Search

The main objective of this study is the serial evaluation of clinical and laboratory changes in cats with experimentally-induced acute pancreatitis (AP). A total of 16 clinically healthy young adult DSH cats were included in the study. Acute pancreatitis was induced in 10 (AP group) by infusing oleic acid into the pancreatic duct, while the other 6 cats served as sham-operated controls (C group). Depression or lethargy, anorexia, abdominal pain, and a palpable mass in the anterior abdomen were the main clinical findings in cats of the AP group. Laboratory data revealed non-regenerative anaemia, mild hypoalbuminaemia, transient hyperglycaemia, and hypertriglyceridaemia. Serum lipase activity, feline trypsin-like immunoreactivity (fTLI) concentration, and feline pancreatic lipase immunoreactivity (fPLI) concentration increased significantly within the first 24 hours after the infusion of oleic acid, with time-dependent positive correlation. In both group of cats urine amylase-creatinine ratio and amylase-creatinine clearance ratio were increased. In cats of the AP group, abdominal ultrasonography revealed a hypoechogenic and enlarged pancreas, along with peripancreatic fluid accumulation. Peritoneal effusion was consistent with a sterile exudate, with higher lipase but lower amylase activities when compared to their serum counterparts in all AP group cats. The induction of AP was confirmed by pancreatic histopathology. AP is characterized by vague symptomatology. Increases in serum fTLI and fPLI concentrations occur early in cats with pancreatitis. In general the two analytes run a parallel course, but fPLI may be increased longer than fTLI. The measurement of lipase activity in the peritoneal fluid could be considered diagnostic.

Background: Rac1 GTPase, a member of the Ras‐related Rho GTPase family, is the major Rac isoform present in platelets and has been shown to be involved in cell actin cytoskeleton reorganization and adhesion. Agonists that induce platelet secretion and aggregation also activate Rac1 GTPase, raising the possibility that Rac1 GTPase may be involved in regulation of platelet function. Objectives: To rigorously define the role of Rac1 in platelet regulation. Methods: We have used a dual approach of gene targeting in mice and pharmacologic inhibition of Rac1 by NSC23766, a rationally designed specific small molecule inhibitor, to study the role of Rac1 in platelet function. Results: Platelets from mice as well as human platelets treated with NSC23766 exhibited a significant decrease in: (i) active Rac1 species and phosphorylation of the Rac effector, p21‐activated kinase; (ii) expression of P‐selectin and secretion of adenosine triphosphate induced by thrombin or U46619; and (iii) aggregation induced by adenosine 5′‐diphosphate, collagen, thrombin and U46619, a stable analog of thromboxane A2. NSC23766 did not alter the cAMP or cGMP levels in platelets. Consistent with the requirement of Rac1 for normal platelet function, the bleeding times in Rac1–/–mice or mice given NSC23766 were significantly prolonged. Conclusions: Our data show that deficiency or inhibition of Rac1 GTPase blocks platelet secretion. The inhibition of secretion, at least in part, is responsible for diminished platelet aggregation and prolonged bleeding times observed in Rac1knockout or Rac1 inhibitor‐treated mice.

Domestic cats with small intestinal disease may develop cobalamin deficiency because of reduced small intestinal uptake of this vitamin. This study assessed the impact of cobalamin deficiency on biochemical and clinical findings in cats with intestinal disease. Nineteen pet cats, all with severe hypocobalaminemia (≤100 ng/L) and histories of gastrointestinal signs, were studied. Cats received cobalamin, 250 μg SC once weekly, for 4 weeks. Biochemical indices of cobalamin availability (eg, serum methylmalonic acid, homocysteine, and cysteine concentrations), serum feline trypsinlike immunoreactivity (fTLI) and serum folate concentrations, and clinical findings were recorded at the start of the study and after 4 weeks of cobalamin therapy. Serum methylmalonic acid (MMA) concentrations (median; range) decreased after cobalamin supplementation (5,373.0; 708.5–29,329.0 versus 423.5; 214.0–7,219.0 nmol/L, P<.0001). Serum homocysteine concentrations were not significantly altered (mean ± SD 8.2 ± 2.9 versus 10.3 ± 4.5 μmol/L, P= .1198), whereas cysteine concentrations increased significantly (122.3 ± 38.8 versus 191.5 ± 29.4 μmol/L, P< .0001). Mean body weight increased significantly after cobalamin therapy (3.8 ± 1.1 versus 4.1 ± 1 kg, P< .01), and the average body weight gain was 8.2%. Significant linear relationships were observed between alterations in serum MMA and fTLI concentrations and the percentage body weight change (P< .05 for both, Pearson r2= 0.26 and 0.245, respectively). Mean serum folate concentration decreased significantly (mean ± SD 19 ± 5 μg/L versus 15.4 ± 6.2 μg/L, P< .001). Reduced vomiting and diarrhea were observed in 7 of 9 and 5 of 13 cats, respectively. These results suggest that cobalamin supplementation in cats with small intestinal disease and severe hypocobalaminemia is associated with normalization of biochemical test results and improvements in clinical findings in most affected cats.

Pressure ulcer incidence and sacral skin temperature (T"s) were measured in hospitalized neurologically impaired Thai patients (n = 17) positioned supine and then laterally. Pressure ulcer incidence within 2 weeks of admission was 47%. Regardless of reclining position, mean sacral T"s in subjects who developed a pressure ulcer was higher (p < .01) than those who did not develop an ulcer. The data suggest that T"s may increase at least 1.2 ^oC 24-96 hr before sacral pressure ulcer development. Sacral T"s may be an objective predictor of sacral pressure ulcer development in hospitalized neurologically impaired Thai patients.

Serum feline trypsinogen‐like immunoreactivity (fTLI) concentrations and abdominal ultrasound have facilitated the noninvasive diagnosis of pancreatitis in cats, but low sensitivities (33% and 20–35%, respectively) have been reported. A radioimmunoassay has been validated to measure feline pancreatic lipase immunoreactivity (fPLI), but the assay's sensitivity and specificity have not been established. In human beings, the sensitivity of computed tomography (CT) is high (75–90%), but in a study of 10 cats, only 2 had CT changes suggestive of pancreatitis. We prospectively evaluated these diagnostic tests in cats with and without pancreatitis. In all cats, serum was obtained for fTLI and fPLI concentrations, and pancreatic ultrasound images and biopsies were acquired. Serum fPLI concentrations (P<.0001) and ultrasound findings (P= .0073) were significantly different between healthy cats and cats with pancreatitis. Serum fTLI concentrations (P= .15) and CT measurements (P= .18) were not significantly different between the groups. The sensitivity of fTLI in cats with moderate to severe pancreatitis was 80%, and the specificity in healthy cats was 75%. Feline PLI concentrations were both sensitive in cats with moderate to severe pancreatitis (100%) and specific in the healthy cats (100%). Abdominal ultrasound was both sensitive in cats with moderate to severe pancreatitis (80%) and specific in healthy cats (88%). The high sensitivities of fPLI and abdominal ultrasound suggest that these tests should play an important role in the noninvasive diagnosis of feline pancreatitis. As suggested by a previous study, pancreatic CT is not a useful diagnostic test for feline pancreatitis.

Controversy exists over the diagnosis of idiopathic small intestinal bacterial overgrowth (SIBO) in dogs and some clinicians use the term antibiotic‐responsive diarrhea (ARD) in preference. However, whether such terms are interchangeable is not clear. To examine the relationship between duodenal bacterial numbers and a clinical response to antibiotics, SIBO and ARD were defined by nonoverlapping criteria. Quantitative duodenal juice bacteriology and indirect serum biochemical tests were used to assess small intestinal bacterial populations in 30 dogs with gastrointestinal disorders, including 9 with ARD. Serum total unconjugated bile acid (TUBA) concentrations were measured in all dogs, serum folate and cobalamin concentrations were measured in 29 of 30 dogs, and quantitative culture of duodenal juice was performed in 22 of 30 dogs. Serum TUBA concentrations also were measured in samples from 38 control dogs. Twenty of 22 affected (clinical) dogs in which quantitative bacteriology was performed were classified as having SIBO (>105colony‐forming units of total bacteria per milliliter of duodenal juice), but bacterial numbers did not differ significantly between dogs with ARD and dogs with other disorders. Increased folate (19/29), decreased cobalamin (16/ 29), or a combination (9/29) were common, but increased TUBA concentrations were documented in only 5 of 30 clinical dogs. Again, no significant differences were observed between dogs with ARD and those with other disorders, and a similar proportion (5/38) of controls had abnormally high TUBA concentrations. Finally, no significant differences were noted when duodenal bacteriology and TUBA concentrations were assessed before and during antibiotic therapy. These results question the utility of quantitative duodenal juice bacteriology and indirect biochemical marker tests for SIBO in the investigation of canine gastrointestinal disorders.

Reductions in cardiac sarcoplasmic reticulum calcium-ATPase (Serca2a) levels are thought to underlie the prolonged calcium (Ca2+) transients and consequent reduced contractile performance seen in human cardiac hypertrophy and heart failure. In freshly isolated cardiac myocytes from rats with monocrotaline-induced right ventricular hypertrophy we found reduced sarcoplasmic reticulum Serca2a expression and prolonged Ca2+transients, characteristic of hypertrophic cardiac disease.

We propose a simple method of assessing the chemical consequences of mixing at interfaces between cool neutral gas with ionized material. It is shown that the temperature within the interface has a significant effect; at low temperatures (∼ 10 K) the chemistry is significantly suppressed, but for temperatures above 100 K the chemistry initiated by proton/O-atom charge exchange gives rise to a rich and abundant chemistry. The chemical state of the cool neutral gas has an important consequence for the interface. A mainly molecular cool gas can give rise to a high ionization state in the interface; this high ionization state appears stable against parameter variations. C2H is one of the most easily observed molecules having a high abundance in some interfaces.

We report that chloromethyl-X-rosamine (MitoTracker Red), a mitochondrion-selective fluorescent probe, has a strong photosensitising action. Photoirradiation of intact cells loaded with chloromethyl-X-rosamine induces depolarisation of the inner mitochondrial membrane and swelling of mitochondria, subsequently resulting in apoptosis. We have studied human osteosarcoma 143B TK-(rho+) cells and the derived (rho)0 206 cell line devoid of mitochondrial DNA. Colony formation tests revealed that chloromethyl-X-rosamine itself has no toxicity to either cell line in the concentration range 100–250 nM (unless photoirradiated). Chloromethyl-X-rosamine has potent phototoxicity such that almost quantitative cell killing was achieved at light doses of >2 J/cm2. These photodamaged cells initially showed swollen degenerative mitochondria and, later, uptake of propidium iodide in their apoptotic nuclei was observed. When cells were loaded with chloromethyl-X-rosamine (100 nM) and imaged by laser scanning confocal microscopy, photoirradiation by the laser beam under routine scanning conditions was sufficient to induce mitochondrial damage in both cell lines. This was evidenced by a rapid decrease of fluorescence intensity of co-loaded rhodamine 123 (indicative of mitochondrial depolarisation). Globular swelling of mitochondria took place within 15 minutes, imaged by the residual fluorescence of chloromethyl-X-rosamine itself, which also markedly decreased in intensity after imaging. Mitochondrial membrane depolarisation of cells loaded with chloromethyl-X-rosamine after photoirradiation using a measured dose of visible light was independently confirmed in 143B TK- and (rho)0 206 cells, by the significant decrease in uptake into cells of [3H]methyltriphenylphosphonium ions. Photoactivation of chloromethyl-X-rosamine in 143B TK-(rho+) cells, whose mitochondria had previously been loaded with calcein, caused rapid release of the mitochondrially entrapped calcein into the cytosol and nucleus. This major change in permeability of the mitochondrial inner membrane could not be prevented by cyclosporin A. Immunohistochemical study of cytochrome c revealed its diffuse redistribution into the cytoplasm in chloromethyl-X-rosamine-loaded cells after irradiation, as opposed to its specific mitochondrial localisation in non-irradiated cells. As a photosensitiser specifically targeted to mitochondria, and also a reporter of membrane potential and morphology, chloromethyl-X-rosamine may provide versatile new applications in studies of mitochondrial roles in cell death.

Abstract: A new method is introduced designed to facilitate study of matrix elements in complex β-decay through the measurement of asymmetric β-emission from a low-temperature polarised source. The method involves coincident detection of the β-emission with subsequent γ-transitions to tag the β-spectrum component. The method is demonstrated by application to the decay of 140La.

This study investigates whether endotoxin-induced contractile dysfunction is associated with a defect in the modulation of calcium homeostasis and the potential mechanisms involved. Treatment of rats in vivo with endotoxin significantly decreased the magnitude of contractile transients in electrically stimulated left ventricular papillary muscle isolated after an equilibration period of 6 hours. Although no significant difference was found in the peak intracellular calcium concentration ([Ca2+]i) between the endotoxin-treated and control groups, resting [Ca2+]iwas significantly elevated in the endotoxin-treated group, producing a smaller Ca2+transient (basal-peak difference) in this group. Pretreatment of rats with dexamethasone prevented the endotoxin-induced decrease in peak tension and inhibited the elevation in resting [Ca2+]i, with a resultant maintenance of Ca2+transient magnitude. Similar observations were made during stimulation of the muscles by the β-adrenoceptor agonist, isoprenaline. These results show that endotoxin-induced reduction of cardiac contractile performance is mediated, at least in part, by elevating resting [Ca2+]i, and a glucocorticoid protected from these negative effects. While endotoxin reduces the magnitude of the Ca2+transient it does not alter peak [Ca2+]iavailability. Further investigation is required to determine whether endotoxin decreases contractile performance by reducing the sensitivity of cardiac myofilaments to calcium.

There are two ways that water ice can form in the interstellar medium: H2O molecules can form in the gas phase and then freeze out onto dust grain surfaces, or O and OH can be converted at the surfaces of grains to form H2O, which is then retained. Bergin et al.(1998) have recently shown that shocks passing through interstellar clouds sufficiently frequently can make the first method effective. However, we present results from a similar chemical model which indicate that this requires significant optical shielding because of the high ionization fraction in regions exposedto a high UV flux. We deduce, therefore, that grain surface reactions probably represent the main source of H2O ice on lines of sight with visual extinction up to about 6 magnitudes to an embedded source or 12 magnitudes to a background object.

Recombinant retroviral vectors have been used to transfer a variety of genetic sequences into hematopoietic stem cells. Although transfer and expression of foreign genetic sequences into reconstituting stem cells is one approach to somatic gene therapy, few studies have shown long lasting phenotypic changes in recipient mice in vivo. In this study, we show successful transfer of a methotrexate-resistant cDNA (DHFRr) into reconstituting hematopoietic stem cells using a retroviral vector, FrDHFR', in which the DHFR cDNA is expressed off a hybrid Friend/Moloney long term repeat. Both primary and secondary recipients transplanted with bone marrow cells infected with this recombinant retrovirus show improved survival and protection from methotrexate-induced marrow toxicity when compared with control animals. These data suggest that retroviral-mediated gene transfer of DHFRrcDNA leads to a stable change in the phenotype of hematopoietic stem cells and progeny derived from those cells in vivo after bone marrow transplantation. Gene transfer using recombinant retroviral vectors seems to be one rational approach to establishing chemotherapy-resistant bone marrow cells.

Retroviral-mediated gene transfer of human adenosine deaminase (hADA) provides a model system for the development of somatic gene therapy as a therapy for diseases of bone marrow-derived cells. We have previously demonstrated that hADA can be observed in all hematopoietic lineages in a minority of mice transplanted with bone marrow cells infected with a simplified retroviral vector, ZipPGK-ADA. Here we report a majority of mice (six of eight) demonstrate expression of hADA in the peripheral blood at least 6 months after transplantation with bone marrow infected with this simplified retroviral vector, which contains no selectable marker. The failure to express hADA in two of eight mice was associated with the absence of the recombinant retroviral provirus in DNA prepared from bone marrow cells of these mice apparently due to failure to efficiently infect the reconstituting hematopoietic stem cell. In an effort to preselect bone marrow stem cells containing proviral integrations, we incorporated the selectable marker neo phosphotransferase (NEO) into a retroviral vector encoding hADA, N2/ZipPGK-ADATKNEO, and used G418 selection of infected bone marrow cells before transplantation. In contrast to the simplified retroviral vector, hADA expression in these recipients was short lived (< 8 weeks), despite the continued presence of intact provirus in DNA prepared from bone marrow of these mice. To determine whether the preselection of bone marrow using G418 was responsible for the lack of sustained hADA expression, we repeated the infection with the N2/ZipPGK-ADATKNEO vector but omitted the G418 selection step. Again, the majority of recipient mice failed to express hADA long term, although the continued presence of provirus in DNA prepared from peripheral blood cell mononuclear cells was clearly demonstrated. Finally, we demonstrate clonal fluctuation of infected stem cells, and observe a temporal correlation between cessation of expression of hADA and the emergence of a dominant stem cell clone between 14 and 20 weeks posttransplantation in one recipient. These data suggest that inclusion of a second transcriptional unit that includes neo phosphotransferase sequences in this simplified vector is associated with decreased expression of the nonselectable ADA sequences.

We have investigated the dependences of the fractional abundances of a number of species on the selective depletions of elemental carbon, nitrogen, oxygen and sulphur, as well as metals. Of the most commonly observed species, HCO+is the one having the fractional abundance that is least sensitive to the selective elemental depletions and for this reason is a particularly appropriate species to study in efforts to diagnose collapse dynamics in star formation from the analysis of infall signatures in spectral line profiles. We suggest that a combination of molecules could be the focus of observations aimed at the investigation of collapse dynamics. Comparisons of the profiles of spectral features of these species would also give insight into the behaviours of the selective depletions of the different elements in the infalling gas.

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. rhlL-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 rhlL-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.

This paper reviews the application of hybrid open-microstrip technology to millimeter-wave (30- 110 GHz) military subsystems. The relevant circuit techniques, active and passive devices, and construction methods are considered. Examples of components/subsystems are discussed.

Despite the fact that over the past 20 years the importance of information skills tuition has been increasingly recognised, many students are still reaching tertiary education with few, if any, of the skills required to consistently find and use information. Much work has been done to compensate for this lack of skills in Higher Education, but in Further Education (FE), until recently, little systematic effort has been put into solving the problem. At the same time, FE colleges are undergoing major changes in adopting a modular approach, and courses are becoming far more student-centred with an increasing emphasis on independent learning and project work. However, library instruction in FE tends to be very basic and few students receive any lessons in information handling. This paper portrays the results of a survey into the information skills abilities of students in Scottish colleges of Further Education. The focus is on students studying for the National Certificate in Business Studies because of their above average library use. Results suggest that students have particular problems related to the cognitive skills required to analyse project requirements, even before they enter the library. These problems are quantified, and the paper concludes that the information skills abilities of students in Scottish Further Education colleges are well below desired standards if students are to be expected to produce independent project work.

Interleukin-11 (IL-11) is a multifunctional cytokine, derived from bone marrow stromal cells, that stimulates proliferation of stem/progenitor precursor cells in the small intestinal crypts and accelerates recovery of intestinal mucosa after cytoablative therapy. This study evaluates whether IL-11 can improve the function and structure of the small intestine and enhance adaptation in an experimental model of short bowel syndrome. After 90% small bowel resection, 32 Sprague-Dawley rats were divided randomly into eight experimental groups of four animals each. Four groups were treated with IL-11 (125 μg/kg twice daily, subcutaneously), and the four control groups were treated with a similar volume (0.1%) of bovine serum albumin (BSA). The animals were weighed daily and were killed on day 2, 4, 6, or 8; remnant small bowel was evaluated for villus height and crypt cell mitosis. The body weight of the animals that received IL-11 was significantly greater at the beginning of postoperative day 4 in comparison to that of the BSA groups (P< .01 during days 5 to 7). The rats that had IL-11 also had significantly greater villus height and crypt cell mitotic rates (P< .05). These observations suggest that IL-11 has a trophic effect on the small bowel during the adaptive phase that follows massive bowel resection and may be useful in the treatment of short bowel syndrome.

A recombinant retrovirus (DHFR*-SVADA) in which human adenosine deaminase (ADA) cDNA is transcribed from an internal SV40 promoter was used to infect murine hematopoietic stem and progenitor cells. Human ADA enzyme was not expressed in infected primary murine pluripotent stem cell-derived spleen or progenitor colonies (CFU-GM, CFU-Mix, BFU-E). In contrast, human ADA enzyme activity was readily detected in progenitor colonies derived from immortalized multipotent factor-dependent cells. The level of human enzyme was near endogenous murine enzyme levels and was equivalent in undifferentiated stem cells and differentiated myeloid, erythroid, and mixed colonies. These results indicate that cellular properties other than the stage of differentiation are important in determining the expression of foreign sequences introduced by retrovi-ruses. Cell lines that are immortalized but still capable of induced differentiation may contain factors that abrogate blocks to expression that are manifested in primary hematopoietic stem cells.

We report on operational experience with and experimental performance of the SLD barrel Cherenkov Ring Imaging Detector from the 1992 and 1993 physics runs. The liquid (C/sub 6/F/sub 14/) and gas (C/sub 5/F/sub 12/) radiator recirculation systems have performed well, and the drift gas supply system has operated successfully with TMAE for three years. Cherenkov rings have been observed from both the liquid and gas radiators. The number and angular resolution of Cherenkov photons have been measured, and found to be close to design specifications.<>

Interleukin-11 (IL-11) is a bone marrow (BM) stromal-derived growth factor that has been shown to stimulate murine myeloid and lymphoid cells both in vitro and in vivo and to inhibit adipogenesis in a murine fibroblast cell line. We have studied the effects of IL-11 on highly purified human BM stem and progenitor cells and on human long-term marrow cultures (LTMC). Adipocyte differentiation is an integral component of murine and human LTMC. IL-11 stimulates myeloid growth as a single cytokine when added to highly enriched CD34+, HLA-DR+bone marrow cells. IL-11 stimulated no growth in the more primitive CD34+, HLA-DR−population even in the presence of additional cytokines. IL-11 addition to human LTMC resulted in the expansion of myeloid and mixed, but not erythroid, progenitor populations. IL-11 dramatically increased the adherent cell populations, including both stromal cells and macrophages. Treated cultures also showed marked inhibition of fat accumulation in the adherent cells due in part to a block in the differentiation of preadipocytes to adipocytes, as shown by RNA analysis using adipocyte-specific markers. These data show that IL-11 stimulates a more differentiated, although multipotential, progenitor cell in human BM and that LTMC provide a useful model for studying the effects of this cytokine in the context of the hematopoietic microenvironment.

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 interieukin-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.

The new, fluorescent Ca2+ indicator, fura-2, promises to expand our understanding of the role of subcellular changes in Ca2+ underlying cell function. During an investigation of the role of Ca2+ in the polarization response of human polymorphonuclear leukocytes to formyl-methionyl-leucyl-phenylalanine, we found that fura-2 trapped by cells incubated with the acetoxy-methyl ester of fura-2, F2-AM, yielded measurements of Ca2+ that were depressed at rest and during the response to formyl-methionyl-leucyl-phenylalanine. Fura-2, trapped by the cells, exhibited a spectrum in the presence of saturating Ca2+ that differed from that of fura-2 free acid. We have shown that the cellular fluorescence can be spectrally decomposed into two components: one with Ca2+ sensitivity identical to fully deesterified fura-2, and another which is Ca2+-insensitive. The Ca2+-insensitive component appears to be more fluorescent than F2-AM as well as spectrally different from F2-AM. The insensitive form probably results from incomplete deesterification of F2-AM by the cells. In order to accurately measure Ca2+ in polymorphonuclear leukocytes, it is imperative to check for the presence of Ca2+-insensitive fluorescence. The contribution of Ca2+-insensitive fura-2 fluorescence can be assessed routinely from spectral data obtained by calibration of intracellular fura-2 with known [Ca2+] using ionomycin. The end-of-experiment calibration step not only ensures accurate [Ca2+] measurements in polymorphonuclear leukocytes and in other cell types that display Ca2+-insensitive, contaminating fluorescence but also yields the spectral characteristics of the insensitive species.

An elucidation of the interaction between the bone marrow microenvironment and hematopoietic stem cells is critical to the understanding of the molecular basis of stem cell self renewal and differentiation. This interaction is dependent, at least in part, on direct cell to cell contact or cellular adhesion to extracellular matrix proteins. Long-term bone marrow cultures (LTMC) provide an appropriate microenvironment for maintenance of primitive hematopoietic stem cells and a means of analyzing this stem cell-stromal cell interaction in vitro. Although LTMC have been successfully generated from murine and human bone marrow, only limited success has been reported in a primate system. In addition, few permanent stromal cell lines are available from nonmurine bone marrow. Because the primate has become a useful model for large animal bone marrow transplant studies and, more specifically, retroviral-mediated gene transfer analysis, we have generated immortalized bone marrow stromal cell lines from primate bone marrow using gene transfer of the Simian virus large T (SV40 LT) antigen. At least one stromal cell line has demonstrated the capacity to maintain early hematopoietic cells in long-term cultures for up to 4 weeks as measured by in vitro progenitor assays. Studies were undertaken to characterize the products of extracellular matrix biosynthesis and growth factor synthesis of this cell line, designated PU-34. In contrast to most murine bone marrow-derived stromal cell lines capable of supporting hematopoiesis in vitro that have been examined, the extracellular matrix produced by this primate cell line includes collagen types I, III, and V, and fibronectin rather than collagen type IV and laminin. Growth factor production analyzed through RNA blot analysis, bone marrow cell culture data, and factor-dependent cell line proliferation assays includes interleukin-6 (IL-6), IL-7, granulocyte-macrophage colony-stimulating factor (GM-CSF), G-CSF, M-CSF, leukemia inhibitory factor, and a novel cytokine designated IL-11. This immortalized primate bone marrow stromal cell line may be useful in 'maintaining early progenitor cells for experimental manipulation without the loss of reconstituting capacity and as a potential source of novel hematopoietic growth factors.

We have examined the effects of recombinant human interleukin-11 (rhlL-11) on the recovery of peripheral blood cell counts and proliferation of progenitors and hematopoietic stem cells (day 12 colony-forming units-spleen—CFU-S12) in vivo using a mouse bone marrow (BM) and spleen cell transplantation model. Recovery of leukocytes was accelerated in animals receiving daily administration of rhlL-11 (100 µg/kg/d) and reached normal levels by day 14 posttransplantation. This increased total leukocyte count reflected mainly an increase in neutrophils. Neutropenia (absolute neutrophil count [ANC] < 1,500) was present in control transplant mice for 14 to 15 days, while in the rhlL-11-treated group, neutrophils recovered to normal by days 8 to 10 and continued to increase until day 19. Animals treated with rhlL-11 had only 1 day with ANC demonstrated <500. Correspondingly, rhlL-11 treatment increased granulocyte-macrophage progenitors (CFU-GM) derived from both spleen and BM cells. Higher doses of IL-11 increased CFU-GM nearly threefold and CFU-Mix fourfold to fivefold, while increasing burst-forming units-erythroid to a lesser degree. BM and spleen cellularity were both increased in IL-11-treated mice, but no increase in CFU-S12was noted. In addition, in vivo daily administration of IL-11 increased peripheral platelet counts by threefold over control transplant mice at day 10 posttransplantation during the postirradiation platelet nadir. Further treatment led to platelet counts higher than normal 18 days posttransplantation when control animals had just attained normal platelet counts. IL-11 can accelerate the recovery of the peripheral blood leukocytes, mainly neutrophils, and platelets in transplant mice, effects that may be clinically useful in future applications for BM transplantation and chemotherapy-related cytopenias.

A description is given of the detector system which forms the large angle spectrometer and vertex detector of the EMC spectrometer. The apparatus is used in the NA9 experiment which studies the complete hadronic final state from the interaction of high energy muons.

The construction of the Cherenkov Ring Imaging Detector (CRID) for the SLD (Stanford Linear Collider Large Detector) experiment at the SLAC (Stanford Linear Accelerator Center) Linear Collider and the testing of its components is reported. Results are included from testing the drift boxes, liquid radiator trays, and mirrors for the barrel CRID. The development of the support systems essential for the operation of the CRID, namely, gas and liquid recirculation systems and monitoring, are discussed.<>

The National Aeronautics and Space Administration (NASA) Psyche mission will visit the 226‐km diameter main belt asteroid (16) Psyche, our first opportunity to visit a metal‐rich object at close range. The unique and poorly understood nature of Psyche offers a challenge to the mission as we have little understanding of the surface morphology and composition. It is commonly accepted that the main evolutionary process for asteroid surfaces is impact cratering. While a considerable body of literature is available on collisions on rocky/icy objects, less work is available for metallic targets with compositions relevant to Psyche. Here we present a suite of impact experiments performed at the NASA Ames Vertical Gun Range facility on several types of iron meteorites and foundry‐cast ingots that have similar Fe‐Ni compositions as the iron meteorites. Our experiments were designed to better understand crater formation (e.g., size, depth), over a range of impact conditions, including target temperature and composition. We find that the target strength, as inferred from crater sizes, ranges from 700 to 1,300 MPa. Target temperature has measurable effects on strength, with cooled targets typically 10–20% stronger. Crater morphologies are characterized by sharp, raised rims and deep cavities. Further, we derive broad implications for Psyche's collisional evolution, in light of available low resolution shape models. We find that the number of large craters (>50 km) is particularly diagnostic for the overall bulk strength of Psyche. If confirmed, the number of putative large craters may indicate that Psyche's bulk strength is significantly reduced compared to that of intact iron meteorites. Many iron meteorites are thought to be remnants of the cores of melted asteroids. Some cores may have been exposed by collisions during the earliest days of Solar System history, with a few survivors possibly found today in the main asteroid belt. National Aeronautics and Space Administration (NASA) Psyche mission will be the first spacecraft to visit asteroid (16) Psyche, an object thought to be representative of these metallic asteroids. Impacts onto (16) Psyche in the past may therefore be able to tell us about the history and nature of this body. To this end, we performed high‐speed impact experiments into metallic targets in order to understand how crater formation differs from rocky bodies. These experiments revealed that impact craters into metal targets are deeper and have sharper rims than on their rocky counterparts. These results will be crucial for interpreting both the bulk properties of Psyche's interior and the modification of Psyche's surface when the Psyche mission reaches its target. We quantify morphological properties of cratering in metallic targets.We derive implications for the Psyche mission.

Absorption of ultrafast laser pulses in a III–V semiconductor heterostructure is used to excite a coherent optical phonon population. The dephasing and decay of the phonon wavepacket is directly monitored on a femtosecond timescale. Localised injection of phonons within a 2D electron gas is shown to reveal strong interactions whose strength can be tuned using the laser wavelength. Experiments utilizing this source of high-wavevector acoustic phonons are discussed.

Rho GTPases (Rho, Rac, and Cdc42) act as molecular switches to regulate cellular functions. We have recently generated a mouse deficient in Rac 2, a GTPase which is expressed only in hematopoietic cells (Roberts et al. Immunity, 1999), and shown defects in PMN movement and other actin-based functions in vivoand in vitro. We now demonstrate that Rac 2 is expressed in purified hematopoietic progenitor/stem cells (HSC) (lin−,c−kit+Sca-1+). Analysis of the function of Rac 2−/− HSC shows multiple abnormalities in vitroand in vivo. Adhesion to the extracellular matrix molecule fibronectin via integrin VLA-4 is significantly reduced. Surprisingly, movement of progenitor/stem cells in response to SDF-1 is increased in both modified Boyden chamber assays and in quantitative time-lapsed video imaging. This increased movement is associated with increased size of filopodia, and increased f-actin content, reminiscent of enhanced activity of Cdc42, suggesting cross-talk between GTPase pathways. Importantly, these abnormalities are mirrored by changes in cell movement in vivo. G-CSF-induced mobilization of hematopoietic progenitor cells is enhanced nearly 3-fold in Rac 2−/− mice, while the augmentation of G-CSF mobilization by concurrent administration of antibodies to VLA-4 is reduced, further supporting the role of Rac 2 in VLA-4 function in stem/progenitor cells localization in the bone marrow microenvironment. These data demonstrate that Rac 2 is critical for normal adhesion and movement of HSC, and suggest that deficiency of Rac 2 in these cells may lead to altered activity of other GTPases, particularly Cdc42.

The mechanism of hematopoietic progenitor cell (HPC) migration and mobilization from bone marrow (BM) to peripheral blood (PB) has not yet been clearly defined. Since we have previously reported that absence of the Rho GTPase Rac2 results in defective movement and adhesion of mast cells and neutrophils, we speculated that the Rac2 protein might play a pivotal role in this process. Since previous reports have implicated the integrin VLA4 in HPC mobilization, we measured adhesion of purified HPC (mature cell lineage antigen negative (Lin−) c-kit+Sa-1+ cells isolated from Rac2−/− and wild type (WT) mice) to the VLA4 binding site, CS-1, on the recombinant HPC to this peptide was observed (21±5% in Rac2−/− cells, vs 61±8% in WT cells, P<0.001). In vitroanalysis of directed movement (modified Boyden chambers) by these cells towards stromal cell-derived factor-1 (SDF-1) demonstrated that, in contrast to the response of differentiated hematopoietic cells, migration of Rac2−/− HPC was increased 4-fold, compared with WT cells (12% versus 3% of cells migrated, respectively, P < 0.01). Increased movement was confirmed by time-lapsed video microscopy. Scanning electron microscopy showed that filopodia generated by these cells following stimulation with SDF-1 were much longer in Rac2−/− HPC compared with WT cells. In addition, Rac2−/− HPC was found to have increased f-actin content relative to WT. HPC movement in vivowas investigated by analysis of mobilization in Rac2−/− versus WT mice. Injection of 250μg of G-CSF/kg/day over 5 days resulted in mobilization of 2.8-fold more progenitors into PB of Rac2−/− mice compared with WT. Simultaneous injection of anti-VLA4 antibody with G-CSF into Rac2−/− mice resulted in significantly reduced augmentation of mobilization, compared with WT mice (in vitrocolony forming cells in PB=50% of WT; day 8 CFU-S in PB=69% of WT), consistent with defective VLA4-mediated function in Rac2−/− HPC. These data demonstrate that Rac2 is essential for normal adhesion and controlled movement of HPC, both in vivoand in vitro, and implicate this GTPase downstream of the integrin VLA-4. The data suggest that lack of Rac 2 in HPC may be associated with increased activity of other GTPases, possibly Cdc42.

La présente analyse rapporte les données intégrées des études de phase 3 PIONEER I & II évaluant la tolérance et l’efficacité de l’adalimumab chez les patients atteints d’hidrosadénite suppurée (HS) modérée à sévère.

Fanconi anemia (FA) is a genetic syndrome characterized by almost uniform development of aplastic anemia. Current therapies for patients lacking HLA-identical sibling hematopoietic stem cell (HSC) donors have shown high morbidity and mortality in clinical trials. Genetic correction of FA HSC using viral vectors has been demonstrated in animal models. However, harvesting of sufficient CD34+ cells at the time that HSC therapy is clinically indicated is difficult due to the severe bone marrow hypoplasia that accompanies pancytopenia. We have opened two phase I clinical trials that seek to determine if potentially useful numbers of CD34+ cells can be collected early in the course of the disease (collection study) and if these cells, once corrected, can engraft without cytoreduction and demonstrate proliferative advantage in vivoover un-corrected cells (gene transfer study). These studies are being conducted with approval by the FDA/NIH-RAC/Institutional IRB and monitored by an independent DSMB. To date, 4 FA patients have undergone a 20 ml/kg bone marrow harvest (BMH) with an average of 1.3x106CD34+ cells/kg (range 0.3–2.9x106CD34+ cells/kg) collected, suggesting that collection of adequate numbers of cells will be challenging, even early in the disease. In the gene transfer study, 3 FA patients with genotype A (FAA) have enrolled, meeting eligibility criteria of FAA, no evidence of malignancy and a minimum of 1x105viable CD34+ cells/kg for ex vivoculture and gene transfer. BMHs from the 3 patients (2 fresh and one previously cryopreserved) were CD34+ cell selected using the CliniMACS device. Despite collection before significant pancytopenia, an average of only 5x105CD34+ cells/kg (range 1.5–10x105CD34+ cells/kg) was purified from these 3 cases representing ~10% of the expected yield from normal individuals. These cells underwent ex vivogene transfer using cytokine prestimulation in serum-free medium followed 2 exposures to a GALV-MSCV-FANCA vector. Transduction efficiency of the final products determined by real-time PCR analysis of CD34+-derived progenitors averaged 48% (range 40–62%). Equivalent efficiency of correction of mitomycin C hypersensitivity in progenitor cells confirmed this analysis at the functional level. Nucleated cell recovery after ex vivomanipulation was 82–110% of input nucleated cells using freshly harvested bone marrow derived CD34+ cells (N=2). However, despite good CD34+ cell recovery and viability after CD34+ selection, only 6% of input nucleated cells were recovered utilizing CD34+ cells purified from the previously cryopreserved bone marrow and these cells were not re-infused. In the two patients who did receive gene corrected cells, the total cell dose re-infused was 2.5–3.5x105nucleated cells/kg, reflecting the low number of initial CD34+ cells placed in culture. One patient is now 6 months post re-infusion with no evidence of gene marking observed in her PB or BM. The second patient had detectable FAA vector sequences in her PB early post-infusion (+4 weeks) but had none detected +8 weeks. The data suggest that while gene transfer efficiency in the clinical setting has been significantly improved, collection and expansion (either in vitroor in vivo) of adequate number of HSC may be critical to the success of genetic correction attempts in FA.

We have previously demonstrated that the hematopoietic cell specific Rho GTPase Rac2 plays not only an essential role in regulating multiple actin-based functions of bone marrow derived mast cells, such as adhesion to fibronectin, migration, and degranulation but is also critical to mast cell survival. To determine the extent of involvement of signaling pathways in increased apoptosis due to Rac2 deficiency, we examined the activation of PI-3Kinase and its downstream effector, Akt, in response to SCF-stimulation. Although, PI-3Kinase activation in response to SCF stimulation was identical in Rac2−/− compared with wild-type (wt) mast cells, the activation of Akt was completely abolished in Rac2−/− mast cells. Unexpectedly, the level of the proapoptotic protein, Bad was significantly elevated (∼10-fold) in Rac2−/− cells in comparison to wt. In contrast, the level of anti-apoptotic protein Bcl-Xl was significantly reduced (∼10-fold) in Rac2−/− cells in comparison to wt. To confirm that enhanced apoptosis in mast cells was the direct result of Rac2 deficiency, Rac2−/− mast cells were transduced with a retrovirus expressing a wt Rac2 cDNA. As expected, Rac2−/− cells expressing Rac2 as a transgene demonstrated complete correction of all actin-based functions and normal survival of mast cells in response to SCF-stimulation. In addition, the activation of Akt and the level of Bad and Bcl-Xl protein were restored to wt levels. In summary, these data demonstrate that Rac2 not only plays an essential role in hematopoietic cell actin-mediated functions, but also plays a critical role in maintaining the survival of these cells by regulating the activation of Akt and the balance between pro- and anti-apoptotic proteins, Bad and Bcl-Xl.