FBXW7 regulates DISC1 stability via the ubiquitin-proteosome system.

Disrupted in schizophrenia 1 (DISC1) is a multi-functional scaffolding protein that has been associated with neuropsychiatric disease. The role of DISC1 is to assemble protein complexes that promote neural development and signaling, hence tight control of the concentration of cellular DISC1 in neurons is vital to brain function. Using structural and biochemical techniques, we show for we believe the first time that not only is DISC1 turnover elicited by the ubiquitin proteasome system (UPS) but that it is orchestrated by the F-Box protein, FBXW7. We present the structure of FBXW7 bound to the DISC1 phosphodegron motif and exploit this information to prove that disruption of the FBXW7-DISC1 complex results in a stabilization of DISC1. This action can counteract DISC1 deficiencies observed in neural progenitor cells derived from induced pluripotent stem cells from schizophrenia patients with a DISC1 frameshift mutation. Thus manipulation of DISC1 levels via the UPS may provide a novel method to explore DISC1 function.

A dust-obscured massive maximum-starburst galaxy at a redshift of 6.34.

Massive present-day early-type (elliptical and lenticular) galaxies probably gained the bulk of their stellar mass and heavy elements through intense, dust-enshrouded starbursts--that is, increased rates of star formation--in the most massive dark-matter haloes at early epochs. However, it remains unknown how soon after the Big Bang massive starburst progenitors exist. The measured redshift (z) distribution of dusty, massive starbursts has long been suspected to be biased low in z owing to selection effects, as confirmed by recent findings of systems with redshifts as high as ~5 (refs 2-4). Here we report the identification of a massive starburst galaxy at z = 6.34 through a submillimetre colour-selection technique. We unambiguously determined the redshift from a suite of molecular and atomic fine-structure cooling lines. These measurements reveal a hundred billion solar masses of highly excited, chemically evolved interstellar medium in this galaxy, which constitutes at least 40 per cent of the baryonic mass. A 'maximum starburst' converts the gas into stars at a rate more than 2,000 times that of the Milky Way, a rate among the highest observed at any epoch. Despite the overall downturn in cosmic star formation towards the highest redshifts, it seems that environments mature enough to form the most massive, intense starbursts existed at least as early as 880 million years after the Big Bang.

The suppression of star formation by powerful active galactic nuclei.

The old, red stars that constitute the bulges of galaxies, and the massive black holes at their centres, are the relics of a period in cosmic history when galaxies formed stars at remarkable rates and active galactic nuclei (AGN) shone brightly as a result of accretion onto black holes. It is widely suspected, but unproved, that the tight correlation between the mass of the black hole and the mass of the stellar bulge results from the AGN quenching the surrounding star formation as it approaches its peak luminosity. X-rays trace emission from AGN unambiguously, whereas powerful star-forming galaxies are usually dust-obscured and are brightest at infrared and submillimetre wavelengths. Here we report submillimetre and X-ray observations that show that rapid star formation was common in the host galaxies of AGN when the Universe was 2-6 billion years old, but that the most vigorous star formation is not observed around black holes above an X-ray luminosity of 10(44) ergs per second. This suppression of star formation in the host galaxy of a powerful AGN is a key prediction of models in which the AGN drives an outflow, expelling the interstellar medium of its host and transforming the galaxy's properties in a brief period of cosmic time.

GnRH agonist for protection against ovarian toxicity during chemotherapy for early breast cancer: the Anglo Celtic Group OPTION trial.

BACKGROUND: Chemotherapy-induced premature ovarian insufficiency (POI) impacts fertility and other aspects of women's health. The OPTION trial tested whether administration of a gonadotropin-releasing hormone agonist during chemotherapy for early breast cancer reduced the risk of POI. PATIENTS AND METHODS: This was a prospective, randomized, parallel group study of the gonadotropin-releasing hormone agonist goserelin administered before and during chemotherapy for breast cancer with stage I-IIIB disease. The primary outcome was amenorrhoea between 12 and 24 months after randomization, supported by elevated follicle stimulating hormone concentrations to give an additional analysis as rate of POI. RESULTS: A total of 227 patients were randomized and the primary analysis was conducted on 202 patients. Goserelin reduced the prevalence of amenorrhoea between 12 and 24 months to 22% versus 38% in the control group (P = 0.015) and the prevalence of POI to 18.5% versus 34.8% in the control group (P = 0.048). Follicle stimulating hormone concentrations were also lower in all women treated with goserelin at both 12 and 24 months (P = 0.027, P = 0.001, respectively). The effect of goserelin was not statistically significant in women >40 years. Assessment of the ovarian reserve using anti-Müllerian hormone showed a marked fall in both groups during treatment to median values of 5% of pretreatment levels in the control group and 7% in the goserelin group, which were not significantly different between groups. CONCLUSION: This study shows that goserelin reduced the risk of POI in women treated with chemotherapy for early breast cancer, with particular efficacy in women aged ≤40 years old. The degree of ovarian protection also seems limited and the clinical significance for fertility and longer term prevention of estrogen deficiency-related outcomes needs to be determined.

A randomized, double-blind phase II study evaluating cediranib versus cediranib and saracatinib in patients with relapsed metastatic clear-cell renal cancer (COSAK).

BACKGROUND: Preclinical work suggests SRC proteins have a role in the development of resistance to vascular endothelial growth factor (VEGF) targeted therapy in metastatic clear-cell renal cancer (mRCC). This hypothesis was tested in this trial using the SRC inhibitor saracatinib and the VEGF inhibitor cediranib. PATIENTS AND METHODS: Patients with disease progression after ≥1 VEGF-targeted therapy were eligible to participate in this double-blind, randomized (1:1) phase II study. The study compared the combination cediranib 30 mg once daily (o.d.) and saracatinib 175 mg o.d. (CS) (n = 69) or cediranib 45 mg o.d. and placebo o.d. (C) (n = 69). Archived tissue was used for biomarker analysis [SRC, focal adhesion kinase (FAK), von Hippel-Lindau, protein tyrosine phosphatase 1b and hypoxia-inducible factor 2α : n = 86]. The primary end point was progression-free survival (PFS) by RECIST v1.1. RESULTS: Between 2010 and 2012, 138 patients were randomized across 16 UK sites. The characteristics of the two groups were well balanced. Partial responses were seen in 13.0% for C and 14.5% for CS (P > 0.05). There was no significant difference in PFS [5.4 months (3.6-7.3 months) for C and 3.9 (2.4-5.3 months) for CS; hazard ratio (HR) 1.18 (0.94-1.48)] or overall survival (OS) [14.2 months (11.2-16.8 months) for C and 10.0 (6.7-13.2 months) for CS; HR 1.28 (1.00-1.63)]. There was no significant difference in the frequency of key adverse events, dose reductions or drug discontinuations. None of the biomarkers were prognostic for PFS or OS. FAK overexpression correlated with an OS benefit [HR 2.29 (1.09-4.82), P > 0.05], but not PFS, for CS. CONCLUSIONS: Saracatinib did not increase the efficacy of a VEGF-targeted therapy (cediranib) in this setting. Biomarker analysis did not identify consistent predictive biomarkers. CLINICALTRIALSGOV: NCT00942877.

Sensitivity testing of trypanosome detection by PCR from whole blood samples using manual and automated DNA extraction methods.

Automated extraction of DNA for testing of laboratory samples is an attractive alternative to labour-intensive manual methods when higher throughput is required. However, it is important to maintain the maximum detection sensitivity possible to reduce the occurrence of type II errors (false negatives; failure to detect the target when it is present), especially in the biomedical field, where PCR is used for diagnosis. We used blood infected with known concentrations of Trypanosoma copemani to test the impact of analysis techniques on trypanosome detection sensitivity by PCR. We compared combinations of a manual and an automated DNA extraction method and two different PCR primer sets to investigate the impact of each on detection levels. Both extraction techniques and specificity of primer sets had a significant impact on detection sensitivity. Samples extracted using the same DNA extraction technique performed substantially differently for each of the separate primer sets. Type I errors (false positives; detection of the target when it is not present), produced by contaminants, were avoided with both extraction methods. This study highlights the importance of testing laboratory techniques with known samples to optimise accuracy of test results.

Movement ecology of an endangered mesopredator in a mining landscape.

BACKGROUND: Efficient movement and energy expenditure are vital for animal survival. Human disturbance can alter animal movement due to changes in resource availability and threats. Some animals can exploit anthropogenic disturbances for more efficient movement, while others face restricted or inefficient movement due to fragmentation of high-resource habitats, and risks associated with disturbed habitats. Mining, a major anthropogenic disturbance, removes natural habitats, introduces new landscape features, and alters resource distribution in the landscape. This study investigates the effect of mining on the movement of an endangered mesopredator, the northern quoll (Dasyurus hallucatus). Using GPS collars and accelerometers, we investigate their habitat selection and energy expenditure in an active mining landscape, to determine the effects of this disturbance on northern quolls. METHODS: We fit northern quolls with GPS collars and accelerometers during breeding and non-breeding season at an active mine site in the Pilbara region of Western Australia. We investigated broad-scale movement by calculating the movement ranges of quolls using utilisation distributions at the 95% isopleth, and compared habitat types and environmental characteristics within observed movement ranges to the available landscape. We investigated fine-scale movement by quolls with integrated step selection functions, assessing the relative selection strength for each habitat covariate. Finally, we used piecewise structural equation modelling to analyse the influence of each habitat covariate on northern quoll energy expenditure. RESULTS: At the broad scale, northern quolls predominantly used rugged, rocky habitats, and used mining habitats in proportion to their availability. However, at the fine scale, habitat use varied between breeding and non-breeding seasons. During the breeding season, quolls notably avoided mining habitats, whereas in the non-breeding season, they frequented mining habitats equally to rocky and riparian habitats, albeit at a higher energetic cost. CONCLUSION: Mining impacts northern quolls by fragmenting favoured rocky habitats, increasing energy expenditure, and potentially impacting breeding dispersal. While mining habitats might offer limited resource opportunities in the non-breeding season, conservation efforts during active mining, including the creation of movement corridors and progressive habitat restoration would likely be useful. However, prioritising the preservation of natural rocky and riparian habitats in mining landscapes is vital for northern quoll conservation.

Suspected tick-borne flavivirus meningoencephalomyelitis in dogs from the UK: six cases (2021).

OBJECTIVES: Tick-borne encephalitis virus and louping ill virus are neurotropic flaviviruses transmitted by ticks. Epidemiologically, tick-borne encephalitis is endemic in Europe whereas louping ill's predominant geographical distribution is the UK. Rarely, these flaviviruses affect dogs causing neurological signs. This case series aimed to describe the clinical, clinicopathological, and imaging findings, as well as the outcomes in six dogs with meningoencephalitis and/or meningomyelitis caused by a flavivirus in the UK in 2021. MATERIALS AND METHODS: Observational retrospective case-series study. Clinical data were retrieved from medical records of dogs with positive serological or immunohistochemical results from three different institutions from spring to winter 2021. RESULTS: Six dogs were included in the study. All dogs presented an initial phase of pyrexia and/or lethargy followed by progressive signs of spinal cord and/or intracranial disease. Magnetic resonance imaging showed bilateral and symmetrical lesions affecting the grey matter of the thalamus, pons, medulla oblongata, and thoracic or lumbar intumescences with none or mild parenchymal and meningeal contrast enhancement. Serology for tick-borne encephalitis virus was positive in five dogs with the presence of seroconversion in two dogs. The viral distinction between flaviviruses was not achieved. One dog with negative serology presented positive immunohistochemistry at post-mortem examination. Three dogs survived but presented neurological sequelae. Three dogs were euthanased due to the rapid progression of the clinical signs or static neurological signs. CLINICAL SIGNIFICANCE: These cases raise awareness of the presence of tick-borne encephalitis as an emergent disease or the increased prevalence of louping ill virus affecting dogs in the UK.

Where have we come from and where are we going? The paediatric HIV programme in Johannesburg, South Africa, from 2004 to 2018: A retrospective analysis of programme trends.

BACKGROUND: The paediatric HIV treatment programme in South Africa (SA) has grown since its inception in 2004. Despite this impressive scale-up of antiretroviral therapy (ART) in children, the proportion of children started on ART and retained in care remains unacceptably low, with only 47% of the 340 000 HIV-positive children in SA on ART in 2020. Johannesburg is one of the districts in SA with the largest number of children living with HIV who are not on ART, and is a priority district for paediatric case finding and retention. OBJECTIVES: To describe the dynamics of the paediatric HIV programme in Johannesburg, SA. METHODS: A secondary analysis was conducted on patient-level HIV treatment data from TIER.Net, the nationally mandated HIV/ART database. Children aged <15 years who received ART between January 2004 and June 2019 at public health facilities in Johannesburg were included. We reported the number of children on ART and the number who entered and exited the programme by age group over time, and analysed the trends of these indicators. RESULTS: By December 2018, 7 630 children aged <15 years remained in Johannesburg's paediatric ART programme: 82.5% were aged 5 - <15 years, with 54.1% of these being 10 - <15 years old. During the study period, 19 850 children were newly initiated on ART. New initiations slowed from 2013, to range from 1 172 to 1 373 yearly. In 2018, 34.2% of initiators were aged <1 year, 24.2% 1 - <5 years and 41.6% 5 - <15 years. Despite these initiations, the number of children on ART only grew by 97 in 2018, owing to programme losses. In 2018, 924  children (12.1%) aged out, 35 (0.5%) died and 983 (12.9%) were lost to follow-up (LTFU), the latter having increased from 10.7% in 2017. Of children who aged out of the paediatric ART programme, 56.3% remained in care at the same facility. CONCLUSION: Early in the SA ART roll-out, many children were found to be HIV infected and started on ART. This number started to slow in 2013, after which the growth rate of the paediatric HIV programme also began to slow. Scale-up of methods for identifying older children with HIV is needed. While ageing out of the paediatric programme is a consideration, the number of children LTFU remains unacceptably high. Further interrogation of barriers to paediatric retention is needed to help realise the Joint United Nations Programme on HIV and AIDS (UNAIDS) 90:90:90 goals for children in SA.

Primary healthcare workers at risk during COVID-19: An analysis of infections in HIV service providers in five districts of South Africa.

BACKGROUND: Protecting healthcare workers (HCWs) from COVID-19 is a global priority. Anova Health Institute (Anova) is the PEPFAR (US President's Emergency Plan for AIDS Relief) District Support Partner for the Johannesburg, Cape Town, Sedibeng, Capricorn and Mopani districts in South Africa, operating in public sector primary healthcare facilities. At the time of the emergence of COVID-19, Anova employed close to 4 000 people: 41% community health workers (CHWs), 23% data staff, 20% nurses and doctors, 12% management/support and 5% allied HCWs. OBJECTIVES: To describe rates of COVID-19 diagnosis in Anova-employed HCWs in five districts. METHODS: Employees exposed to, tested for or diagnosed with COVID-19 were required to report the event. These reports were compiled into a database to monitor the impact of COVID-19 on the workforce. We kept a timeline of key events occurring at national and district level, including Anova's policies and their implementation, that was used to describe organisational response. We described the number of confirmed cases, cumulative incidence rates and testing rates, broken down by district and job category. We estimated expected deaths and the effect on time off work. RESULTS: Of Anova employees, 14% (n=562) were diagnosed with COVID-19 by the end of September 2020. Cumulative incidence was highest in Sedibeng (29%) and lowest in Mopani (5%). All HCWs experienced high incidences: data staff 17%, allied HCWs 16%, CHWs 14%, nurses and doctors 13%, and management/support 11%. At the peak of the epidemic, for 5 weeks, >5% of employees were unable to work owing to exposure or infection, significantly disrupting service delivery. The additional administrative burden on managers was substantial. CONCLUSIONS: It is critical that all cadres of HCWs are protected in the workplace, including in primary care settings, where better structuresare needed to perform risk assessments and conduct outbreak investigations. CHWs and data staff may be at higher risk owing to poor infrastructure, limited power to negotiate working conditions, and limited experience of infection prevention and control. Their working conditions must be improved to reduce their risk.

Effect of COVID-19 on HIV, tuberculosis, and prevention of mother-to-child transmission of HIV indicators in Mopani district, South Africa.

BACKGROUND: To prevent the spread of SARS-CoV-2, many countries instituted lockdown measures. As the virus was initially slow to spread to rural areas in South Africa, Mopani district in Limpopo Province did not experience a high incidence of COVID-19 until the second wave in December 2020. Until then, lockdown measures were more likely than SARS-CoV-2 infections to have an adverse impact on health services. OBJECTIVES: To analyse HIV, tuberculosis (TB) and prevention of mother-to-child transmission of HIV (PMTCT) indicator trends in Mopani during the COVID-19 lockdown and two COVID-19 waves. METHODS: Using monthly data from the District Health Information System from February 2019 to December 2020, we conducted a retrospective review of data elements and indicators that fall into the following domains: primary healthcare head count (HC), HIV, antiretroviral treatment (ART), PMTCT and TB. Aggregated data were analysed, and an interrupted time series analysis was conducted. We assessed percentage changes between the January - March 2020 and April - June 2020 periods, and differences in means for the period April - December 2019 v. the period April - December 2020 were assessed for statistical significance. RESULTS: At the start of the first wave in April 2020, a statistically significant decline of 10% was recorded for total HC utilisation rates (p=0.1). We also found declines of 665 HIV tests (from 1 608 to 942) and 22 positive HIV tests (from 27 to 4) for children between the ages of 18 months and 14 years (p=0.05), with no recovery. Significant declines were also recorded for children aged <15 years starting (change from 35 to 21) and remaining (change from 4 032 to 3 986) on ART, as well as for adults starting ART (change from 855 to 610) at the onset of the first wave (p=0.01). No significant change was detected in PMTCT and TB indicators during the first wave. Pronounced decreases in HC were recorded in December, during the country's second wave (change from 237 965 to 227 834). CONCLUSION: Declines were recorded for most indicators in Mopani, with HC being affected the most at the start of the first wave and not showing any significant recovery between waves. Strategies are required to mitigate the effects of future COVID-19 waves and encourage positive health-seeking behaviour.

The formation of cluster elliptical galaxies as revealed by extensive star formation.

The most massive galaxies in the present-day Universe are found to lie in the centres of rich clusters. They have old, coeval stellar populations suggesting that the bulk of their stars must have formed at early epochs in spectacular starbursts, which should be luminous phenomena when observed at submillimetre wavelengths. The most popular model of galaxy formation predicts that these galaxies form in proto-clusters at high-density peaks in the early Universe. Such peaks are indicated by massive high-redshift radio galaxies. Here we report deep submillimetre mapping of seven high-redshift radio galaxies and their environments. These data confirm not only the presence of spatially extended regions of massive star-formation activity in the radio galaxies themselves, but also in companion objects previously undetected at any wavelength. The prevalence, orientation, and inferred masses of these submillimetre companion galaxies suggest that we are witnessing the synchronous formation of the most luminous elliptical galaxies found today at the centres of rich clusters of galaxies.

Human diseases: clues to cracking the connexin code?

The vertebrate gap junctions formed by the connexin family of transmembrane proteins came to the attention of geneticists in 1993 with the identification of mutations linked to a form of demyelinating neuropathy. Since then, several other genetic disorders have been linked to mutations in specific connexin genes. Also, different diseases can result from different mutations in the same connexin gene. In addition, specific connexin knockout mice have surprising phenotypes. This is leading cell biologists to look afresh at connexins and their involvement in intercellular communication through gap junctions, a process that seems central to coordinating cell function within tissues. Here, we comment on how genetic studies are giving a new impetus to the cell biology of gap junctions.

New suggestions for the mechanical control of bone remodeling.

Bone is constantly renewed over our lifetime through the process of bone (re)modeling. This process is important for bone to allow it to adapt to its mechanical environment and to repair damage from everyday life. Adaptation is thought to occur through the mechanosensitive response controlling the bone-forming and -resorbing cells. This report shows a way to extract quantitative information about the way remodeling is controlled using computer simulations. Bone resorption and deposition are described as two separate stochastic processes, during which a discrete bone packet is removed or deposited from the bone surface. The responses of the bone-forming and -resorbing cells to local mechanical stimuli are described by phenomenological remodeling rules. Our strategy was to test different remodeling rules and to evaluate the time evolution of the trabecular architecture in comparison to what is known from micro-CT measurements of real bone. In particular, we tested the reaction of virtual bone to standard therapeutic strategies for the prevention of bone deterioration, i.e., physical activity and medications to reduce bone resorption. Insensitivity of the bone volume fraction to reductions in bone resorption was observed in the simulations only for a remodeling rule including an activation barrier for the mechanical stimulus above which bone deposition is switched on. This is in disagreement with the commonly used rules having a so-called lazy zone.

Characterization of compounds on nicotinic acetylcholine receptor alpha7 channels using higher throughput electrophysiology.

Alpha7 nicotinic acetylcholine receptor channels are important ligand-gated ion channels that are fast desensitizing, cation selective and have been implicated in the pathophysiology of schizophrenia and Alzheimer's disease. We report here high quality alpha7 parallel patch clamp recordings using the QPatch automated patch clamp system. The QPatch patch clamps up to 48 cells in parallel with the same high fidelity as conventional patch clamp. EC(50) and IC(50) values were comparable to values obtained with conventional patch clamp. The EC(50) value for acetylcholine (ACh) on the QPatch with area under the curve (AUC) analysis was 26microM compared to a value of 29microM determined from conventional patch clamp experiments. Sequential additions of ACh can be made with minimal decay of the peak amplitude. The competitive alpha7 antagonist methyllycaconitine (MLA) blocked currents with an IC(50) value of 0.25nM which is similar to published IC(50) values for MLA. Finally, two different classes of positive allosteric modulators represented by PNU-120596 and NS-1738 elicited characteristic responses, thus allowing accurate characterization of modulation and measurements of potency. These results demonstrate that alpha7 nicotinic acetylcholine receptor channels can be studied reliably in a higher throughput, parallel manner with the QPatch automated patch clamp system.

Mechanical behavior of idealized, stingray-skeleton-inspired tiled composites as a function of geometry and material properties.

Tilings are constructs of repeated shapes covering a surface, common in both manmade and natural structures, but in particular are a defining characteristic of shark and ray skeletons. In these fishes, cartilaginous skeletal elements are wrapped in a surface tessellation, comprised of polygonal mineralized tiles linked by flexible joints, an arrangement believed to provide both stiffness and flexibility. The aim of this research is to use two-dimensional analytical models to evaluate the mechanical performance of stingray skeleton-inspired tessellations, as a function of their material and structural parameters. To calculate the effective modulus of modeled composites, we subdivided tiles and their surrounding joint material into simple shapes, for which mechanical properties (i.e. effective modulus) could be estimated using a modification of traditional Rule of Mixtures equations, that either assume uniform strain (Voigt) or uniform stress (Reuss) across a loaded composite material. The properties of joints (thickness, Young's modulus) and tiles (shape, area and Young's modulus) were then altered, and the effects of these tessellation parameters on the effective modulus of whole tessellations were observed. We show that for all examined tile shapes (triangle, square and hexagon) composite stiffness increased as the width of the joints was decreased and/or the stiffness of the tiles was increased; this supports hypotheses that the narrow joints and high tile to joint stiffness ratio in shark and ray cartilage optimize composite tissue stiffness. Our models also indicate that, for simple, uniaxial loading, square tessellations are least sensitive and hexagon tessellations most sensitive to changes in model parameters, indicating that hexagon tessellations are the most "tunable" to specific mechanical properties. Our models provide useful estimates for the tensile and compressive properties of 2d tiled composites under uniaxial loading. These results lay groundwork for future studies into more complex (e.g. biological) loading scenarios and three dimensional structural parameters of biological tilings, while also providing insight into the mechanical roles of tessellations in general and improving the design of bioinspired materials.

The novel gene glaikit, is expressed during neurogenesis in the Drosophila melanogaster embryo.

A novel gene glaikit (gkt) has been identified which is expressed in the delaminating neuroblasts of the D. melanogaster embryonic central nervous system. At the earliest stages of embryonic development the expression of glaikit was ubiquitous, but by the time the neuroblasts are delaminating gkt expression became restricted to neuroblasts and a few ganglion mother cells. The gkt gene has no characterized homologues and encodes no previously described protein motifs. There are, however, evolutionary conserved predicted genes present in S. pombe, S. cerevisiae and C. elegans. Ectopic neuroblasts induced in either Notch or Delta mutant backgrounds also showed expression of glaikit.

The resolution, enrichment, and organization of normal bone marrow high proliferative potential colony-forming cell subsets on the basis of rhodamine-123 fluorescence.

Cell sorting on the basis of rhodamine-123 (Rh123) fluorescence has been used in conjunction with negative immunomagnetic selection to analyze the high proliferative potential colony-forming cell (HPP-CFC) compartment of normal murine bone marrow and to resolve and enrich HPP-CFC subpopulations responsive to different combinations of the hemopoietic growth factors interleukin 1 alpha (IL-1 alpha), interleukin-3 (IL-3), and colony-stimulating factor 1 (CSF-1). HPP-CFC with a specific requirement for IL-1 alpha plus IL-3 plus CSF-1 in order to proliferate were resolved and enriched on the basis of their low Rh123 retention (Rh-dull), whereas HPP-CFC that grew in the presence of IL-3 plus CSF-1, IL-3 alone, or CSF-1 alone were Rh-bright. Further addition of IL-1 alpha to IL-3 plus CSF-1 stimulated few additional HPP-CFC in the Rh-bright fraction. Our data confirm the value of Rh123 as a probe for the dissection and analysis of the primitive hemopoietic stem cell (PHSC) compartment. These data also show that the Rh123 staining characteristics of IL-1 alpha plus IL-3 plus CSF-1-responsive HPP-CFC are consistent with the hypothesis that these HPP-CFC are closely related to PHSC with long-term reconstituting capacity in vivo and that they are among the most primitive progenitors yet detected in clonal agar culture.

Accelerated establishment of murine long-term bone marrow cultures by addition of stem cell factor.

The effect of stem cell factor (SCF) on the establishment of hematopoietic activity in murine long-term bone marrow cultures (LTBMC) was investigated by addition of SCF to (a) normal LTBMC from the onset of culture and (b) pre-established irradiated bone marrow stroma inoculated with lineage negative (Lin-) primitive hematopoietic progenitor cells enriched on the basis of low rhodamine-123 uptake (Rh-dull). Hematopoietic activity was established more rapidly in LTBMC grown in the presence of SCF (70 ng/mL), and the typical decline in cellularity and progenitor cell content during the first weeks of culture was not observed. SCF also promoted the rapid expansion of progenitor cells derived from Lin-, Rh-dull primitive hematopoietic cells inoculated onto irradiated preestablished bone marrow stroma. The data demonstrate that exogenous SCF augments hematopoietic activity in LTBMC, and that the levels of endogenous SCF elaborated in LTBMC may be suboptimal for expansion of hematopoietic cells.