Diurnal expression of the thrombopoietin gene is regulated by CLOCK.

BACKGROUND: Most physiologic processes exhibit diurnal fluctuations controlled by the circadian regulation of sleep-wake behavior and feeding cycles. In addition, many cell types express endogenous circadian rhythms that affect cell-specific processes. Independent reports support the hypothesis that thrombopoietin (TPO) is under circadian control. OBJECTIVES: The current study tested the hypothesis that CLOCK, a circadian transcription factor, may regulate Thpo, the gene encoding TPO. METHODS: Circadian gene expression patterns were analyzed in mice and in human cell lines, Small interfering RNA was used to knock down CLOCK expression in cell lines, and gene expression was also examined in Clock(Δ19/Δ19) mutant mice. RESULTS: It was found that there was a diurnal rhythm in the expression of Thpoin vivo in mice, and that this was associated with concomitant rhythms of protein abundance. Thpo was rhythmically expressed in human cell lines, consistent with the gene being directly or indirectly regulated by the circadian clock. Silencing of CLOCK in the Huh7 human hepatoma cell line led to a significant reduction in the rhythmicity of Thpo expression. The expression of Mpl in murine marrow also displayed diurnal rhythmicity in vivo. In Clock(Δ19/Δ19) mutant mice, Thpo and Mpl expression was disrupted and there was an increase in the number of mature megakaryocytes, but no change in the ploidy distribution within the megakaryocyte population. CONCLUSIONS: These findings establish that Clock regulates Thpo and Mpl expression in vivo, and demonstrate an important link between the body's circadian timing mechanisms and megakaryopoiesis.

Nephron-sparing surgery for bilateral Wilms' tumours: a single-centre experience with 23 cases.

INTRODUCTION: The challenge of management with bilateral Wilms' tumours is the eradication of the neoplasm, while at the same time preserving renal function. Surgical management with a variety of nephron-sparing techniques, combined with chemotherapy and occasionally supplemented by transplantation has evolved over the last 30 years to achieve remarkable success. We document the experience of a single centre in a developing country. MATERIAL AND METHODS: Twenty-three bilateral Wilms' tumours were seen in our service between 1981 and 2007. Treatment was, in most cases, according to National Wilms' Tumour Study Group protocols, with initial bilateral biopsy, neoadjuvant chemotherapy, and tumourectomy. Technique of nephrectomy included full mobilization of the tumour-involved kidney, topical cooling with slush ice, vascular exclusion, tumour resection and reconstruction of the remnant kidney. RESULTS: Twelve patients are alive and free of disease one to 15 years after treatment, all with well-preserved renal function (lowest glomerular filtration rate was 65 ml/min per (1.73 m 2 ). None of the survivors have hypertension. Eleven have died (two of unrelated disease) including six of the seven with spread outside the kidney. All three with unfavourable histology are alive. Four of the five metachronous presentations are alive, as are eight of 12 patients with synchronous bilateral tumours who presented since 2000. CONCLUSIONS: Appropriate chemotherapy and nephron-sparing surgery can achieve good results with preservation of adequate renal function in nearly all cases. Unfavourable histology did not have a reduced survival in our series. Metastatic spread outside the kidney had a poor prognosis.

Food-entrained rhythmic expression of PER2 and BMAL1 in murine megakaryocytes does not correlate with circadian rhythms in megakaryopoiesis.

BACKGROUND: Circadian rhythms control a vast array of biological processes in a broad spectrum of organisms. The contribution of circadian rhythms to the development of megakaryocytes and the regulation of platelet biology has not been defined. OBJECTIVES: This study tested the hypothesis that murine megakaryocytes exhibit hallmarks of circadian control. METHODS: Mice expressing a PER2::LUCIFERASE circadian reporter protein and C57BI/6 mice were used to establish if megakaryocytes expressed circadian genes in vitro and in vivo. Mice were also subjected to 3 weeks on a restricted feeding regime to separate food-entrained from light-entrained circadian rhythms. Quantitative real time polymerase chain reaction (PCR), flow cytometry and imunohistochemistry were employed to analyse gene expression, DNA content and cell-cycle behavior in megakaryocytes collected from mice over a 24-h period. RESULTS: Megakaryocytes exhibited rhythmic expression of the clock genes mPer2 and mBmal1 and circadian rhythms in megakaryopoiesis. mPer2 and mBmal1 expression phase advanced 8 h to coincide with the availability of food; however, food availability had a more complex effect on megakaryopoiesis, leading to a significant overall increase in megakaryocyte ploidy levels and cell-cycle activity. CONCLUSIONS: Normal megakaryopoiesis requires synchrony between food- and light-entrained circadian oscillators.

Expression of activin subunits and receptors in the developing human ovary: activin A promotes germ cell survival and proliferation before primordial follicle formation.

The formation of the essential functional unit of the ovary, the primordial follicle, occurs during fetal life in humans. Factors regulating oogonial proliferation and interaction with somatic cells before primordial follicle formation are largely unknown. We have investigated the expression, localisation and functional effects of activin and its receptors in the human fetal ovary at 14-21 weeks gestation. Expression of mRNA for the activin betaA and betaB subunits and the activin receptors ActRIIA and ActRIIB was demonstrated by RT-PCR. Expression of betaA mRNA increased 2-fold across the gestational range examined. Activin subunits and receptors were localised by immunohistochemistry. The betaA subunit was expressed by oogonia, and the betaB subunit and activin receptors were expressed by both oogonia and somatic cells. BetaA expression was increased in larger oogonia at later gestations, but was low in oocytes within newly formed primordial follicles. Treatment of ovary fragments with activin A in vitro increased both the number of oogonia present and oogonial proliferation, as detected by bromodeoxyuridine (BrdU) incorporation. These data indicate that activin may be involved in the autocrine and paracrine regulation of germ cell proliferation in the human ovary during the crucial period of development leading up to primordial follicle formation.

Developmental changes in expression of myeloid cell leukemia-1 in human germ cells during oogenesis and early folliculogenesis.

The regulation of germ cell number in the developing ovary is central to female reproduction. Members of the Bcl-2 family of proapoptotic and antiapoptotic proteins have been implicated in this process in rodents. We investigated the expression of Mcl-1, Bcl-2, Bax, and BAD at 13-21 gestational wk in the human fetal ovary and of Mcl-1 in the adult ovary. mRNA expression of Mcl-1 and its short form Mcl-1s, Bcl-2, Bax, and BAD was demonstrated in fetal ovary by RT-PCR. Hybridization array analysis suggested a selective increase in Mcl-1 expression between 14 and 18 wk gestation, which was confirmed by quantitative PCR. There was a corresponding change in the expression of Mcl-1 protein, detected by immunohistochemistry, from germ cells at the periphery of the ovary at 14-16 wk to the largest germ cells, including oocytes within newly formed primordial follicles, at 21 wk. Mcl-1 was also expressed by oocytes of primordial and preantral follicles in the adult. Bax and BAD immunostaining was detected in both somatic and germ cells in the fetal ovary, whereas Bcl-2 was restricted to somatic cells: no changes in expression were observed. Apoptotic cells, detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling, were observed in all fetal ovaries but were infrequent. These results confirm that Bcl-2 family members are differentially expressed in several cell types within the developing human ovary. Increased mRNA expression and the changing distribution of Mcl-1 in germ cells as they develop into primordial follicles as well as persistence in the growing oocyte in the adult may indicate an important role for this survival/antiapoptotic factor throughout germ cell development and maturation.

Expression and localization of inhibin alpha, inhibin/activin betaA and betaB and the activin type II and inhibin beta-glycan receptors in the developing human testis.

Inhibins and activins have roles in the regulation of cell proliferation and differentiation in a variety of tissues. This study investigated the distribution of the three inhibin/activin subunits (alpha, betaA and betaB) and their receptors in the human testis between week 13 and week 19 of gestation using RT-PCR and immunohistochemistry. mRNA for all three subunits and for the activin type II receptors ActRIIA and ActRIIB was detected at all stages of gestation examined. Sertoli cells showed intense immunostaining for the alpha subunit and some staining for the betaB subunit, whereas only the betaB subunit was detected in gonocytes. No betaA subunit staining was detected within the tubules. All three subunits were localized to interstitial Leydig cells. Cells of the rete testis and the epididymal epithelium also showed immunostaining for betaB; however, staining for the other subunits was weak or absent. Peritubular cells showed intense immunostaining for the beta-glycan inhibin receptor, which was also localized to interstitial cells, but was not detected within the tubular compartment, rete testis or epididymal epithelium. ActRIIA was detected in gonocytes and in interstitial cells; ActRIIB was distributed widely. These data indicate that fetal Leydig and Sertoli cells have the potential to produce both activins and inhibins, whereas gonocytes may produce only activin B. The distribution of activin and inhibin receptors implies that the intratubular compartment and developing duct system are sites of action of activin B but not inhibin at this stage of development, whereas both activins and inhibins may be involved in the development and function of the peritubular and interstitial cells.

Disseminated strongyloidiasis in a child with lymphoblastic lymphoma.

PURPOSE: This report describes a case of disseminated strongyloidiasis in a child receiving chemotherapy for T-cell lymphoblastic lymphoma. PATIENT AND METHODS: A 10-year-old boy became severely ill with disseminated strongyloidiasis 4 weeks after starting chemotherapy for T-cell lymphoblastic lymphoma. He responded to treatment with supportive care, antibiotics, and albendazole but required ivermectin to eradicate the strongyloides infection. CONCLUSION: Disseminated strongyloidiasis is a severe, life-threatening complication of Strongyloides stercoralis infection that can occur in patients on immunosuppressive therapy, particularly when this therapy includes corticosteriods. In endemic areas, screening patients due to undergo immunosuppressive treatment and appropriate antistrongyloides treatment may be life saving. Ivermectin is the treatment of choice.

Sacrococcygeal germ-cell tumours--the Red Cross War Memorial Children's Hospital experience, 1980-1996.

OBJECTIVE: To document the experience of Red Cross War Memorial Children's Hospital in the treatment of sacrococcygeal germ-cell tumours. PATIENTS: Twenty-seven patients with sacrococcygeal germ-cell tumours were treated in our hospital from 1980 to 1996. DESIGN: A retrospective review of these patients' records was undertaken. RESULTS: There were 19 female and 8 male patients. Seventeen (63%) presented in the neonatal period, 13 on the first day of life. Complete surgical resection of the tumour was achieved in all patients with mature or immature teratomas (20 patients) and in 2 neonates with malignant tumours. The first of these 2 neonates, with a malignant teratoma, was not given chemotherapy and remains well 10 years later. The second, with a yolk-sac tumour, also received no initial chemotherapy. He relapsed at the age of 9 months and was successfully treated with repeat excision and chemotherapy. All 5 patients first diagnosed after the age of 1 year had malignant tumours. These patients had incomplete surgical resection (3) or biopsy only (2), and 3 were successfully treated with chemotherapy. One patient relapsed with yolksac tumour after initial complete resection of a mature teratoma. She was successfully treated with repeat surgery and chemotherapy.

The role of Epstein-Barr virus in Hodgkin's disease from different geographical areas.

Recent studies have suggested that Epstein-Barr virus (EBV) may play a role in the aetiology of Hodgkin's disease. To determine the role of EBV in childhood Hodgkin's disease in different geographical areas, immunohistochemical staining and in situ hybridisation were used to analyse latent membrane protein 1 (LMP 1) and small nuclear non-transcribed RNAs (EBER-1) respectively. Testing for EBV within the Reed-Sternberg and Hodgkin's cells was carried out in childhood Hodgkin's disease from 10 different countries. The proportion of LMP 1 positive cases varied significantly, being 50% of cases from the United Kingdom (38/75), South Africa (9/18), Egypt (7/14), and Jordan (8/16), 60% from the United Arab Emirates (6/10), 70% from Australia (11/16), 81% from Costa Rica (34/42), 88% from Iran (7/8), 90% from Greece (20/22), and 100% of the 56 cases from Kenya. A sensitive polymerase chain reaction based EBV strain typing technique was established using archival tissues. EBV strain type 1 was shown to be predominant in childhood Hodgkin's disease from the United Kingdom, South Africa, Australia, and Greece. Type 2 was predominant in Egypt. EBV strain types 1 and 2 were both detected in some cases of childhood Hodgkin's disease in the United Kingdom, Costa Rica, and Kenya. The high incidence of EBV and the presence especially in developing countries of dual infection with both strain types 1 and 2 may reflect socioeconomic conditions leading to malnutrition induced immunological impairment. The possibility of HIV infection also needs to be explored.

Presentation and outcome of 25 cases of Fanconi's anemia.

Twenty-five children with Fanconi's anemia (FA) who attended the Paediatric Haematology Clinic of Red Cross Children's Hospital over the past 20 years were retrospectively reviewed. There was a female predominance with 17 girls and 8 boys in the group. The clinical features and laboratory data are enumerated. An unusually high prevalence of gastrointestinal anomalies (20%) was found. Seventeen children received an adequate trial of androgen therapy and in 12 the initial hemoglobin concentration increased by more than 2 g/dl or return to normal. Most patients subsequently required intermittent courses of androgens but three were able to stop treatment and maintain a normal hemoglobin concentration for from 3.5 to 20 years. Ten patients remain alive, of whom five had less severe hematological problems and did not require any treatment. The mean period of follow-up of the survivors is 8.7 years. Thirteen patients have died, with a mean time of 5.5 years from diagnosis to death. Of the deaths, two were due to malignant disease and one resulted from cerebral hemorrhage. The other 10 children died of confirmed or suspected infections. Treatment options of FA are discussed.

Reduction of neonatal blood eliminates the error induced by oxygen saturation in the spectrophotometric measurement of carboxyhaemoglobin.

Blood carboxyhaemoglobin (COHb) is a sensitive index of haemolysis and has been used in assessing the cause of different types of neonatal jaundice. Although the introduction of automated spectrophotometry provides rapid and accurate measurement in adult blood, in neonates oxygenated foetal haemoglobin (HbF) is thought to interfere with COHb measurement. In an attempt to eliminate this problem, the haemoglobin in neonatal blood was reduced with sodium dithionite. Cord blood from 50 infants was measured before and after reduction using an IL-282 co-oximeter; COHb levels fell after reduction. A significant positive correlation was found between apparent COHb% and oxygenation of cord blood. In contrast, no significant correlation was found between these parameters in adult blood where COHb values remained the same or rose slightly after reduction. In 20 healthy non-icteric neonates the mean reduced blood COHb value was not significantly different from the mean COHb value of 23 healthy non-smoking adults. We suggest that COHb in neonatal blood can be simply and accurately measured by the IL-282 co-oximeter provided that the blood is fully reduced.

Haemoglobin Köln in Cape Town. A case report.

A 7-year-old patient presented with an acute haemolytic episode. Investigation showed the presence of an unstable haemoglobin (Hb), identified as Hb Köln. No other members of the family were affected. This disorder has occasionally been seen as a spontaneous mutation. This is the first report of Hb Köln in a South African family, although other unstable Hb variants have been described.

Haemoglobin E variants: a clinical, haematological and biosynthetic study of 4 South African families.

The clinical, haematological and biosynthetic features of subjects with Hb E variants are described. An association with red cell hypochromia and microcytosis was confirmed, although this was not invariable in Hb E trait. Imbalanced globin chain synthesis was found in the majority of Hb E carriers. A patient doubly heterozygous for Hb E and Hb S, a condition we have not previously seen reported, had a benign clinical course with minor haematological changes, despite a relatively large amount of Hb S (67%).

Microcytic anaemia and haemoglobinopathy in Cape Town children.

Patients with thalassemia as well as those with iron deficiency typically have red cell microcytosis and hypochromia. In view of the large number of children with microcytic anaemia or an isolated microcytosis seen at the Red Cross War Memorial Children's Hospital, the frequency with which a low red cell mean corpuscular volume (MCV) was associated with the presence of thalassaemia or with an abnormal haemoglobin was investigated. Of 730 patients with an MCV of 60 fl or less, 46 (6.4%) were found to carry the beta-thalassaemia gene and 20 children (2.7 %) had an abnormal haemoglobin, most commonly haemoglobin E. The prevalence of thalassaemia was greatest among Coloured patients and abnormal haemoglobins were found exclusively in this group of children. The implications of these findings are discussed.

The effect of glucose-6-phosphate dehydrogenase deficiency on the severity of neonatal jaundice in Cape Town.

A study was made of 3718 newborn infants with jaundice in excess of physiological levels. Prematurity, haemolytic disease, haematomas or infections were present in 1278 patients. Of the remaining 2440 neonates, 137 were deficient in glucose-6-phosphate dehydrogenase (G-6-PD) and 2303 had idiopathic hyperbilirubinaemia. Exchange transfusion was necessary in 59 (42,7%) of the patients with G-6-PD deficiency and in 426 (18,5%) of those with idiopathic hyperbilirubinaemia. Kernicterus occurred in 3 infants (2,2%) with G-6-PD deficiency and in 3 (0,13%) with idiopathic hyperbilirubinaemia. These findings indicate that G-6-PD deficiency contributes significantly to the severity of neonatal jaundice in the population group studied and should be regarded as a potentially dangerous condition.

The histochemical nitroblue tetrazolium reduction test in newborn infants.

Normal values for the nitroblue tetrazolium (NBT) test in full-term, premature and small-for-gestational-age infants are presented. The NBT reduction was elevated in all groups of babies studied. There was no statistical difference between the groups and the range was wide. Infected babies also showed elevated NBT reduction. However, 60% of the results obtained in the infected group fell within the same range as those obtained in normal neonates. This renders the test unhelpful in the diagnosis of bacterial infection in the neonatal period.