Abstract
Concurrent Session
8:30 AM
Friday, January 29, 1999
The Effects Of The Anti-Leukotriene Zileuton Upon Neutrophil Recruitment In X-Linked Chronic Granulomatous Disease Knock-Out Mice Exposed To Sterile Aspergillus Fumigatus Hyphae
Chronic Granulomatous Disease (CGD) is a recessive disease caused by defects in the neutrophil respiratory burst oxidase that produces superoxide, an important precursor for microbicidal oxidants. CGD patients suffer from increased microbial and fungal infections as well as inflammatory granulomas that may represent persistent lesions in response to debris normally cleared by oxidant-dependent mechanisms, such as Aspergillus fumigatus hyphae (Morgenstern et. al., J Exp. Med 185, 207, 1997). An experimental model for CGD is a knock-out mouse with a mutation in the X-linked gene for gp91phox that codes for the larger subunit of the respiratory burst oxidase cytochrome b. We have previously shown that the inflammatory exudate induced by intraperitoneal (I.P.) injection of thioglycollate in X-CGD mice had increased numbers of neutrophils compared to wild-type (WT). Experimental evidence suggests oxidants are important for degradation of leukotrienes, including leukotriene B4 (LTB4), a potent neutrophil chemoattractant. We hypothesized that increased numbers of X-CGD neutrophils may be recruited by an accumulation of chemoattractants in the absence of their superoxide-dependent clearance or inactivation. In the current study Zileuton, a 5-Lipoxygenase inhibitor, was used to inhibit the production of LTB4 via the arachidonic acid pathway. We investigated the effect of Zileuton on the inflammatory exudate induced by an I.P. injection of sterilized A. fumigatus hyphae (50 µg) in WT and X-CGD C57BL/6J mice. 30 minutes before injection and 3 hours after, either Zileuton (100 mg/kg suspended in 0.2% hydroxypropyl methylcellulose, HPMC) or 0.2% HPMC alone was administered by gavage. After 6 hours, when neutrophils were present in maximal numbers, mice were sacrificed and peritoneal cavities lavaged to assess exudate formation with total cell counts and histologic analysis of cellular composition. In mice administered the HPMC vehicle alone, the X-CGD mice (n=9, 6.03X106 ± 3.56X105) compared to WT (n=13, 6.15X105 ± 6.42X105) had a 10-fold increase in neutrophil numbers (p<0.0001). Zileuton decreased the numbers of exudate neutrophils isolated from WT (n=14, 2.56 X105 ± 1.23X105) and X-CGD mice (n=8, 1.40X106 ± 6.42X105), however neutrophils were still increased by 5-fold in X-CGD relative to WT mice (p<0.0001). These results suggest that leukotriene-elicited neutrophil chemotaxis of X-CGD in response to sterile A. fumigatus is an important mechanism for recruiting increased numbers of neutrophils in murine X-CGD, although other mechanisms may also contribute.
Altered Leukotriene Synthesis By Neutrophils From Patients With Sickle Cell Disease.
The acute chest syndrome, a major cause of morbidity and mortality in sickle cell disease (HbSS) patients, is characterized by pulmonary sequestration and inflammation. Leukotrienes (LT), potent mediators of vasoreactivity, play an important role in the modulation of pulmonary hemodynamics. LTE4 is a pulmonary vasoconstrictor, while LTB4 induces chemotaxis and adhesion of neutrophils to vascular endothelium. Neutrophils produce leukotrienes in response to various stimuli. We have documented an abnormal leukotriene metabolism in patients with sickle cell disease. The role of leukotrienes in the pathogenesis of the acute chest syndrome associated with sickle cell disease is not known. In the current study we investigated the production of leukotrienes by neutrophils of patients with sickle cell disease in comparison with normal controls. The neutrophils from HbSS patients in steady state, and healthy homozygous HbAA controls were isolated by dextran sedimentation and the purified cells were studied with or without stimuli. The production of LTB4 and LTE4 was quantitated by a specific ELISA and the values are expressed as mean ± SEM/2.5x107 cells. LTB4 production was higher in HbAA vs. HbSS cells (1.41±0.34 vs. 0.10±0.06; p=0.01). Both showed an increase in LTB4 production with time; only the HbSS cells produced significantly more LTB4 at 30 min (p=0.02). LTE4 production was significantly elevated at 10 min vs. 30 min in controls (0.68 ±0.12 vs. 0.16 ±0.03; p=0.02), while HbSS neutrophils produced a constant amount of LTE4 (0.23±0.07 vs. 0.26 ±0.09). HbSS neutrophils were more responsive to stimulation with FMLP (a synthetic tripeptide with characteristics to naturally occurring bacterial oligopeptide) than control cells and produced more LTB4 and LTE4. An increase in LTE4 production occurred with time in response to FMLP stimulation in control cells. These data suggest that, once activated, the neutrophils from HbSS patients have an exaggerated production of leukotrienes. We speculate that this alteration in leukotriene metabolism and its resultant adverse effect on the endothelium may contribute to the acute chest syndrome.
Carbon Monoxide Production And Upregulation Of Heme Oxygenase Activity In Mice After Heme Administration.
Heme oxygenase (HO) catalyzes the rate-limiting step in the predominant pathway for heme degradation leading to the equimolar production of Fe(II), carbon monoxide (CO), biliverdin, and, ultimately, bilirubin. We are developing a hemolytic mouse (Balb-C) model for studying in vivo efficacy of metalloporphyrin inhibitors of HO to suppress bilirubin production, which is measured by total body CO excretion rate (V'eCO) with a flow-through chamber system. The V'eCO of each mouse in a 50-mL chamber, supplied with 30 mL air/min, was monitored by measuring chamber exit air for CO by gas chromatography. The basal V'eCO was established before mice (20.9±3.1g) were injected via the tail vein with 30-µmol heme arginate/kg. At various times during the V'eCO measurement period, animals were sacrificed for determination of hepatic and splenic HO activity by the gas chromatographic CO method. We found that the V'eCO of untreated animals (58 ± 10 µL CO/hr/kg) did not change during the 6-h monitoring period. However, the V'eCO of heme-treated animals started to increase 15±5 min after injection, and after 78±15 min, reached a peak V'eCO of 202±27 µL CO/hr/kg. The V'eCO returned to baseline after 6 h. During this period, splenic HO activity did not change significantly from baseline (555±82 µmol CO/hr/mg FW tissue). In contrast, the basal hepatic HO activity (141±29 µmol CO/hr/mg FW tissue) began to increase approximately 1.5 h after heme injection, reached a plateau at 2.5±1 h of 719±75 µmol CO/hr/mg FW tissue, then slowly diminished from 6 h to 12 d when 138% of baseline HO activity, or 19% of peak HO activity, remained. We conclude that the mouse is an attractive hemolytic model for the study of HO inhibitors because it displays a relatively rapid and reproducible V'eCO profile in comparison to the rat model. Furthermore, we have improved the HO activity assay so that the activity in organs weighing as little as 50 mg can be easily determined. We demonstrate here for the first time that hepatic HO activity upregulation occurs quickly and shortly (1.5 h) after heme injection and remained at a high level for days, whereas the injected heme was metabolized rather quickly. In contrast, splenic HO activity was not increased because this organ apparently is maximally active.
Clinical Severity Score Predicts Early Mortality In Thrombotic Thrombocytopenic Purpura-Hemolytic Uremic Syndrome (Ttp-Hus)
TTP and HUS are severe disorders characterized by microangiopathic hemolytic anemia and thrombocytopenia. Untreated, the short term mortality is estimated at 90%. Prognostic variables for predicting response and survival have been difficult to validate because of the relatively small sample sizes of previous analyses. We performed a retrospective cohort analysis on 126 consecutive patients with an established diagnosis of TTP-HUS treated principally with plasma exchange. These patients had been referred to the Sacramento Medical Foundation Blood Center and the University of California Davis Medical Center between 1978 and 1998. To standardize disease involvement, patients were assigned a previously described Clinical Severity Score (Rose, Am J Med 83:437, 1987) based on four clinical and laboratory parameters, if available, at the time of presentation. The Severity Score incorporates the neurologic, renal, and hematologic abnormalities and is the sum of all the parameters, with a range of 0-8 points. We also determined the effect of therapeutic plasma exchange on 30-day mortality, response rate, and overall survival. 122 patients (97%) received plasma exchange as principal treatment, with a mean of 9 exchanges and a mean cumulative infused volume of 43,040 ± 77,662 mL of fresh frozen plasma. There were 56% complete responders and 21% partial responders for an overall response proportion of 77%. Overall 30-day mortality was 10.3% (n=13). Relapse rate was 12.8%. Univariate analysis demonstrated that a higher Clinical Severity Score at the time of diagnosis increased the risk of 30-day mortality with an odds ratio of 2.5 and a p-value of 0.0067. In conclusion, we have confirmed that early, aggressive plasma exchange therapy results in both high response and survival rates in this large cohort of TTP-HUS patients. We have likewise shown that the Clinical Severity Score may be a useful prognostic variable in predicting 30-day mortality.
Frequent Methylation Of P15 5' Region In Leukemic And Preleukemic Disorders, And Reduction Of P15 Methylation Levels By 5-Aza-Deoxycytidine Treatment Is Correlated With Clinical Remission.
Abberant CpG island methylation of genes involved in cell proliferation and differentiation has been demonstrated in numerous cancers. The tumor suppressor genes (TSG), p15 and p16, are negative cell cycle regulators that control the progression through the G1 phase of the cell cycle. Earlier studies have shown that these TSGs can be inactivated by mutation, deletions, and methylation in many primary cancers. Methylation of the 5′ CpG island of these important TSGs was correlated with inactivation of expression; and 5-aza-deoxycytidine (5-aza-CdR), a cytosine analog that inhibits DNA methylation, can re-induce expression. The methylation status of p15 andp16 in acute myelogenous leukemia (AML), acute lymphocytic leukemia (ALL), and the preleukemic disorder, myelodysplastic syndrome (MDS), were examined by a quantitative bisulfite based PCR assay, Methylation Sensitive Single Nucleotide Primer Extension (Ms-SNuPE). Methylation of p15 was detected in the DNAs from the bone marrows and peripheral blood of 100% (5/5) MDSs, 89% (8/9) AMLs, and 100% (2/2) ALLs at diagnosis which was above the levels found in the WBC DNAs from healthy donors. In contrast, p16 5′ CpG island methylation was absent or low in these leukemic and preleukemic DNA samples by Ms-SNuPE. Since MDSs evolve into AMLs in a majority of the cases (40-60%) which also showed frequent p15 methylation, DNAs from nineteen MDS patients undergoing a clinical trial in Europe with 5-aza-CdR were also examined. Clinical improvement or remission was detected in 10/19 (53%) of these treated MDS cases, and 10 patients had reduced p15 methylation detected by Ms-SNuPE through the course of treatment. Reduced p15 5′ region methylation was correlated with clinical response (partial or complete remission) in 6/10 (60%) patients, although improvement of MDSs also occurred in the absence of reduction in p15 DNA methylation levels. Trilineage improvement of all three hematopoietic lineages affected in MDS was induced by 5-aza-CdR, and reduction in p15 methylation levels may be one factor associated with the clinical response to the treatment. Hypermethylation of p16, in contrast, is infrequent in MDSs and leukemias and may not play a role in the pathogenesis of these hematologic disorders.
Immune Gene Therapy For Acute Lymphoblastic Leukemia.
Acute Lymphoblastic Leukemia (ALL) is the most frequent type of cancer in children and high risk groups present with less than a 30 % survival chance. Pre-B ALL cells are inefficient antigen presenting cells, allowing the leukemia to evade the patient's immune system. We are studying the potential of a cell vaccine to increase immunogenicity of leukemia cells such that rejection of minimal residual discase after chemotherapy can be achieved by the patient's immunc system. To study this, two approaches are being applied. A murine model of ALL has been established using a BALB/c pre-B cell line (BM185) engineered to express the p185 oncogene BCR-ABL. This model mimics Philadelphia chromosome positive ALL; when injected into mice, BM185 develops into a rapidly growing leukemia. BM185 cells were engineered to express various immunomodulators to augment the host anti-leukemic immune response. BM185 cells expressing CD80 were completely rejected in 50% of mice, allowing longterm disease-free survival. Disease-free survival was correlated with the development of cytotoxic T cells against BM185. Currently, we are studying CD40L which activates antigen presenting cells. Preliminary studies indicate enhanced survival of mice receiving BM185 cells expressing CD40L when compared to the wild type BM185 cell line. Additionally, studies are being done with human dendritic cells in vitro. Conditions have been established to generate large numbers of dendritic cells from cord blood and bone marrow. We are currently studying gene transfer of the BCR-ABL oncogene into dendritic cells to enginecr these cells to present antigenic determinants of the BCR-ABL protein to the immune system. In the future, these studies will translate into an immune gene therapy clinical trial for ALL.
Interleukin-15 Increases Cd16/56 Receptor Expression And Motile Shape Change In Neonatal Cells In Long Term Cultures.
Newborn infants have a higher susceptibility to viral infections partially due to a deficiency in natural killer (NK) function as compared to normal adults. Previously, we showed that interleukin (IL)-15 increases NK number (by CD16/56 expression), and function in cytotoxicity assays in short term (1 week) cultures in neonatal (cord) cells. We now examine the effect of IL-15 in longer term cultures of cord cells. Mononuclear cells (MNC) were separated from umbilical cord blood obtained from newborns and incubated at 37°C for up to 12 weeks with IL-15. Flow cytometry assays were performed to assess surface receptor expression. The table shows a mean percentage ± standard error of cells expressing both CD16 and CD56 with IL-15 concentrations in ng/ml.
Number in parentheses = N; *p<0.05 compared to no cytokines.
CD16/56 expression was greatly, diminished in the absence of cytokines, but the addition of IL-15 (1 ng/ml) increased expression to >40% by 6 weeks. In addition, the total number of cells expressing CD56 (with or without CD16 expression) increased to >90% when exposed to IL-15 (10 ng/ml) for 7 weeks or longer (not shown). Cord MNC exposed to IL-15 tended to exhibit an elongate shape with pseudopod formation characteristic of a motile cell, and the percentage of motile cells increased with increasing concentrations of IL-15 (not shown). IL-15 also greatly expanded the number of cord MNC in culture with up to a 100-fold increase in the cell population by 6 weeks in 10 ng/ml IL-15. Thus, IL-15 can stimulate and expand cord MNC in long term cultures that potentially might be useful in treatment of immunodeficient infants.