of Langerhans, often contain small numbers of alpha and/or beta cells. It is suggested that these clusters of endocrine cells may in some way be antigenically different from those resident in the principal islets and thus serve as an additional target for the immune system in rats bearing the BB genome. The development of overt diabetes requires a massive destruction of beta cells within the islets of Langerhans. Two forms of diabetes mellitus emerge in untreated animals. The more common, designated unstable diabetes, is severe and lethal unless treated with insulin. Less commonly, a stable type of diabetes mellitus ensues for which insulin therapy is not mandatory. In each, the concentration of pancreatic immunoreactive insulin is profoundly decreased, although relatively greater amounts are present in the stable form. Unstable diabetic rats demonstrate a reduction in the concentration of pancreatic immunoreactive glucagon and somatostatin, suggesting that alpha and delta cells also sustain injury in this model of insulin-dependent diabetes mellitus.