Leukemia inhibitory factor upregulates cytokine expression by a murine stromal cell line enabling the maintenance of highly enriched competitive repopulating stem cells

Attempts to maintain or expand primitive hematopoietic stem cells in vitro without the concomitant loss of their differentiative and proliferative potential in vivo have largely been unsuccessful. To investigate this problem, we compared the ability of three cloned bone marrow (BM) stromal cell lines to support the growth of primitive Thy- 1lo Sca-1+H-2Khi cells isolated by fluorescence-activated cell sorting from the BM of Ly-5.2 mice treated 1 day previously with 5-fluo- rouracil. Sorted cells were highly enriched in cobblestone area-forming cells (CAFC), but their frequency was dependent on the stromal cell lines used in this assay (1 per 45 cells on SyS-1; 1 per 97 cells on PA6). In the presence of recombinant leukemia inhibitory factor (LIF), CAFC cloning efficiency was increased to 1 per 8 cells on SyS-1 and 1 per 11 cells on PA6, thus showing the high clonogenicity of this primitive stem cell population. More primitive stem cells with competitive repopulating potential were measured by injecting the sorted cells into lethally irradiated Ly-5.1 mice together with 10(5) radioprotective Ly-5.1 BM cells whose long-term repopulating ability has been "compromised" by two previous cycles of marrow transplantation and regeneration. Donor-derived lymphocytes and granulocytes were detected in 66% of animals injected with 50 sorted cells. To quantitate the maintenance of competitive repopulating units (CRU) by stromal cells, sorted cells were transplanted at limiting dilution before and after being cultured for 2 weeks on adherent layers of SyS-1, PA6, or S17 cells. CRU represented 1 per 55 freshly sorted cells. CRU could be recovered from cocultures supported by all three stromal cell lines, but their numbers were approximately-sevenfold less than on day 0. In contrast, the addition of LIF to stromal cultures improved CRU survival by 2.5-fold on S17 and PA6 cells (approximately two-fold to threefold decline), and enabled their maintenance on SyS-1. LIF appeared to act indirectly, because alone it did not support the proliferation of Thy- 1lo Sca-1+H-2Khi cells in stroma-free cultures. Polymerase chain reaction (RT-PCR) analysis revealed that Interleukin-1beta (IL-1 beta) IL-2, IL-6, granulocyte-colony stimulating factor, granulocyte macrophage-colony stimulating factor, transforming growth factors, LIF, and Steel Factor (SLF) mRNAs were upregulated in SyS-1 within 1 to 6 hours of LIF-stimulation. To determine if increased expression of SLF by LIF-stimulated SyS-1 cells could account for their capacity to support stem cells, sorted calls were cocultured on simian CV-E cells that were transfected with an expression vector encoding membrane-bound SLF, or supplemented with soluble SLF. In both cases, SLF synergized with IL-6 produced endogenously by CV-E cells enabling CAFC growth equivalent to that on LIF-stimulated SyS-1. CAFC development on LIF- stimulated SyS-1 could also be completely abrogated by an anti-SLF antibody. These data provide evidence for a role of LIF in the support of long-term repopulating stem cells by indirectly promoting cytokine expression by BM stroma. Furthermore, we have used quantitative assays to show a maintenance of CRU numbers, with retention of in vivo function following ex vivo culture.     

Recombinant human factor IX: replacement therapy, prophylaxis, and pharmacokinetics in canine hemophilia B

Recombinant human factor IX (rFIX) has been expressed in transduced cultured cell systems since 1985. Because there has been limited in vivo testing of rFIX in hemophilia B subjects, this study was undertaken using the severe hemophilia B canines of the Chapel Hill strain. Three groups of hemophilic dogs received either 50, 100, or 200 IU/kg of rFIX. As a control, a fourth group of hemophilic dogs received 50 IU/kg of a high purity, plasma-derived human FIX (pdFIX). The coagulant and hemostatic effects of rFIX and pdFIX were similar with all comparative dosing regimens. Based on activity data, the elimination half-life of rFIX was 18.9 +/- 2.3 hours and pdFIX was 17.9 +/- 2.1 hours. A prophylactic regimen administering rFIX daily resulted in a continuous therapeutic level of plasma FIX and was accompanied by a two-fold increase in recovery levels by day 5, compared to that observed with administration of a single bolus. The mechanisms of the high to complete recovery of FIX with the prophylactic regimen could depend not only on the degree of saturation of the vascular endothelial binding sites but also on the altered dynamics of the balance of FIX distribution between the intravascular and extravascular compartments. The pharmacokinetic (PK) parameters for rFIX and pdFIX were similar. However, the relative PK values for V1 and V5s of both products on day 5 differed greatly from day 1 and may reflect the changing equilibrium of FIX between compartments with elevated levels of plasma FIX. Neutralizing antihuman FIX antibodies resulting from human FIX antigen being administered to FIX deficient dogs were observed beginning at 14 days. The antigenicity of rFIX and pdFIX appeared to be comparable. Despite the very different procedures used for production of rFIX and pdFIX products, in vivo testing in hemophilia B dogs showed the functional behavior of these products is similar; they are highly effective for replacement therapy and for prophylaxis.     

Colonic and jejunal motor disturbances after colonic antigen challenge of sensitized rat

BACKGROUND & AIMS: Inflammation in the colon may alter motility in the proximal gut and potentiate clinical symptoms. The aim of this study was to characterize the effect of colonic anaphylaxis on local (colonic) and remote (small intestinal) motility and identify the mechanism and mediators involved. METHODS: Rats were sensitized by intraperitoneal injection of 10 microg egg albumin and surgically prepared with electrodes in jejunum and colon and a colostomy tube. Colonic and jejunal myoelectric activity were recorded in fasted animals before and after colonic antigen challenge without and then after pretreatment with specific antagonists. RESULTS: Colonic antigen challenge of sensitized rats was associated with significant (1) increase in colonic myoelectric spike activity, (2) disruption of fasting jejunal motility and initiation of aborally propagating spike complexes, and (3) increase in plasma rat mast cell protease II levels with a decrease in granulated mast cells in colon but not jejunum. The myoelectric disturbance in both colon and jejunum was inhibited significantly by pretreatment with atropine and hexamethonium, doxantrazole, cyclooxygenase, and lipoxygenase inhibitors. Methysergide inhibited only the jejunal disturbance. CONCLUSIONS: Colonic antigen challenge of sensitized animals results in local mast cell activation and the release of mediators that modulate neural pathways to initiate both a local colonic and a remote jejunal myoelectric disturbance. (Gastroenterology 1997 Jun;112(6):1996-2005)     

Precision of navigated and conventional open-wedge high tibial osteotomy in a cadaver study

High tibial osteotomy (HTO) is an established treatment option for isolated medial osteoarthritis in young and active patients. One important factor for success of this procedure is the degree of correction of the weight-bearing line. Computer-assisted navigation systems are believed to improve the precision of axis correction through intraoperative real-time monitoring. This study investigates the precision of correction of the weight-bearing line in open-wedge HTO with and without a navigation system.Nineteen legs of well-preserved human cadaver were randomly assigned to navigated (n = 10) or conventional (n = 9) HTO. In order to achieve a sufficient amount of correction in all legs the weight-bearing line was aimed at 80 percent of the width of the tibial plateau.The mean deviation of the weight-bearing line from the desired 80 percent was 1 percent in the navigated and 8.6 percent in the conventional operated legs (p = 0.002). The weight-bearing line of all navigated but only 5 of the 9 conventional operated legs was within a ± 5 percent tolerance level (p = 0.33).Navigated open-wedge HTO achieved better correction of the weight-bearing line than the conventional method in human cadaver legs. Future studies have to prove this advantage in a clinical setting and it''s effect on patient outcome.     

C2orf37 mutational spectrum in Woodhouse–Sakati syndrome patients

Alazami AM, Schneider SA, Bonneau D, Pasquier L, Carecchio M, Kojovic M, Steindl K, de Kerdanet M, Nezarati MM, Bhatia KP, Degos B, Goh E, Alkuraya FS. C2orf37 mutational spectrum in Woodhouse–Sakati syndrome patients. Woodhouse–Sakati syndrome (WSS) is a rare autosomal recessive disorder that encompasses hypogonadism, deafness, alopecia, mental retardation, diabetes mellitus and progressive extrapyramidal defects. The syndrome is caused by mutation of the C2orf37 gene. Here we studied a cohort of seven new cases from three ethnic backgrounds, presenting with the hallmarks of WSS, in an effort to extend the mutational spectrum of this disorder. Genetic analysis revealed a novel mutation in each of the four families investigated, of which three were nonsense mutations and the fourth was a splice site ablation. We also examined a separate collection of 11 cases presenting with deafness and dystonia, two constituents of WSS, but found no pathogenic changes. This study doubles the number of known mutations for this disorder, confirms that truncating mutations in C2orf37 are the only known cause of WSS, and suggests that mutations in this gene do not contribute significantly to cases presenting with isolated elements of WSS such as deafness and dystonia. The lack of correlation between clinically expressivity of WSS and the site of the eight truncating mutations strongly supports that they are equally null, while the intrafamilial variability argues for an important role of modifiers in this disease.     

Neonatal immune system activation with lipopolysaccharide enhances behavioural sensitization to the dopamine agonist, quinpirole, in adult female but not male rats

Administration of the bacterial cell wall component, lipopolysaccharide (LPS), stimulates the immune and endocrine systems inducing an acute phase of sickness and stress responses in adult and neonatal rats. Neonatal LPS exposure has been shown to alter a variety of behavioural and physiological processes in the adult animal. Early developmental stress, such as maternal separation, causes similar acute as well as long-term behavioural changes in adults, including altered sensitivity to drugs of abuse. Moreover, results of studies have shown evidence of a direct link between immune activation and sensitivity to dopamine-based drugs of abuse. The current study examined the effects of neonatal LPS treatment on subsequent locomotor sensitization to the dopamine (D(2)/D(3)) agonist, quinpirole, in adult rats as an index of drug sensitivity. Male and female Long-Evans rats were treated systemically with either LPS (50microg/kg) or saline (0.9%) on postnatal days 3 and 5. Locomotor sensitization was then examined in the adult rats (postnatal day 70). Animals were injected with quinpirole (0.5mg/kg, s.c.) or saline every other day for a total of 10 injections and locomotor activity was assessed for 60min immediately following injections 1, 2, 4, 6, and 10. Animals also received a 'challenge' injection of 0.5mg/kg quinpirole 28 days after injection 10, to assess persistence of behavioural sensitization. Locomotor activity progressively increased with repeated administration of quinpirole, indicating locomotor sensitization in all of the drug-treated groups. There was an overall sex difference, with females showing significantly greater sensitization than males. Moreover, neonatal LPS treatment potentiated both the level and the rate of development of locomotor sensitization to quinpirole administration in females, but not in males. Thus, the current study revealed that neonatal exposure to bacterial infection increases dopamine (D(2)/D(3)) agonist sensitivity in a sex-specific manner. These findings have important implications for the sexually dimorphic development of addictions to both natural and artificial rewards.     

Cardiovascular abnormalities in end stage renal failure: the effect of anaemia or uraemia?

Plasma interleukin-8 (IL-8) concentrations were measured in patients with atopic dermatitis. Plasma IL-8 was not detected in 25 controls (0/25), in allergic rhinitis (0/20), or in bronchial asthma during remission (0/13), while low concentrations of IL-8 were detectable in a few patients with urticaria (1/19), contact dermatitis (4/17), and bronchial asthma at the time of attack (6/16). In contrast, IL-8 was detectable in most cases of atopic dermatitis (41/52). Moreover, IL-8 concentrations were significantly higher in severe than in mild or moderate atopic dermatitis. IL-8 concentrations decreased as atopic dermatitis was improved by treatment, and IgE production in vitro was also decreased while serum IgE concentrations remained unchanged. IL-8 measurement may be a useful tool for the study of the pathogenesis and clinical course of atopic dermatitis.     

Travel and changes in routine do not increase the risk of sudden infant death syndrome

We investigated the relationship between travel and changes in routine and the sudden infant death syndrome (SIDS) among 485 SIDS cases compared with 1800 randomly selected control infants. There was no increased risk of SIDS with travel. Special events, such as christenings, were not associated with an increased risk of SIDS. However, visits to and by friends or relatives were associated with a significantly reduced risk of SIDS after controlling for potential confounders (odds ratios = 0.70; 95% confidence interval = 0.52, 0.96). These findings may indicate less social support in SIDS cases.