Antennapedia homeobox peptide enhances growth and branching of embryonic chicken motoneurons in vitro.

Spinal motoneuron development is regulated by a variety of intrinsic and extrinsic factors. Among these, a possible role for homeoproteins is suggested by their expression in the motoneuron at relatively late stages. To investigate their possible involvement in motoneuron growth, we adapted a novel technique recently developed in this laboratory, based on the ability of the 60 amino acid-long homeobox of Antennapedia (pAntp) to translocate through the neuronal membrane and to accumulate in the nucleus (Joliot, A. H., C. Pernelle, H. Deagostini-Bazin, and A. Prochiantz. 1991. Proc. Natl. Acad. Sci. USA. 88:1864-1868; Joliot, A. H., A. Triller, M. Volovitch, C. Pernelle, and A. Prochiantz. 1991. New Biol. 3:1121-1134). Motoneurons from E5 chicken spinal cord were incubated with pAntp, purified by panning on SC1 antibody and plated on polyornithine/laminin substrata without further addition of pAntp. After 24 h, neurite outgrowth was already extensive in controls. In cultures of motoneurons that had been preincubated with 10(-7) M pAntp, neurite length was doubled; a similar effect was obtained using postnatal muscle extracts. Morphological analysis using a neurofilament marker specific for axons indicated that the homeobox peptide enhances primarily axonal elongation and branching. To test the hypothesis that the biological activity of pAntp involves its specific attachment to cognate homeobox binding sites present in the genome, we generated a mutant of pAntp called pAntp40P2, that was still able to translocate through the motoneuron membrane and to reach the nucleus, but had lost the specific DNA-binding properties of the wild-type peptide. Preincubation of pAntp40P2 with purified motoneurons failed to increase neurite outgrowth. This finding raises the possibility that motoneuron growth is controlled by homeobox proteins.

Antenatal depressed mood and child cognitive and physical growth at 18-months in South Africa: a cluster randomised controlled trial of home visiting by community health workers.

AIM: To examine the child outcomes at 18-months post-birth of a population cohort of women with antenatal depressed mood, half of whom were randomly chosen to receive perinatal home visits from community health workers during pregnancy. METHOD: Pregnant women in 24 neighbourhoods (98% participation) were randomised by neighbourhood to: (1) standard clinic care (SC; 12 neighbourhoods; n = 594) or (2) the Philani Intervention Program, a home visiting intervention plus standard care (12 neighbourhoods; n = 644). The physical and cognitive outcomes of children of mothers with antenatally depressed mood (Edinburg Perinatal Depression Scale >13) in the intervention condition were compared at 18-months post-birth to children of mothers without depressed mood in pregnancy in both conditions. RESULTS: More than a third of mothers had heightened levels of antenatal depressed mood (35%), similar across conditions. Antenatal depressed mood was significantly associated with being a mother living with HIV, using alcohol and food insecurity. At 18-months, the overall cognitive and motor scale scores on the Bayley Scales of Development were similar. However, 10.3% fewer children of mothers with antenatal depressed mood in the intervention condition had cognitive scores on the Bayley Scales that were less than 85 (i.e., s.d. = 2 lower than normal) compared with children of mothers with antenatal depressed mood in the SC condition. Intervention children of mothers with antenatal depressed mood were also significantly less likely to be undernourished (Weight-for-Age Z-scores < -2). CONCLUSION: Cognitive development and child growth among children born to mothers with antenatal depressed mood can be improved by mentor mother home visitors, probably resulting from better parenting and care received early in life.

Stimulation of human epidermal differentiation by delta-notch signalling at the boundaries of stem-cell clusters.

BACKGROUND: Human epidermis is renewed throughout life from stem cells in the basal layer of the epidermis. Signals from the surrounding keratinocytes influence the differentiation of the stem cells, but the nature of the signals is unknown. In many developing tissues, signalling mediated by the transmembrane protein Delta1 and its receptor Notch1 inhibits differentiation. Here, we investigated the role of Delta-Notch signalling in postnatal human epidermis. RESULTS: Notch1 expression was found in all living epidermal layers, but Delta1 expression was confined to the basal layer of the epidermis, with highest expression in those regions where stem cells reside. By overexpressing Delta1 or Delta(T), a truncated form of Delta1, in primary human keratinocytes and reconstituting epidermal sheets containing mixtures of Delta-overexpressing cells and wild-type cells, we found that cells expressing high levels of Delta1 or Delta(T) failed to respond to Delta signals from their neighbours. In contrast, wild-type keratinocytes that were in contact with neighbouring cells expressing Delta1 were stimulated to leave the stem-cell compartment and initiate terminal differentiation after a few rounds of division. Delta1 promoted keratinocyte cohesiveness, whereas Delta(T) did not. CONCLUSIONS: We propose that high Delta1 expression by epidermal stem cells has three effects: a protective effect on stem cells by blocking Notch signalling; enhanced cohesiveness of stem-cell clusters, which may discourage intermingling with neighbouring cells; and signalling to cells at the edges of the clusters to differentiate. Notch signalling in epidermal stem cells thus differs from other progenitor cell populations in promoting, rather than suppressing, differentiation.

Maintenance of neuroepithelial progenitor cells by Delta-Notch signalling in the embryonic chick retina.

BACKGROUND: Neurons of the vertebrate central nervous system (CNS) are generated sequentially over a prolonged period from dividing neuroepithelial progenitor cells. Some cells in the progenitor cell population continue to proliferate while others stop dividing and differentiate as neurons. The mechanism that maintains the balance between these two behaviours is not known, although previous work has implicated Delta-Notch signalling in the process. RESULTS: In normal development, the proliferative layer of the neuroepithelium includes both nascent neurons that transiently express Delta-1 (Dl1), and progenitor cells that do not. Using retrovirus-mediated gene misexpression in the embryonic chick retina, we show that where progenitor cells are exposed to Dl1 signalling, they are prevented from embarking on neuronal differentiation. A converse effect is seen in cells expressing a dominant-negative form of Dl1, Dl1(dn), which we show renders expressing cells deaf to inhibitory signals from their neighbours. In a multicellular patch of neuroepithelium expressing Dl1(dn), essentially all progenitors stop dividing and differentiate prematurely as neurons, which can be of diverse types. Thus, Delta-Notch signalling controls a cell's choice between remaining as a progenitor and differentiating as a neuron. CONCLUSIONS: Nascent retinal neurons, by expressing Dl1, deliver lateral inhibition to neighbouring progenitors; this signal is essential to prevent progenitors from entering the neuronal differentiation pathway. Lateral inhibition serves the key function of maintaining a balanced mixture of dividing progenitors and differentiating progeny. We propose that the same mechanism operates throughout the vertebrate CNS, enabling large numbers of neurons to be produced sequentially and adopt different characters in response to a variety of signals. A similar mechanism of lateral inhibition, mediated by Delta and Notch proteins, may regulate stem-cell function in other tissues.

Neurotrophic activity of a homeobox peptide.

Homeoproteins are well known for their role in defining the shape of organs during early development. The late expression of some homeogenes in the nervous system suggests that they might have other, additional functions, possibly in neurite growth and target recognition. The 60 amino acid-long peptide corresponding to the homeobox of Antennapedia (pAntp) translocates through the membrane of neurons in culture and reaches their nuclei. This process is followed by an enhanced morphological differentiation of the neurons. Internalization by neurons is four-fold that observed with fibroplasts. This difference is abolished upon treatment with Endo-N which specifically cleaves alpha,2-8 bonds in polysialic acid. To understand the mode of action of the peptide, we constructed three mutants modified in their capacity to specifically bind promoters and/or to translocate through the cell membrane. The biological properties of the mutants demonstrate that the neurotrophic action of pAntp requires its internalization and integrity of its specific DNA-binding capacity.

Promoter-specific regulation of gene expression by an exogenously added homedomain that promotes neurite growth.

pAntp, a 60 amino acid long peptide corresponding to the homeodomain of the Drosophila Antennapedia protein, translocates through neuronal membranes when added exogenously to neurons in culture, where it accumulates in the nucleus and promotes neurite outgrowth. We proposed that the peptide, once internalized, may compete for homeoprotein DNA binding sites. To investigate this point, we have produced a permanent fibroblast cell line which carries a luciferase reporter gene under the control of a 93 bp genomic region of the HOXD9 promoter with binding sites for homeoproteins. Externally added pAntp specifically down-regulates the expression of the reporter gene, suggesting that the neurotrophic effects observed previously are mediated by direct binding of pAntp to homeoprotein target sites.

Sensory organ generation in the chicken inner ear: contributions of bone morphogenetic protein 4, serrate1, and lunatic fringe.

The chicken inner ear is a remarkably complex structure consisting of eight morphologically distinct sensory organs. Unraveling how these sensory organs are specified during development is key to understanding how such a complex structure is generated. Previously, we have shown that each sensory organ in the chicken inner ear arises independently in the rudimentary otocyst based on Bone morphogenetic protein 4 (Bmp4) expression. Here, we compare the expression of Bmp4 with two other putative sensory organ markers, Lunatic Fringe (L-fng) and chicken Serrate1 (Ser1), both of which are components of the Notch signaling pathway. L-fng and Ser1 expression domains were asymmetrically distributed in the otic cup. At this early stage, expression of L-fng is similar to Delta1 (Dl1), in an anteroventral domain apparently corresponding to the neurogenic region, while Ser1 is expressed at both the anterior and posterior poles. By the otocyst stage, the expression of both L-fng and Ser1 largely coincided in the medial region. All presumptive sensory organs, as identified by Bmp4 expression, arose within the broad L-fng- and Ser1-positive domain, indicating the existence of a sensory-competent region in the rudimentary otocyst. In addition, there is a qualitative difference in the levels of expression between L-fng and Ser1 such that L-fng expression was stronger in the ventral anterior, whereas Ser1 was stronger in the dorsal posterior region of this broad domain. This early difference in expression may presage the differences among sensory organs as they arise from this sensory competent zone.

Combined use of pleural adenosine deaminase with lymphocyte/neutrophil ratio. Increased specificity for the diagnosis of tuberculous pleuritis.

UNLABELLED: Increased pleural fluid adenosine deaminase (ADA) activity is classically associated with tuberculous pleuritis. However, increased activity can also occur in a number of other diseases and this may negatively affect the diagnostic utility of ADA measurements and decrease its specificity for the diagnosis of tuberculosis (TB). The presence of ADA in pleural fluids reflects the cellular immune response in the pleural cavity and in particularly, the activation of T lymphocytes. Different disease entities are typically associated with the presence of particular types of leukocytes. OBJECTIVE: To determine whether the combined use of ADA activity and differential cell counts would provide a more efficient means for diagnosing tuberculous pleurisy than the use of ADA levels alone. METHODS: Biochemistry, cytology, and microbiology studies were performed on 472 consecutive pleural fluids. ADA and differential cell counts were determined on all exudative effusions. RESULTS: ADA activity in tuberculous effusions was significantly higher than in any other diagnostic group (p < 0.005). At a level of 50 U/L, the sensitivity, specificity, positive predictive value (ppv), negative predictive value (npv), and efficiency for the identification of TB were calculated at 91%, 81%, 84%, 89%, and 86%, respectively. When the additional requirement of a lymphocyte neutrophil ratio of 0.75 or greater was included, the sensitivity, specificity, ppv, npv, and efficiency for the identification of TB were calculated at 88%, 95%, 95%, 88%, and 92%, respectively. CONCLUSION: ADA, especially when combined with differential cell counts and lymphocyte/neutrophil ratios, remains a useful test in the diagnosis tuberculous pleuritis.

Sequential half-body irradiation as salvage therapy in chemotherapy-resistant multiple myeloma.

Fifteen patients with a median age of 58 years, having multiple myeloma resistant to conventional combinations of cytotoxic drugs, received sequential half-body irradiation as salvage therapy. Response was obtained in 53% (n = 8: group 1); this was objective in 40% (n = 6), being defined as 50% or greater reduction in paraprotein, clearance of light chains from the urine, or an unequivocal decrease in tumor bulk on an adequate marrow trephine biopsy; a further 13% (n = 2) just failed to meet these criteria but nevertheless had excellent subjective response. Median survival was 24 months. No objective or subjective improvement occurred in 47% (n = 7: group 2); median transient survival was 4 months. Short-term toxicity was limited to transient nausea in 30% (n = 5) and protracted pancytopenia in about one-half of the patients (n = 7), who remain dependent on intermittent RBC transfusions. Morbidity is only moderate, and the response rate of 53% in refractory patients suggests that sequential half-body irradiation has a definite place in managing patients with end-stage disseminated myelomatosis.

No emergence of Echinococcus multilocularis in foxes in Flanders and Brussels anno 2007-2008.

Echinococcus multilocularis is highly endemic in red foxes in southern Belgium (region of Wallonia), especially in the higher located forested areas. The north of Belgium, including the regions of Flanders and Brussels, is more urbanized and has been colonized entirely by red foxes since the 1980s. A temperospatial analysis of compiled epidemiological data from 1996 to 2003 predicted a northwest spread of the cestode from Wallonia and the Netherlands towards Flanders and Brussels (Prev. Vet. Med. 2006, 76, 137-150). In 2007-2008, none of 187 examined foxes from the north tested positive (<2.8%, α = 0.01), compared to 1.7% in 1996-1999. This suggests that the parasite is not emerging in the examined area and the endemic region has not significantly extended northwest during the last decade. The possible reasons are discussed in the article, including the relatively low altitude, milder climate or low abundance of suitable intermediate hosts. The low prevalence in foxes and the generally low infection rate in humans imply that the risk for public health in Flanders and Brussels is limited anno 2007-2008.

Multiple blastic transformations in the course of chronic granulocytic leukaemia.

A 14-year-old boy with Philadelphia chromosome-positive chronic granulocytic leukaemia underwent morphologically distinctive blastic transformation on 3 separate occasions. The first was clearly lymphoblastic, the second was associated with meningeal leukaemia and was morphologically biphenotypic, expressing mixed myeloid and lymphoid features, while the third was characterised by extreme thrombocytosis and megakaryoblastic proliferation. The classical t(9:22) Philadelphia chromosome remained constant throughout the illness, but the additional cytogenetic changes that occurred with the first transformation were abolished with response to conventional therapy for lymphoblastic leukaemia. A total of 19 months of good-quality life was achieved, but on the third occasion, the boy died from intracranial haemorrhage before treatment could be initiated.

Survey of the health and nutrition status of a squatter community in Khayelitsha.

The findings of a nutrition and health survey in Site B, a squatter area in Khayelitsha close to Cape Town, are reported. Of the children under 6 years, 16.8% were found to be under weight for age, 23.5% were stunted and 2.5% wasted, indicating a serious nutritional crisis in this community. Children with a low-birth-weight had a 3 times greater risk of being under weight for age and a 2 times greater risk of being stunted than children with birth-weights greater than 2,500 g. Of the children born outside Cape Town, 21.9% were under weight for age compared with 13.5% of children born in Cape Town. Of the pre-school children, 4.2% had completed or were on antituberculosis treatment compared with 2% of the children in the age group 6-18 years and 3.2% of adults. Sixty per cent of the pre-school children with tuberculosis were under weight for age. Half the adult population was fully employed, and 22% of households had no wage earners. Assuming literacy after 4 years of schooling, 76% of the adults were literate, but only 2.5% had completed Standard 10. Women were generally better qualified than men.

The cost effectiveness of the Philani Nutrition Day Centre in Crossroads squatter camp, Cape Town.

One hundred and seventeen children aged 0-7 years who had been treated at Philani Nutrition Day Centre were sought in Crossroads squatter camp near Cape Town after discharge; 61 were traced and complete records were obtained for 42. Twenty-seven (64,2%) of the 42 were below the third percentile (weight for age, National Center for Health Statistics standards) at the time of admission to the centre and 3 (7,1%) were still below it at discharge 1-26 (mean 7,8) months later. Six (14,3%) were below the third percentile when they were traced 1-23 months (mean 10,3 months) later. Total attendances from 1982 to 1983 were estimated at 12 300 child-days. Expenditure on the centre for this period was estimated at R29 759, of which R20 282 (68,2%) was spent on salaries, R6 340 (21,3%) on food and R3 137 (10,5%) on sundries (including R2 500 on drugs). The estimated cost per child per day's attendance was R2,42. The duration of treatment was estimated to be 80 attendances for a severe and 32 attendances for a moderate case of undernutrition, giving an estimated cost of between R194 and R73 per child for complete treatment. We conclude that this expenditure: (i) benefited most of the children who were originally below the third percentile; (ii) sustained children not actually admitted to the centre by feeding them or their siblings, probably resulting in there being more food available at home; and (iii) prevented the (costly) admission to hospital of some children.

Adenosine deaminase estimations in the differentiation of pleural effusions.

Adenosine deaminase (ADA) estimations were performed on the pleural fluid from 368 effusions. The mean (+/-SD) ADA concentration in tuberculous effusions was 92,11 +/- 37,05 U/l, and these values were found to be highly statistically different from the 23,23 +/- 13,15 U/l in secondary malignant tumours of the pleura, the 34,86 +/- 14,2 U/l in mesotheliomas, and the 23,81 +/- 15,07 U/l in pulmonary embolism. The ADA values of 64,3 +/- 44,95 U/l in lymphoproliferative disorders were less significantly different. No statistical difference could be found between values in the tuberculous group and the ADA levels of 97,57 +/- 82 U/l found in para-infective effusions, but these could be distinguished from each other by microscopic examination of the pleural fluid. The importance of ADA estimations in the diagnosis and differentiation of tuberculous effusions is discussed and the role of microscopy is emphasized.

Notch signaling in the development of the inner ear: lessons from Drosophila.

The sensory patches in the ear of a vertebrate can be compared with the mechanosensory bristles of a fly. This comparison has led to the discovery that lateral inhibition mediated by the Notch cell-cell signaling pathway, first characterized in Drosophila and crucial for bristle development, also has a key role in controlling the pattern of sensory hair cells and supporting cells in the ear. We review the arguments for considering the sensory patches of the vertebrate ear and bristles of the insect to be homologous structures, evolved from a common ancestral mechanosensory organ, and we examine more closely the role of Notch signaling in each system. Using viral vectors to misexpress components of the Notch pathway in the chick ear, we show that a simple lateral-inhibition model based on feedback regulation of the Notch ligand Delta is inadequate for the ear just as it is for the fly bristle. The Notch ligand Serrate1, expressed in supporting cells in the ear, is regulated by lateral induction, not lateral inhibition; commitment to become a hair cell is not simply controlled by levels of expression of the Notch ligands Delta1, Serrate1, and Serrate2 in the neighbors of the nascent hair cell; and at least one factor, Numb, capable of blocking reception of lateral inhibition is concentrated in hair cells. These findings reinforce the parallels between the vertebrate ear and the fly bristle and show how study of the insect system can help us understand the vertebrate.

Megakaryoblastic transformation in myeloproliferative disorders.

Megakaryoblastic transformation, with cells showing characteristic morphology and ultrastructural cytochemistry, developed in two patients with agnogenic myeloid metaplasia or myelofibrosis, and in one with chronic granulocytic leukemia. From the onset of the leukemic phase, all three have followed a relatively benign clinical course, surviving for 48, 29, and 24 months, respectively; only hydroxyurea has been necessary to control thrombocytosis. This experience emphasizes that it is possible for a patient to have an improved quality of life with minimum chemotherapy. In contrast, megakaryoblastic leukemia or acute myelofibrosis, where splenomegaly and a leukoerythroblastic blood picture are typically absent, responds poorly to any form of chemotherapy, and survival is short.

Neurotrophic activity of the Antennapedia homeodomain depends on its specific DNA-binding properties.

In previous reports we have demonstrated that the 60-aa peptide corresponding to the homeodomain of the Drosophila protein Antennapedia (pAntp) translocates through the membrane of neurons in culture, accumulates in neuronal nuclei, and promotes neurite growth. To analyze the importance of specific pAntp DNA-binding properties in this phenomenon we have constructed three mutant versions of pAntp that differ in their ability to translocate through the membrane and to bind specifically the cognate sequence for homeodomains present in the promoter of HoxA5. We demonstrate that removing two hydrophobic residues of the third helix inhibits pAntp internalization and suppresses its neurotrophic activity. We also show that pAntp neurotrophic activity is lost when mutations are introduced in positions preserving its penetration and nuclear accumulation but abolishing its capacity to bind specifically the cognate DNA-binding motif for homeoproteins. Our results strongly suggest that pAntp neurotrophicity requires both its internalization and its specific binding to homeobox cognate sequences. We propose that homeoproteins might regulate important events in the morphological differentiation of the postmitotic neuron.

Neurotrophic activity of an homeobox peptide.

The sixty aminoacid-long peptide corresponding to the homeobox of Antennapedia (pAntp) translocates through the membrane of neurons in culture and reaches their nuclei. This process is followed by an enhanced morphological differentiation of the neurons. Internalization by neurons is 4-fold that observed with fibroblasts. This difference is abolished upon treatment with Endo-N which specifically cleaves alpha, 2-8 bounds in polysialic acid (PSA). To understand the mode of action of the peptide, the authors constructed three mutants modified in their capacity to specifically bind promoters and/or to translocate through the cell membrane. The biological properties of the mutants demonstrate that the neurotrophic action of pAntp requires its internalization and the integrity of its specific DNA-binding capacity.

Superior vena cava syndrome caused by an intrathoracic plasmacytoma.

A case of an extramedullary intrathoracic plasmacytoma causing superior vena cava syndrome is described. Review of the literature on intrathoracic plasmacytomas and superior vena cava syndrome revealed that no similar cases have been described to date. The initial presentation, management, and response to treatment are described.

Use of adenosine deaminase as a diagnostic tool for tuberculous pleurisy.

BACKGROUND: A statistical audit of adenosine deaminase (ADA) in pleural effusions was undertaken. METHODS: ADA analysis, cytological and microbiological examinations, and differential cell counts were performed on 462 pleural fluid samples. RESULTS: ADA activity in tuberculous effusions was higher than in any other diagnostic group. At a level of 50 U/l the sensitivity and specificity for the identification of tuberculosis was 90% and 89%, respectively. CONCLUSIONS: ADA activity remains a useful test in the evaluation of pleural effusions.