Warren P. Bishop

Initial clinical experience with allopurinol‐thiopurine combination therapy in pediatric inflammatory bowel disease

Background: Thiopurines are a mainstay of immunomodulator therapy in inflammatory bowel disease (IBD). Despite their efficacy, some patients may have a poor response due to inability to achieve adequate levels of the active metabolite, 6‐thioguanine (6‐TGN). Others experience hepatotoxicity, which correlates with excessive 6‐methylmercaptopurine (6‐MMP) levels. Two adult studies have demonstrated successful manipulation of thiopurine metabolism with allopurinol, a xanthine oxidase inhibitor, to achieve more optimal thiopurine levels. The aim was to retrospectively characterize the utility of allopurinol to optimize thiopurine metabolite levels in pediatric IBD patients. Methods: Thirteen patients received allopurinol daily (100 mg in patients ≥30 kg and 50 mg <30 kg), and their thiopurine dose was simultaneously reduced to 25%–50% of the previous maintenance dose. Metabolite levels and other screening labs were checked 2–4 weeks later. Results: The mean azathioprine dose was decreased from 148.1 to 59.6 mg daily (60% of the mean original dose). The mean 6‐TGN level increased from 173 to 303 pmol/8 × 108 red blood cell count (RBC) (P = 0.03), and the mean 6‐MMP level decreased from 7888 to 2315 pmol/8 × 108 RBC (P < 0.001). Elevated transaminase levels improved or resolved in all patients. Two patients experienced reversible neutropenia. At the conclusion of the study 9 patients (69%) remained on combination therapy with a mean duration of follow‐up of 162.8 ± 119.2 days. Conclusions: Combination therapy successfully shunted thiopurine metabolites to a more favorable pattern. Reversible neutropenia was the most common side effect (2 patients). Long‐term prospective studies are needed in this patient population.

Effect of okadaic acid on apo B and apo A-I secretion by CaCo-2 cells

The effect of protein phosphorylation on the synthesis and secretion of apo B and apo A-I by CaCo-2 cells was investigated. Okadaic acid, a potent inhibitor of protein serine/threonine phosphatases 1 and 2A, caused a significant increase in total cellular protein phosphorylation. Apo B-48 was phosphorylated in control cells and this was increased significantly in the presence of okadaic acid. Under the experimental conditions, the phosphorylation of apo B-100 or apo A-I was not observed. No evidence of tyrosine phosphorylation of apo B-100, B-48, or apo A-I was found. Okadaic acid did not change the amount of apo B mass within cells but apo B mass secreted into the basolateral medium was decreased by 40%. Apo A-I mass within cells or in the basolateral medium was unaffected by okadaic acid. Despite causing an 18% decrease in total protein synthesis, okadaic acid did not alter the rate of synthesis of apo B-100, apo B-48, or apo A-I. Cellular turnover of labeled apo B-100 in cells incubated with okadaic acid was similar to controls, whereas apo B-48 and apo A-I turnover were slowed by okadaic acid. Compared to controls, however, 1 microM okadaic acid caused a 75% and 50% decrease in the secretion of newly synthesized apo B-100 and apo B-48, respectively, while decreasing labeled apo A-I secretion by 35%. In contrast to apo A-I mRNA levels, which were not altered by okadaic acid, apo B mRNA levels were significantly decreased by the polyether fatty acid. Despite differences observed in the phosphorylation state of apo B-100 and apo B-48, okadaic acid decreased the secretion of both forms of apo B without altering their synthesis. Okadaic acid, by increasing cellular protein phosphorylation, significantly disrupts the secretory processing of apo B by CaCo-2 cells.

Mucosal morphology in isolated bowel segments: importance of exposure to luminal contents

An isolated bowel segment (IBS) is a loop of intestine that has been freed from its mesenteric attachment after the development of vascular collaterals between the antimesenteric surface of the gut and the host organ. Surgical creation of such artificially vascularized isolated bowel segments is of interest to researchers for a variety of studies, and may be useful in the treatment of short bowel syndrome, allowing longitudinal division of the remaining small bowel to double its length. We created four surgical variants to study the ability of the collateral blood supply to maintain mucosal integrity in the presence or absence of normal luminal contents. In all groups, a collateral blood supply was created in a 5- to 7-cm segment of adult rat jejunum by hepatoenteropexy (Iowa model II). In Thiry-Vella (T-V) and isolated bowel segment (IBS) rats, this segment was exteriorized at both ends to exclude luminal contents. Control and IBS in continuity (IBS-C) loops were left in continuity. The mesentery of IBS and IBS-C rats was divided 5 weeks later, leaving the experimental segment entirely dependent on the collateral circulation. All animals were harvested at 7 weeks after the initial surgery. Tissues were analyzed for mucosal weight, protein content per centimeter of bowel, length of villi, depth of crypts, DNA content, and sucrase activity. We found that segments retaining luminal continuity had significantly higher mucosal weight and DNA content per centimeter of bowel compared with exteriorized loops.

Peroxynitrite inhibits epidermal growth factor receptor signaling in Caco-2 cells

Intestinal mucosa serves as an important barrier that may be disrupted by inflammation. A complex system of cellular and humoral factors, including epidermal growth factor (EGF), maintains the integrity of this barrier. We hypothesized that peroxynitrite, generated in inflamed intestinal epithelium, can alter the EGF receptor function by nitrating tyrosine residues and blocking ligand-activated tyrosine autophosphorylation. Caco-2 cells or A431 cell membranes were treated with peroxynitrite or its decomposed form. Cell proliferation was measured by [3H]thymidine uptake. Immunoblot and immunoprecipitation were used to assess the tyrosine phosphorylation and nitration. Binding of epidermal growth factor to its receptor was detected by affinity labeling with 125I-EGF. Peroxynitrite inhibited EGF-induced Caco-2 cell proliferation and binding of EGF to its receptor in a concentration-dependent manner. Peroxynitrite abolished EGF-stimulated receptor autophosphorylation and nitrated EGF receptor tyrosine residues. Peroxynitrite generated during inflammation may disrupt the EGF-induced signaling in intestinal epithelial cells.

Interruption of a transforming growth factor α autocrine loop in Caco-2 cells by antisense oligodeoxynucleotides

BACKGROUND & AIMSnWe have previously shown that Caco-2 cell proliferation is driven by basolateral membrane epidermal growth factor receptors. The aim of this study was to investigate whether autocrine production of transforming growth factor alpha (TGF-alpha) activates these receptors and stimulates proliferation using antisense oligodeoxynucleotides.nnnMETHODSnCaco-2 cells grown on microporous membranes or Jurkat cells were exposed to conventional or 5 cholesterol-modified oligodeoxynucleotides synthesized with random, antisense, or missense base sequences. Indices of proliferation were measured, including [3H]thymidine or [3H]uridine uptake for studies of short-term stimulation and the methylthiotetrazole assay as an index of cell number increase over longer periods. Secretion of TGF-alpha by cells was detected using a soft agar bioassay.nnnRESULTSnIncubation with antisense oligodeoxynucleotides inhibited TGF-alpha secretion compared with controls. Random and missense oligodeoxynucleotides had no effect on proliferation. The TGF-alpha antisense oligodeoxynucleotides markedly inhibited proliferation, an effect that was abolished by adding TGF-alpha to the medium. Oligonucleotides had no effect on Jurkat cells, a lymphocytic cell line lacking epidermal growth factor receptors. Cholesterol-modified oligodeoxynucleotides were more effective and specific than unmodified oligodeoxynucleotides.nnnCONCLUSIONSnCaco-2 cell proliferation is driven by autocrine stimulation of epidermal growth factor receptors by TGF-alpha. This mechanism may be effectively inhibited by antisense oligodeoxynucleotides, particularly those modified by the 5 attachment of cholesterol.

Overexpression of Fas-ligand by neuroblastoma: A novel mechanism of tumor-cell killing

BACKGROUND/PURPOSEnBinding of Fas ligand (Fas-L) to the membrane-bound Fas receptor incites a series of intracellular events that results in programmed cell death or apoptosis. Although this apoptotic phenomenon plays a key role in down-regulating cytotoxic T cells, the authors have shown previously that pancreatic beta cells (bTC) overexpressing Fas-L paradoxically undergo accelerated rejection that is dependent on a Fas/Fas-L interaction. This study evaluates whether a neuroblastoma (NB) cell line manipulated to overexpress Fas-L undergoes similar destruction and whether tumor-specific protective immunity can be produced.nnnMETHODSnThe authors transfected NB cells (SK-N-MC) with either mFas-L cloned into a pcDNA3.1/Zeo plasmid vector (NB/Fas-L) or with the vector alone (NB/control). Successful transfection of Fas-L was characterized by reverse transcription polymerase chain reaction (RT-PCR) and the ability of transfectants to induce apoptosis of Fas-sensitive T cells (Jurkat). Expression of Fas and Fas-L in untransfected NB clones was characterized by immunohistochemistry and RNase protection assay (RPA). Apoptosis was measured by FACScan analysis using an Annexin V assay. A total of 3x10(6) NB/control and NB/Fas-L cells were implanted subcutaneously into the hind leg of Balb/C SCID mice. Tumor-specific protective immunity was also tested in this model by inoculating mice with NB/Fas-L before implanting NB/control cells.nnnRESULTSnZeocin resistance and RT-PCR confirmed successful transfection of Fas-L into NB cells. Fas Ligand transfectants induced apoptosis in 17.6%+/-2.9% of Fas-sensitive T cells, whereas controls induced apoptosis in only 2.8%+/-1.2% (P = .01, n = 3). Although Fas appears to be constitutively expressed by NB in low amounts, introduction of Fas-L into NB cells did not induce suicide or affect tumor cell growth in vitro. In vivo, NB cells expressing Fas-L failed to grow in SCID mice (n = 3), whereas controls grew rapidly in all animals until death (n = 3). NB/control cells implanted into the opposite leg of mice that rejected initial NB/Fas-L transfectants also grew rapidly (n = 3) implying no protective immunity.nnnCONCLUSIONSnOverexpression of Fas-L in NB clones targets such cells for rapid destruction even in immune compromised hosts, suggesting potential utility of Fas-L in combating NB. In this SCID mouse model, the observed effect is probably neutrophil mediated and does not provide tumor-specific protective immunity.

Fatal cryptococcosis presenting as hepatobiliary dysfunction in an ALL patient

Although current therapies for acute lymphoblastic leukemia (ALL) in children provide high cure rates, invasive fungal infections remain a significant source of mortality. We report a fatal case of cryptococcosis presenting as hepatic dysfunction in a patient with ALL and Down syndrome. Autopsy results confirmed Cryptococcus septicemia with involvement of lungs, liver, and lymph nodes. The severity of the fungal sepsis and underlying immunosuppression probably contributed to the unusual presentation and fatal outcome. This report highlights the need to consider cryptococcal infection as a cause of sepsis syndrome in immunocompromised patients when bacterial cultures are negative.