Biomedical subjects

D A Williams

Publications and source records attributed to D A Williams.

At least 19 recordsLinked to original sources

PubMed1995

Murine embryonic yolk sac cells promote in vitro proliferation of bone marrow high proliferative potential colony-forming cells.

To examine the influence of the hematopoietic microenvironment on hematopoietic cell proliferation and differentiation during the yolk sac phase of hematopoiesis, we have recently established cell lines from embryonic yolk sac visceral endoderm (YSE) and mesoderm (YSM). In the present experiments, we compared in vitro growth of adult murine bone marrow high proliferative potential colony-forming cells (HPP-CFC) in coculture with YSE- and YSM-derived or adult bone marrow stromal cell lines. Whereas both yolk sac-derived and adult stromal cell lines supported the proliferation of HPP-CFC during coculture, YSE- and YSM-derived cells stimulated a significant increase in total HPP-CFC compared with adult bone marrow stromal cell lines. Conditioned media from both YSE- and YSM-derived cell lines also stimulated the growth of HPP-CFC in vitro, but only in combination with exogenous recombinant hematopoietic growth factors. Although multiple hematopoietic growth factor mRNAs were detected in the yolk sac-derived cells by polymerase chain reaction, only macrophage colony-stimulating factor (M-CSF) activity was detected in conditioned media using an enzyme-linked immunosorbent assay. A neutralizing polyclonal antibody against M-CSF did not diminish the YSE- or YSM-derived cell line conditioned media promotion of HPP-CFC colony formation. These results suggest that murine yolk sac-derived cell lines produce a novel soluble factor(s) that recruits primitive bone marrow hematopoietic cells to grow in vitro in response to a combination of hematopoietic growth factors.

Animals↗

PubMed1995

Interleukin-11 stimulates multilineage progenitors, but not stem cells, in murine and human long-term marrow cultures.

Interleukin-11 (IL-11) is a bone marrow microenvironment-derived growth factor with pleiotropic effects on a variety of hematopoietic cells. To more accurately assess the effects of IL-11 on stem and progenitor compartments within the hematopoietic microenvironment (HM), we added recombinant human (rh) IL-11 to human and murine long-term bone marrow cultures (LTMC) and analyzed primitive (high proliferative potential-colony forming cells [HPP-CFC], long-term culture-initiating cells [LTC-IC], and long-term reconstituting stem cells) and progenitor (day 12 colony forming unit-spleen [CFU-S12], colony forming unit-megakaryocyte [CFU-Mk] and colony forming unit-granulocyte/macrophage [CFU-GM]) compartments throughout the duration of the cultures. rhIL-11 (100 ng/mL) added twice weekly resulted in significantly increased nonadherent (NA) cellularity, CFU-GM, and CFU-Mk production in human LTMC. Addition of rhIL-11 to murine LTMC was associated with a 5- to 40-fold increase in CFU-GM and a four- to 20-fold increase in day 12 CFU-S in NA cells. However, IL-11 had no significant effect on total HPP-CFC concentration and decreased the size of the more primitive stem/progenitor compartment as evidenced by both decreased LTC-IC frequency in human LTMC and decreased frequency of long-term reconstituting stem cells in murine LTMC. These data suggest that IL-11 may increase commitment of stem cells into a multipotential progenitor compartment.

Animals↗

PubMed1995

Retrovirus-mediated expression of a DNA repair protein in bone marrow protects hematopoietic cells from nitrosourea-induced toxicity in vitro and in vivo.

Severe and delayed myelosuppression is a major side effect encountered with the clinical use of nitrosourea-type chemotherapeutic drugs. The DNA repair protein O6-methylguanine DNA methyltransferase (MGMT) has been shown to repair nitrosourea-induced DNA damage. We therefore investigated the effect of expressing MGMT in hematopoietic cells (via retrovirus-mediated gene transfer) on nitrosourea-induced toxicity. A retroviral vector (N2/ZipPGK-MGMT) expressing the human MGMT cDNA from the phosphoglycerate kinase promoter was constructed. Infection of murine bone marrow with the N2/ZipPGK-MGMT retrovirus significantly increased the survival of murine bone marrow-committed progenitor cells following in vitro exposure to N-N'-bis(2-chloroethyl)-N-nitrosourea (BCNU, carmustine). MGMT gene transfer also protected murine hematopoietic cells in vivo in a murine model of BCNU-induced myelosuppression. The infusion of 4-6 x 10(6) N2/ZipPGK-MGMT-transduced bone marrow cells into mice every 2 weeks significantly increased peripheral leukocyte counts, platelet counts, and hematocrits compared to infusions of mock-infected bone marrow cells. In addition, bone marrow-committed progenitor cells from some recipient animals demonstrated increased resistance to BCNU in vitro when analyzed 2.5 months after initial treatment. The integration of the N2/ZipPGK-MGMT provirus in the spleen DNA from these animals correlated with committed progenitor cell resistance to BCNU. These data suggest that MGMT expression in hematopoietic progenitor and precursor cells protects against nitrosourea-induced toxicity and that gene transfer may prove useful in attempts to reduce nitrosourea-induced myelosuppression in the clinical setting.

3T3 Cells↗

PubMed1995

Membrane-bound Steel factor induces more persistent tyrosine kinase activation and longer life span of c-kit gene-encoded protein than its soluble form.

Alternative splicing of exon 6 results in the production of two isoforms of Steel factor (SLF): the membrane-bound and soluble forms. To investigate differences in the kinetics of c-kit tyrosine kinase activated by these two isoforms, we used a stromal cell line (SI/SI4) established from SI/SI homozygous murine embryo fetal liver and its stable transfectants containing either hSCF248 cDNA (including exon 6; secreted form) or hSCF220 cDNA (lacking exon 6; membrane-bound form) as the source of each isoform. Interaction of factor dependent myeloid cell line MO7e with stromal cells producing either isoform resulted in activated c-kit tyrosine kinase and induction of the same series of tyrosine phosphorylated cellular proteins in MO7e cells. However, SI4-h220 (membrane-bound form) induced more persistent activation of c-kit kinase than SI4-h248 (soluble form) did. Flow cytometric analysis and pulse-chase studies using [35S]methionine showed that SI4-h248 induced rapid downmodulation of cell-surface c-kit expression and its protein degradation in MO7e cells, whereas SI4-h220 induced more prolonged life span of c-kit protein. Addition of soluble recombinant human SLF to SI4-h220 cultures enhanced reduction of cell-surface c-kit expression and its protein degradation. Because the kinetics of c-kit inactivation strikingly fits with the protein degradation rates of c-kit under the conditions described above, rapid proteolysis of c-kit protein induced by soluble SLF stimulation may function as a "turn-off switch" for activated c-kit kinase.

Alternative Splicing↗

PubMed1995

Analysis of Ca2+ and Sr2+ activation characteristics in skinned muscle fibre preparations with different proportions of myofibrillar isoforms.

To understand how the coexistence of fast and slow contractile and regulatory systems within single skeletal muscle fibres might affect contractile behaviour, fibre segments from the fast-twitch extensor digitorum longus and predominantly slow-twitch soleus muscle of the adult rat were tied together, either in parallel or in series, and then activated in Ca(2+)- and Sr(2+)-buffered solutions. Experimental force-pCa and force-pSr relations were compared with theoretical force-pCa and force-pSr curves predicted by a model for composite fibres, which accounted for the coexistence of fast and slow myosin within the contractile unit and enabled an estimate to be made of the relative contribution of fast- and slow-twitch elements within the tied-fibre combinations. The contractile behaviour of a fast-twitch and a slow-twitch muscle fibre tied either in series or in parallel, were compared with the force-pCa and force-pSr data predicted from the composite fibre model. Interestingly, the resultant force-pCa(-pSr) curves of the parallel-tied fibre combinations were well fitted with those predicted by the composite model. However, the experimental force-pCa(-pSr) curves of the series-tied fibres were not well fitted by a composite curve based on the known proportion of fast- and slow-twitch fibre components. A total force-length diagram was devised to take into account changes in the length of the fibre segments tied in series during activation, as well as possible differences in fibre diameter. Using this diagram it was possible to explain accurately the Ca2+ and Sr2+ activation curves of known fast- and slow-twitch segments tied in series. The results from this study are important for the interpretation of contractile data obtained from single muscle fibres exhibiting mixed fast- and slow-twitch contractile characteristics. Such muscle fibres have previously been identified in animals affected by muscular diseases (e.g. dystrophy), in mammalian extraocular muscles and in animals subjected to long-term exercise training.

Animals↗

PubMed1995

Forms of inhibition in animal and human learning.

Forms of inhibition were identified in human predictive learning that are qualitatively similar to those identified by P.C. Holland (1984) in rats. When P (positive) signaled the outcome and PN (N = negative) signaled the absence of the outcome, participants learned the discrimination, but the negative cue did not suppress responding to a transfer cue. Post-learning reversal training, in which N was followed by the outcome, did not abolish the original discrimination. These 2 results imply a configural form of inhibition. Negative transfer, which indicated a 2nd, elemental form of inhibition, was observed when neither PN nor N were reinforced during the discrimination stage. Under these conditions, negative transfer and the original discrimination were both abolished by individually pairing N with the outcome. Empirical parallels and differences with the animal conditioning literature are discussed.

Adult↗

PubMed1995

Mouse model of X-linked chronic granulomatous disease, an inherited defect in phagocyte superoxide production.

Chronic granulomatous disease (CGD) is a recessive disorder characterized by a defective phagocyte respiratory burst oxidase, life-threatening pyogenic infections and inflammatory granulomas. Gene targeting was used to generate mice with a null allele of the gene involved in X-linked CGD, which encodes the 91 kD subunit of the oxidase cytochrome b. Affected hemizygous male mice lacked phagocyte superoxide production, manifested an increased susceptibility to infection with Staphylococcus aureus and Aspergillus fumigatus and had an altered inflammatory response in thioglycollate peritonitis. This animal model should aid in developing new treatments for CGD and in evaluating the role of phagocyte-derived oxidants in inflammation.

Alleles↗

PubMed1995

Biphasic effect of angiotensin II on intracellular sodium concentration in rat proximal tubules.

Intracellular Na+ concentration ([Na+]i) was determined using ratiometric measurement of the Na(+)-sensitive fluorescent probe, sodium-binding benzofuran isophthalate (SBFI). Angiotensin II (ANG II, 10(-11)-10(-7) M), applied to the basolateral membrane of rat isolated proximal convoluted tubules, induced a rapid and reversible dose-dependent increase in [Na+]i, which was initiated within 300 ms. A maximal response was observed over the range 10(-9)-10(-7) M ANG II, with an average increase in [Na+]i of 7.4 +/- 1.0 mM. At higher concentrations (10(-6)-10(-5) M) ANG II decreased [Na+]i compared with control (14.2 +/- 0.6 mM). The increase in [Na+]i induced by 10(-9) M ANG II was attenuated by inhibiting the Na+/H+ antiporter with clonidine, whereas HOE-694, a specific blocker of the NHE-1 isoform of the Na+/H+ exchanger, had no effect. The increase in [Na+]i induced by 10(-9) M ANG II was enhanced by inhibition of the Na(+)-HCO3- cotransporter with hydrogen-4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, with an average increase in [Na+]i of 17.1 +/- 6.6 mM. The data provide direct, high time resolution measurements of the effects of ANG II on [Na+]i in the proximal tubule and support the proposition that an increase in transepithelial Na+ reabsorption by ANG II involves stimulation of both an Na+/H+ exchanger and the Na(+)-HCO3- cotransporter.

Angiotensin II↗

PubMed1995

Salmeterol xinafoate in children on high dose inhaled steroids.

BACKGROUND: Current UK and international guidelines on asthma management recommend that, in pediatric patients still symptomatic on treatment with high-dose inhaled corticosteroids, consideration should be given to the introduction of regular twice daily long-acting beta 2-agonists. OBJECTIVE: The purpose of this study was to assess the efficacy and safety of inhaled salmeterol xinafoate 50 micrograms bid via the Diskhaler when added to the existing treatment of children with moderate to severe asthma. METHODS: A 12-week multicenter, double-blind, placebo-controlled, parallel group study was conducted at 78 hospital centers throughout the United Kingdom, involving 210 asthmatic children aged between 4 and 16 years of age. Morning peak expiratory flow (PEF), evening PEF, night-time and daytime symptoms and relief medication usage were recorded daily by the patient or parent over a 12-week treatment period. RESULTS: Compared with placebo, the addition of salmeterol xinafoate to existing high dose inhaled corticosteroid treatment significantly improved mean morning PEF expressed as percent predicted (PEF-PP) during the first 4 weeks of treatment (median increase 6.5 percentage points P < .001). This effect persisted throughout the 12-week treatment period (P < .05). Both groups demonstrated an overall improvement in mean morning PEF-PP, 7.5 percentage points for salmeterol xinafoate and 4 percentage points for placebo. The mean evening PEF-PP followed a similar although less pronounced trend which was significant only during the first 4 weeks of treatment (P = .014). Daytime relief medication and recorded symptoms were reduced significantly in both groups. There was a greater improvement in the number of symptom-free days during the first 4 weeks (P < .01) and the last 4 weeks (P < .05) of treatment for salmeterol xinafoate. The overall incidence and nature of minor adverse events was similar in both groups. CONCLUSIONS: This study demonstrates that the addition of salmeterol xinafoate to inhaled corticosteroid therapy in symptomatic asthmatic children significantly improves morning PEF-PP, and reduces their symptoms and use of relief medication.

Administration, Inhalation↗

PubMed1995

Time cues block the CS, but the CS does not block time cues.

Analysis of conditioned defensive freezing in rats revealed that prior pairings of a tone conditioned stimulus (CS) and footshock in Context 1 at Time 1 failed to give that tone CS the power to block conditioning to Context 2 at Time 2. This failure of an excitatory CS to block conditioning of time cues was not reciprocal. When the stimulus roles were reversed, excitatory time cues blocked conditioning to the tone CS. This asymmetry in blocking is best explained by the notion that time cues have special access to the association-formation mechanism.

Animals↗

PubMed1995

Matrix molecule interactions with hematopoietic stem cells.

We have reviewed some aspects of the production, distribution, and organization of fibronectin in the bone marrow ECM and discussed HSC-fibronectin interactions. Many questions remain. Which isoforms of fibronectin are produced in the bone marrow during ontogeny, normal hematopoiesis, and pathophysiologic challenges to the marrow microenvironment? Do HSCs interact with ED-A- and ED-B-containing fibronectin isoforms the same way they interact with plasma fibronectin? Are different fibronectin isoforms selectively expressed in different regions of the bone marrow, in keeping with the spatial organization of HSC and hematopoietic progenitor cell compartments? Addressing these questions may give us a better understanding of the role of fibronectin in HSC localization, proliferation, and differentiation. Considerable information regarding the synthesis and secretion of other ECM molecules by adherent stromal cells has been derived from in vitro analysis of LTBMCs [3]. As with fibronectin, much remains to be learned of the precise distribution and composition of bone marrow ECM molecules during in vivo development; how in vivo bone marrow ECM secretion, turnover, and remodeling are regulated during normal and pathologic hematopoietic states; and the nature and importance of in vivo cellular interactions that occur between PHSC and individual constituents of the bone marrow ECM.

Animals↗

PubMed1994

Increased survival and multilineage hematopoietic protection from delayed and severe myelosuppressive effects of a nitrosourea with recombinant interleukin-11.

The chloroethylnitrosoureas, such as 1,3-bis(2-chloroethyl)-1-nitrosourea, are alkylating agents which are thought to exert antitumor activity by initiating lethal DNA interstrand cross-links. Although nitrosoureas are among the most active agents against childhood and adult gliomas, the utility of this class of agents has been limited by severe and cumulative myelosuppression, which can be fatal. Nitrosourea-induced myelosuppression in humans is delayed and may continue after withdrawal of the agent. We have developed a murine model which mimics the delayed and cumulative myelosuppression seen in humans receiving nitrosoureas. In this model, we demonstrate that interleukin-11, a stromal-derived hematopoietic growth factor with pleiotropic effects in a number of preclinical ablation models, markedly diminishes nitrosourea-induced pancytopenia and leads to a significant reduction in chemotherapy-related mortality. These data suggest that interleukin-11 could allow significant dose intensification in the treatment of tumors which are nitrosourea sensitive.

Animals↗

PubMed1994

Murine yolk sac endoderm- and mesoderm-derived cell lines support in vitro growth and differentiation of hematopoietic cells.

The mechanisms involved in the induction of yolk sac mesoderm into blood islands and the role of visceral endoderm and mesoderm cells in regulating the restricted differentiation and proliferation of hematopoietic cells in the yolk sac remain largely unexplored. To better define the role of murine yolk sac microenvironment cells in supporting hematopoiesis, we established cell lines from day-9.5 gestation murine yolk sac visceral endoderm and mesoderm layers using a recombinant retrovirus vector containing Simian virus 40 large T-antigen cDNA. Obtained immortalized cell lines expressed morphologic and biosynthetic features characteristic of endoderm and mesoderm cells from freshly isolated yolk sacs. Similar to the differentiation of blood island hematopoietic cells in situ, differentiation of hematopoietic progenitor cells in vitro into neutrophils was restricted and macrophage production increased when bone marrow (BM) progenitor cells were cultured in direct contact with immortalized yolk sac cell lines as compared with culture on adult BM stromal cell lines. Yolk sac-derived cell lines also significantly stimulated the proliferation of hematopoietic progenitor cells compared with the adult BM stromal cell lines. Thus, yolk sac endoderm- and mesoderm-derived cells, expressing many features of normal yolk sac cells, alter the growth and differentiation of hematopoietic progenitor cells. These cells will prove useful in examining the cellular interactions between yolk sac endoderm and mesoderm involved in early hematopoietic stem cell proliferation and differentiation.

Animals↗

PubMed1994

Interleukin-11: a multifunctional growth factor derived from the hematopoietic microenvironment.

IL-11 is a unique growth factor derived from cells making up the HM. Although cloned based on IL-6-like bioactivity, IL-11 and IL-6 have distinct biologic profiles (Table 1). IL-11, like many recently cloned growth factors, has pleiotropic effects on hematopoietic cells presumably depending on the cytokine and cellular environment into which it is introduced. However, some general findings are consistent (Table 2). In addition, IL-11 has significant effects, either primary or secondary, on nonhematopoietic cells, including neurons, small intestine crypt progenitor/stem cells, and preadipocytes. The institution of human trials with IL-11 will provide important information on the pharmacologic effects of IL-11 on human hematopoietic cells in the context of frequently used chemotherapy protocols. The physiologic role(s) of IL-11 are unknown but will become clear (at least in the mouse) with gene targeting experiments underway in several laboratories.

Acute-Phase Proteins↗

PubMed1994

Contractile properties and temperature sensitivity of the extraocular muscles, the levator and superior rectus, of the rabbit.

1. Contractile and fatigue-resistance characteristics, temperature sensitivity (10-37 degrees C) of contraction, and histochemical fibre types were determined for two of the extraocular muscles, the superior rectus and levator palpebrae superioris (levator), of the rabbit. 2. The levator displayed similar contractile characteristics (time to peak, half-relaxation time of twitch response, and twitch-tetanus force ratio) to mammalian fast-twitch limb muscle at room temperature (20 degrees C). However, normalized twitch and tetanic force levels were significantly less than those found in limb muscle. The superior rectus displayed the characteristics of even faster contraction than the levator at 20 degrees C, but generated lower maximum force levels than the levator. 3. The twitch response of the superior rectus showed a biphasic relaxation phase. This response was not due to non-twitch (tonic) fibres present in the superior rectus as it was unaffected by propranolol application during muscle stimulation. 4. The superior rectus and levator displayed significantly less fatigue in the tetanic force response than fast-twitch limb muscles did in response to a fatiguing electrical stimulation protocol. The levator was significantly more fatigue resistant than the superior rectus. 5. The force responses of both extraocular muscles displayed a similar dependence on temperature (10-37 degrees C) to limb skeletal muscles. 6. The superior rectus and levator exhibited a high proportion of fast-twitch muscle fibres (type II) as shown by myosin ATPase staining. Succinate dehydrogenase activity indicated that these muscles showed a high oxidative capacity, with a staining intensity typical of type I or type II A fibres of limb muscles. 7. The results emphasize the morphological and functional complexity of mammalian extraocular muscles. The combination of very fast contractile properties with high oxidative capacity make these muscles well suited to their role in eye/eyelid movement.

Animals↗

PubMed1994

Contractile properties of single skinned fibres from the extraocular muscles, the levator and superior rectus, of the rabbit.

1. The superior rectus and levator palpebrae superioris (levator), two of the extraocular muscles, were dissected from the rabbit and stored in a glycerol-based solution at -20 degrees C in order to prepare single, skinned fibres. 2. The Ca(2+)- and Sr(2+)-activated isometric contractile properties were determined for individual extraocular muscle fibres. Fibres were separated into discrete groups or fibre types on the basis of their physiological characteristics. The superior rectus and levator muscles were both found to consist of fibres which exhibited similar contractile characteristics to fast- and slow-twitch fibres from other mammalian muscle, including type I, type IIA and type IIB fibres. 3. As well as the existence of the normal, classical fibre types in extraocular muscle there were also a large number of fibres from both muscles which exhibited mixed fast- and slow-twitch contractile characteristics within the single contracting unit. Of the fibres sampled, the mixed fibres comprised the second largest population (7/19, 37%) in the levator and the largest fibre population in the superior rectus (11/31, 35.5%). These results are consistent with histochemical and immunohistochemical reports in the literature which suggest the co-existence of fast and slow myosin along the length of the extraocular muscle fibres. 4. Extraocular muscle fibres exhibited lower absolute maximum forces compared with other mammalian limb muscle fibres. However, when corrected for fibre cross-sectional area, the maximum tension development was within the normal range for mammalian limb muscle fibres, except for one group (type IIA) of fast-twitch fibres which exhibited significantly lower maximal tension. 5. The existence of a large proportion of fibres with composite fast- and slow-twitch characteristics highlights the functional and morphological complexity of these muscles. It is postulated that the functional significance of these mixed fibres may be to provide or enhance the resolution for subtle, precise movements of the eye and eyelid.

Animals↗

PubMed1994

Identification and mutation of primary and secondary proteolytic cleavage sites in murine stem cell factor cDNA yields biologically active, cell-associated protein.

Phenotypic abnormalities of melanocytes, germ cells, and hematopoietic cells of Steel mice demonstrate the critical role of stem cell factor (SCF) in development. Production of SCF in the hematopoietic microenvironment as either a membrane-associated or soluble factor leads to pleiotropic effects on hematopoietic stem and progenitor cells and significant effects on the production of erythroid cells. Although the production of these two forms of SCF is highly regulated, the physiologic role(s) of membrane-associated and soluble SCF remain unclear. We have demonstrated that the generation of soluble murine SCF by murine stromal cells derived from the fetal hematopoietic microenvironment is dependent on two distinct proteolytic cleavage sites. The primary site in exon 6 is preferentially utilized in these cells. The secondary site located in exon 7 is utilized only in the absence of the primary site. Proteolytic processing at this secondary site appears to be species-specific, since the human protein sequence diverges at this site, and protein expressed from the human cDNA encoding this site in murine stromal cells remains largely membrane-associated. Site-directed mutagenesis of the murine SCF cDNA encoding both proteolytic cleavage sites leads to the generation of membrane-associated and biologically active SCF on murine stromal cells. These results suggest that the regulation of processing of the secondary proteolytic cleavage site could play a critical role in the function of membrane-associated SCF protein.

Amino Acid Sequence↗

PubMed1994

A bone marrow stromal-derived growth factor, interleukin-11, stimulates recovery of small intestinal mucosal cells after cytoablative therapy.

The proliferation of epithelial cells lining the small intestinal mucosa may be regulated by microenvironmental signals leading to differentiation of precursor cells in the small intestinal crypts. Proliferation of hematopoietic cells within the hematopoietic microenvironment is known to be regulated by a growing number of glycoprotein growth factors in a hierarchial fashion. We studied the effects of administration of the microenvironment-derived hematopoietic growth factor interleukin-11 (IL-11) on mice given combination radiation/chemotherapy. Treatment of such mice with IL-11 led to significantly increased survival and evidence of rapid recovery of the small intestinal mucosa, which is severely damaged by these cytoxic agents. This recovery was associated with an increase in the mitotic index of crypt cells and an increased frequency of staining of these cells with a monoclonal antibody to proliferating cell nuclear antigen, a member of the cyclin family of nuclear antigens.

Animals↗