Barriers and facilitators to HIV prevention and care for Venezuelan migrant/refugee women and girls in Colombia.

Venezuelan migrant and refugee women and girls (VMRWG) face risks of exposure to and infection from HIV and threats of multiple forms of violence (including GBV) during and after migration. Yet, there is a lack of evidence on barriers and facilitators to VMRWGs' access to HIV prevention and care services this population at all stages of their migration. We addressed this evidence gap by conducting a qualitative study composed of fifty-four semi-structured interviews with practitioners (n = 24) and VMRWG (n = 30) in the two largest receiving cities of migrants in Colombia. We sought to identify perceived barriers and facilitators to HIV prevention and care to inform policies and programmatic efforts. Analysis followed a theory-informed approach using the Socioecological Model. Findings describe multi-level barriers to access to HIV prevention and care related to discrimination, gender-based violence, rigid gender norms, lack of information and system fragmentation. Policies that integrate community-based networks and support intersectoral work are pivotal to breach the gaps between services and communities and develop a gender-sensitive approach that tackles the relationship between gender-based violence and HIV risk.

When do adolescent mothers return to school? Timing across rural and urban South Africa.

While adolescent mothers' postpartum return to school offers long-term benefits, returning too early in the absence of sufficient support may impede the healthy development of their children. This study assessed the rates of adolescent mothers' return to school in South Africa (SA) and examined how many months after birth school-aged girls resumed schooling. Two independent studies recruited 1 114 adolescent mothers in Eastern Cape Province and Durban, respectively. Across the two studies, 64.7% (n=649) and 47.8% (n=53) of mothers, respectively, had returned to school at the time of the study. Of these mothers, the majority had returned to school within the first 2 months post partum (study 1: 67.6%; n=439; and study 2: 58.5%; n=31), fewer between 2 and 6 months (16.9%; n=110; and 22.6%; n=12), and after 12 months (2.0%; n=13; and unknown). Our findings indicate a mismatch between SA's national policy recommendations and actual return patterns, showing that a large proportion of mothers returned to school much earlier than advised. This study also highlights a particular need to amend school policies that support early-returners and their children. Additional research on the needs of mother-child dyads and studies on the impact of different timescales on educational and health outcomes are needed to further inform policy and practice regarding adolescent mothers' return to school.

The latency-associated nuclear antigen encoded by Kaposi's sarcoma-associated herpesvirus activates two major essential Epstein-Barr virus latent promoters.

The latency-associated nuclear antigen (LANA) encoded by the Kaposi's sarcoma-associated herpesvirus (KSHV) is expressed in the majority of KSHV-infected cells and in cells coinfected with Epstein-Barr virus (EBV). In coinfected body cavity-based lymphomas (BCBLs), EBV latent membrane protein 1 (LMP1), which is essential for B-lymphocyte transformation, is expressed. EBNA2 upregulates the expression of LMP1 and other cellular genes through specific interactions with cellular transcription factors tethering EBNA2 to its responsive promoters. In coinfected BCBL cells, EBNA2 is not detected but LANA, which is constitutively expressed, contains motifs suggestive of potential transcriptional activity. Additionally, recent studies have shown that LANA is capable of activating cellular promoters. Therefore, we investigated whether LANA can affect transcription from two major EBV latent promoters. In this study, we demonstrated that LANA can efficiently transactivate both the LMP1 and C promoters in the human B-cell line BJAB as well as in the human embryonic kidney 293 cell line. Moreover, we demonstrated that specific domains of LANA containing the putative leucine zipper and the glutamic acid-rich region are highly effective in upregulating these viral promoters, while the amino-terminal region (435 amino acids) exhibited little or no transactivation activity in our assays. We also specifically tested truncations of the LMP1 promoter element and showed that the -204 to +40 region had increased levels of activation compared with a larger region, -512 to +40, which contains two recombination signal-binding protein J kappa binding sites. The smaller, -204 to +40 promoter region contains specific binding sites for the Ets family transcription factor PU.1, transcription activating factor/cyclic AMP response element, and Sp1, all of which are known to function as activators of transcription. Our data therefore suggest a potential role for LANA in regulation of the major EBV latent promoters in KSHV- and EBV-coinfected cells. Furthermore, LANA may be able to activate transcription of viral and cellular promoters in the absence of EBNA2, potentially through association with transcription factors bound to their cognate sequences within the -204 to +40 region. This regulation of viral gene expression is critical for persistence of these DNA tumor viruses and most likely involved in mediating the oncogenic process in these coinfected cells.

Culture in reduced levels of oxygen promotes clonogenic sympathoadrenal differentiation by isolated neural crest stem cells.

Isolated neural crest stem cells (NCSCs) differentiate to autonomic neurons in response to bone morphogenetic protein 2 (BMP2) in clonal cultures, but these neurons do not express sympathoadrenal (SA) lineage markers. Whether this reflects a developmental restriction in NCSCs or simply inappropriate culture conditions was not clear. We tested the growth and differentiation potential of NCSCs at approximately 5% O(2), which more closely approximates physiological oxygen levels. Eighty-three percent of p75(+)P(0-) cells isolated from embryonic day 14.5 sciatic nerve behaved as stem cells under these conditions, suggesting that this is a nearly pure population. Furthermore, addition of BMP2 plus forskolin in decreased oxygen cultures elicited differentiation of thousands of cells expressing tyrosine hydroxylase, dopamine-beta-hydroxylase, and the SA lineage marker SA-1 in nearly all colonies. Such cells also synthesized and released dopamine and norepinephrine. These data demonstrate that isolated mammalian NCSCs uniformly possess SA lineage capacity and further suggest that oxygen levels can influence cell fate. Parallel results indicating that reduced oxygen levels can also promote the survival, proliferation, and catecholaminergic differentiation of CNS stem cells (Studer et al., 2000) suggests that neural stem cells may exhibit a conserved response to reduced oxygen levels.

Competence, specification and commitment in otic placode induction.

The inner ear is induced from cranial ectoderm adjacent to the hindbrain. Despite almost a century of study, the molecular mechanisms of inner ear induction remain obscure. We have identified four genes expressed very early in the anlage of the inner ear, the otic placode. Pax-2, Sox-3, BMP-7 and Notch are all expressed in placodal ectoderm from the 4-5 somite stage (ss) onwards, well before the otic placode becomes morphologically visible at the 12-14ss. We have used these four molecular markers to show that cranial ectoderm becomes specified to form the otic placode at the 4-6ss, and that this ectoderm is committed to a placodal fate by the 10ss. We also demonstrate that much of the embryonic ectoderm is competent to generate an otic placode if taken at a sufficiently early age. We have mapped the location of otic placode-inducing activity along the rostrocaudal axis of the embryo, and have determined that this activity persists at least until the 10ss. Use of the four molecular otic placode markers suggests that induction of the otic placode in birds occurs earlier than previously thought, and proceeds in a series of steps that are independently regulated.

Lineage-restricted neural precursors can be isolated from both the mouse neural tube and cultured ES cells.

We have previously identified multipotent neuroepithelial (NEP) stem cells and lineage-restricted, self-renewing precursor cells termed NRPs (neuron-restricted precursors) and GRPs (glial-restricted precursors) present in the developing rat spinal cord (A. Kalyani, K. Hobson, and M. S. Rao, 1997, Dev. Biol. 186, 202-223; M. S. Rao and M. Mayer-Proschel, 1997, Dev. Biol. 188, 48-63; M. Mayer-Proschel, A. J. Kalyani, T. Mujtaba, and M. S. Rao, 1997, Neuron 19, 773-785). We now show that cells identical to rat NEPs, NRPs, and GRPs are present in mouse neural tubes and that immunoselection against cell surface markers E-NCAM and A2B5 can be used to isolate NRPs and GRPs, respectively. Restricted precursors similar to NRPs and GRPs can also be isolated from mouse embryonic stem cells (ES cells). ES cell-derived NRPs are E-NCAM immunoreactive, undergo self-renewal in defined medium, and differentiate into multiple neuronal phenotypes in mass culture. ES cells also generate A2B5-immunoreactive cells that are similar to E9 NEP-cell-derived GRPs and can differentiate into oligodendrocytes and astrocytes. Thus, lineage restricted precursors can be generated in vitro from cultured ES cells and these restricted precursors resemble those derived from mouse neural tubes. These results demonstrate the utility of using ES cells as a source of late embryonic precursor cells.

Alternative neural crest cell fates are instructively promoted by TGFbeta superfamily members.

How growth factors influence the fate of multipotent progenitor cells is not well understood. Most hematopoietic growth factors act selectively as survival factors, rather than instructively as lineage determination signals. In the neural crest, neuregulin instructively promotes gliogenesis, but how alternative fates are determined is unclear. We demonstrate that bone morphogenic protein 2 (BMP2) induces the basic-helix-loop-helix protein MASH1 and neurogenesis in neural crest stem cells. In vivo, MASH1+ cells are located near sites of BMP2 mRNA expression. Some smooth muscle differentiation is also observed in BMP2. A related factor, transforming growth factor beta1 (TGFbeta1), exclusively promotes smooth muscle differentiation. Like neuregulin, BMP2 and TGFbeta1 act instructively rather than selectively. The neural crest and hematopoietic systems may therefore utilize growth factors in different ways to generate cellular diversity.

A reciprocal cell-cell interaction mediated by NT-3 and neuregulins controls the early survival and development of sympathetic neuroblasts.

Neurotrophin 3 (NT-3) can support the survival of some embryonic sympathetic neuroblasts before they become nerve growth factor dependent. We show that NT-3 is produced in vivo by nonneuronal cells neighboring embryonic sympathetic ganglia. NT-3 mRNA is produced by these nonneuronal cells in vitro and is up-regulated by platelet-derived growth factor, ciliary neurotrophic factor, and glial growth factor 2 (a neuregulin). Nonneuronal cell-conditioned medium promotes survival and induces TrkA expression in isolated sympathetic neuroblasts, and this activity is blocked by anti-NT-3 antibody. Neuroblasts also enhance NT-3 production by nonneural cells. Neuroblasts synthesize several forms of neuregulin, and antibodies to neuregulin attenuate the effect of the neuroblasts on the nonneuronal cells. These data suggest a reciprocal cell-cell interaction, in which neuroblast-derived neuregulins promote NT-3 production by neighboring nonneuronal cells, which in turn promotes neuroblast survival and further differentiation.

Role of environmental signals and transcriptional regulators in neural crest development.

The processes by which undifferentiated cells are assigned to particular fates are far from clear. We review recent work that has examined this problem in the neural crest, a multipotential cell population that gives rise to peripheral neurons in vertebrates. Peripheral neuronal differentiation appears to occur in a series of developmental steps that can be regulated independently by signals in the environment. Furthermore, such steps are reflected by corresponding changes in the pattern of regulatory transcription factor expression in differentiating neural crest cells. The determination of neuronal identity may proceed by a series of parallel regulatory pathways involving transcription factors acting both in cascades and in combinatorial arrays.

The H-2KbtsA58 transgenic mouse: a new tool for the rapid generation of novel cell lines.

The ability to generate expanded populations of individual cell types able to undergo normal differentiation in vitro and in vivo is of critical importance in the investigation of the mechanisms that underly differentiation and in studies on the use of cell transplantation to repair damaged tissues. This review discusses the development of a strain of transgenic mice that allows the direct derivation of conditionally immortal cell lines from a variety of tissues, simply by dissociation of the tissue of interest and growth of cells in appropriate conditions. In these mice the tsA58 mutant of SV40 large T antigen is controlled by the interferon-inducible Class I antigen promoter. Cells can be grown for extended periods in vitro simply by growing them at 33 degrees C in the presence of interferon, while still retaining the capacity to undergo normal differentiation in vivo and in vitro. In addition, it appears that cell lines expressing mutant phenotypes can readily be generated by preparing cultures from appropriate offspring of matings between H-2KbtsA58 transgenic mice and mutant mice of interest.

Differential regulation of transcription factor gene expression and phenotypic markers in developing sympathetic neurons.

We have examined the regulation of transcription factor gene expression and phenotypic markers in developing chick sympathetic neurons. Sympathetic progenitor cells first express the bHLH transcriptional regulator Cash-1 (a chicken achaete-scute homologue), followed by coordinate expression of Phox2, a paired homeodomain protein, and GATA-2, a zinc finger protein. SCG10, a pan-neuronal membrane protein, is first detected one stage later, followed by the catecholaminergic neurotransmitter enzyme tyrosine hydroxylase (TH). We have used these markers to ask two questions: (1) is their expression dependent upon inductive signals derived from the notochord or floor plate?; (2) does their sequential expression reflect a single linear pathway or multiple parallel pathways? Notochord ablation experiments indicate that the floor plate is essential for induction of GATA-2, Phox2 and TH, but not for that of Cash-1 and SCG10. Taken together these data suggest that the development of sympathetic neurons involves multiple transcriptional regulatory cascades: one, dependent upon notochord or floor plate-derived signals and involving Phox2 and GATA-2, is assigned to the expression of the neurotransmitter phenotype; the other, independent of such signals and involving Cash-1, is assigned to the expression of pan-neuronal properties. The parallel specification of different components of the terminal neuronal phenotype is likely to be a general feature of neuronal development.

The characterization of astrocyte cell lines that display properties of glial scar tissue.

The glial scar has been proposed to be a major impediment to regeneration in the adult CNS. Analysis of glial scars in vivo is complicated, however, by the large number of cell types present in such lesions. We have attempted to simplify analysis of the glial scar environment by deriving a series of conditionally immortal astrocyte cell lines that display several properties expressed by glial scar tissue in vitro. The astrocyte lines, which were derived from H-2KbtsA58 transgenic mice, expressed macromolecules associated with glial scars in vivo and were significantly less effective than neonatal astrocytes at promoting neurite outgrowth from postnatal central and peripheral neurons. The astrocyte lines also inhibited migration of oligodendrocyte type-2 astrocyte progenitor cells in vitro. We propose that certain properties shown previously to be expressed by glial scars may be reconstituted in vitro by astrocytes alone.

Repair of demyelinated lesions by transplantation of purified O-2A progenitor cells.

The transplantation of well defined populations of precursor cells offers a means of repairing damaged tissue and of delivering therapeutic compounds to sites of injury or degeneration. For example, a functional immune system can be reconstituted by transplantation of purified haematopoietic stem cells, and transplanted skeletal myoblasts and keratinocytes can participate in the formation of normal tissue in host animals. Cell transplantation in the central nervous system (CNS) has been proposed as a means of correcting neuronal dysfunction in diseases associated with neuronal loss; it might also rectify glial cell dysfunction, with transplanted oligodendrocyte precursor cells eventually allowing repair of demyelinating damage in the CNS. Here we use co-operating growth factors to expand purified populations of oligodendrocyte type-2 astrocyte (O-2A) progenitor cells for several weeks in vitro. When injected into demyelinating lesions in spinal cords of adult rats, created in such a way as to preclude host-mediated remyelination, these expanded populations are capable of producing extensive remyelination. In addition, transplantation of O-2A progenitor cells genetically modified to express the bacterial beta-galactosidase gene gives rise to beta-galactosidase-positive oligodendrocytes which remyelinate demyelinated axons within the lesion. These results offer a viable strategy for the manipulation of neural precursor cells which is compatible with attempts to repair damaged CNS tissue by precursor transplantation.

From chance to choice in the generation of neural cell lines.

Despite the central importance of cell lines in contemporary studies in cellular and molecular biology, many areas of potential investigation remain impeded by the limited number of lines available and by the difficulty in generating new lines of interest. Thus, there has been a constant pressure to develop improved methods for obtaining cell lines of particular interest. This review examines some of the problems associated with in vitro approaches to cell line generation. In addition, two different ways in which transgenic animals can be used to overcome the limitations of in vitro production of cell lines are discussed. In the first approach, specific promoters are utilized to target expression of immortalizing genes to cells of interest. The second approach is concerned with development of a strain of transgenic animals (the H-2KbtsA58 transgenic mouse) designed to obviate the need for identification of cell-type specific promoters, and in which it is theoretically possible to directly generate conditionally immortal cell lines from any tissue of the body by simple dissection and growth of cells in appropriate tissue culture conditions. Finally, approaches are also discussed in which investigations on the control of precursor differentiation have been applied so as to bypass the need for expression of activated immortalizing oncogenes in the generation of large quantities of conditionally immortalized cells with the capacity to undergo normal differentiation in vitro and in vivo.

The cellular measurement of time.

This review describes three biological processes in which there is evidence for single cells being able to measure elapsed time. We describe the work that has led to this view, and review more recent work that has provided new insights into possible mechanisms for the measurement of time.