Oral administration of non-absorbable delayed release 6-mercaptopurine is locally active in the gut, exerts a systemic immune effect and alleviates Crohn's disease with low rate of side effects: results of double blind Phase II clinical trial.

Therapy for Crohn's disease (CD) with thiopurines is limited by systemic side effects. A novel formulation of fixed-dose, delayed-release 6-mercaptopurine (DR-6MP) was developed, with local effect on the gut immune system and minimal absorption. The aim of this study was to evaluate the safety and efficacy of DR-6MP in patients with moderately severe CD compared to systemically delivered 6-mercaptopurine (Purinethol). Seventy CD patients were enrolled into a 12-week, double-blind controlled trial. The primary end-point was the percentage of subjects with clinical remission [Crohn's Disease Activity Index (CDAI) < 150] or clinical response (100-point CDAI reduction). Twenty-six (56·5%) and 13 (54·2%) subjects from the DR-6MP and Purinethol cohorts, respectively, completed the study. DR-6MP had similar efficacy to Purinethol following 12 weeks of treatment. However, the time to maximal clinical response was 8 weeks for DR-6MP versus 12 weeks for Purinethol. A higher proportion of patients on DR-6MP showed clinical remission at week 8. A greater improvement in Inflammatory Bowel Disease Questionnaire (IBDQ) score was noted in the DR-6MP group. DR-6MP led to a decrease of CD62(+) expression on T cells, implying a reduction of lymphocyte adhesion to site of inflammation. DR-6MP was safer than Purinethol, with significantly fewer adverse events (AEs). There was no evidence of drug-induced leucopenia in the DR-6MP group; the proportion of subjects who developed hepatotoxicity was lower for the DR-6MP. Non-absorbable DR-6MP is safe and biologically active in the gut. It is clinically effective, exerting a systemic immune response with low systemic bioavailability and a low incidence of side effects.

Relationships between hypoglycaemia and gastric emptying abnormalities in insulin-treated diabetic patients.

We hypothesize that hypoglycaemia in insulin-treated diabetic patients may result from gastric emptying abnormalities causing insulin and food absorption mismatching. We tested gastric emptying in insulin-treated diabetic patients with unexplained hypoglycaemia and without dyspepsia and in diabetic patients without hypoglycaemia, prospectively. Thirty-one diabetic patients with unexplained hypoglycaemic events within 2 h of insulin injection and 18 insulin-treated diabetic patients without hypoglycaemic events underwent gastric emptying breath tests, glycaemic control and autonomic nerve function. Gastric emptying tests were abnormal in 26 (83.9%) and in four (22.2%) patients with and without hypoglycaemia, respectively (P < 0.001). Gastric emptying was significantly slower in hypoglycaemic diabetic patients (t1/2 139.9 +/- 74.1 vs 77.8 +/- 23.3 and t(lag) 95.8 +/- 80.3 vs 32.84 +/- 16.95 min, P < 0.001 for both comparisons; t-tests). A significant association between hypoglycaemic patients and abnormal values of t1/2 and t(lag) was found (P < 0.001). Gastric emptying abnormalities were more frequent in hypoglycaemic patients. We suggest gastric emptying tests for diabetic patients with unexplained hypoglycaemic events.

Expression of HLA class I antigens in sporadic adenomas and histologically normal mucosa of the colon.

The loss of HLA antigens by neoplastic cells is considered important for tumor growth and metastasis, inasmuch as it may allow tumors to escape immune surveillance. We have observed reduced expression of HLA antigens in sporadic colon cancer and adenomas from familial adenomatous polyposis patients. We now studied the expression of HLA class I antigens in patients with sporadic adenomas, which are precursors of colorectal cancer. Expression of HLA class I antigens was studied by immunohistochemistry in (a) sporadic colon adenomas, (b) histologically normal mucosa distant from the adenomas, (c) histologically normal colonic mucosa from patients with history of sporadic colon adenomas, and (d) colonic mucosa from normal subjects. HLA class I antigen expression was moderately reduced in 56% and severely reduced in 44% of the adenomas; this reduction was significant when compared to controls (P < 0.0001). The reduction of HLA class I expression in adenomas was related to the grade of dysplasia of the adenomas. HLA class I expression of normal appearing mucosa was decreased in 76% of patients with adenoma (P < 0.0001) and in 54% of patients with history of adenoma (P < 0.005) compared to normal controls. These changes were antigen specific, inasmuch as the expression of carcinoembryonic antigen, a surface antigen, was not affected. Our findings suggest that reduced HLA class I expression is an early event in the cell transformation process from normal to neoplastic state, preceding in many cases the onset of histological changes. HLA class I could be potentially used as a premalignant marker in the colon.

Quantitation of antibody binding to cell surface antigens by x-ray fluorescence spectrometry.

An X-ray spectrometric method has been developed to quantitate antibody bindng to whole cell surfaces in order to obtain a distribution of binding within a population of cells. The method involves incubation of target cells with ferritin-labeled antibody. Analysis of prepared samples in a modified transmission electron microscope with an X-ray detector and data analysis equipment, yields quantitative results on the binding of labeled antibody to individual cells. The binding of anti-2,4-dinitrophenol serum to Chinese hamster ovary cells with attached 2,4-dinitrophenol haptens was measured by X-ray spectrometry. Measurements of attached hapten by a radioisotopic marker correlated with the X-ray spectrometric determination of bound antibody. The use of synchronized cells in metaphase and G1 phases of the cell cycle permitted investigations into the binding per unit surface area. The distribution of antibody binding among a given population of cells was related to the surface area of the cells.

Type C Niemann-Pick disease: a murine model of the lysosomal cholesterol lipidosis accumulates sphingosine and sphinganine in liver.

We have determined the levels of free sphingoid bases in livers of normal and cholesterol lipidotic Niemann-Pick type C mice. Hepatic sphingosine and sphinganine levels in affected mice (593 pmol/mg protein) were elevated more than 20-fold when compared to levels in age-matched normal mice (26 pmol/mg protein). Upon fractionation of mutant liver homogenates by differential centrifugation, most of the sphingoid bases sedimented with beta-hexosaminidase in the 9000 x g pellet. Co-sedimentation of sphingoid bases with a lighter beta-hexosaminidase peak in Percoll gradients suggests that these bases accumulate in lipid laden lysosomes. A cytosolic sphinganine kinase is the first enzyme in the degradative pathway of sphingoid base metabolism. Activity of this enzyme was partially deficient in crude mutant liver cytosolic extracts due to the presence of an inhibitory substance. Following molecular sieving of mutant cytosolic extracts on Sepharose 4B, sphinganine kinase, with normal levels of activity, was resolved from a complex higher-molecular-weight inhibitor fraction. The Km values for either sphinganine or ATP-Mg substrates with partially purified sphinganine kinase from normal and mutant mouse liver extracts, were similar. These findings indicate that accumulation of free sphingoid bases is not due to a direct inherent deficiency in the catalytic activity of sphinganine kinase. The possible cause and effect relationship between the accumulation of these endogenous hydrophobic amines and the lesion in intracellular cholesterol trafficking in Niemann-Pick type C disease is discussed.

Type C Niemann-Pick disease: cellular uncoupling of cholesterol homeostasis is linked to the severity of disruption in the intracellular transport of exogenously derived cholesterol.

A uniquely attenuated disruption of cholesterol homeostasis has been characterized in certain Niemann-Pick, type C (NP-C) fibroblasts. Uptake of LDL-cholesterol by cultured fibroblasts derived from two clinically affected brothers with this variant biochemical phenotype led to less intracellular accumulation of unesterified cholesterol than found in other typical cell lines. This limited cholesterol lipidosis in the variant NP-C cells reflected cholesterol processing errors that differed from the cellular lesions in classical NP-C cells in the following ways: (1) a more limited intracellular distribution of the excessive unesterified cholesterol; (2) shorter and more transient delays in the induction of cholesterol-mediated homeostatic responses; and (3) more efficient intracellular transport of exogenously derived cholesterol to the plasma membrane and the endoplasmic reticulum. Activation of acyl-CoA cholesterol acyltransferase (ACAT) was greater than 100-fold in both control and NP-C fibroblasts when cell cultures were preconditioned with 25-hydroxycholesterol, but the subsequent esterification of exogenous non-lipoprotein [3H]cholesterol remained deficient in all NP-C cells. In the variant NP-C cells conditioned with the oxysterol, this esterification of exogenous [3H]cholesterol was less affected than in classical NP-C cultures. The NP-C mutation affects a broad spectrum of metabolic responses related to the processing of exogenously derived cholesterol. Among this pleiotropic array of deficient responses, retarded intracellular cholesterol transport appears most closely linked to the primary mutation. This conclusion is supported by two current observations: (1) the degree to which sterol transport is affected in mutant cells in turn reflects the extent to which cholesterol-homeostatic responses are compromised; and (2) sterol transport remains deficient despite concurrent normal activation of other cellular responses, such as cholesterol esterification.

Safety and tolerability of antagonist anti-human CD40 Mab ch5D12 in patients with moderate to severe Crohn's disease.

BACKGROUND: The ligation of CD40 by CD154 is a critical step in the interaction between APC and T cells. In animals, antagonizing CD40L-CD40 has been shown to reduce the severity of several autoimmune and inflammatory disorders, including experimental colitis. AIM: To investigate tolerability and safety of an antagonist chimeric monoclonal anti-human CD40 antibody (ch5D12) for treatment of Crohn's disease. METHOD: ch5D12 was administrated to 18 patients with moderate to severe Crohn's disease in a single dose, open-label dose-escalation phase I/IIa study. RESULTS: ch5D12 plasma concentrations increased dose-dependently after infusion. Two patients developed an anti-ch5D12 antibody response. Overall response and remission rates were 72 and 22%, respectively with no evidence for a dose-response effect. Treatment with ch5D12 reduced microscopic disease activity and intensity of the lamina propria cell infiltrate, but did not alter percentages of circulating T and B cells. ch5D12 was well tolerated, although some patients experienced headache, muscle aches, or joint pains, which may have been related to the study drug. CONCLUSIONS: Antagonizing CD154-CD40 interactions with ch5D12 is a promising therapeutic approach for remission induction in Crohn's disease.

The murine Niemann-Pick type C lesion affects testosterone production.

We have determined the effects of the Niemann-Pick type C (NPC) lesion, which impairs transport of cholesterol from lysosomes, on the androgenic status of male NPC mice. The mice have low serum testosterone levels resulting from decreased testosterone secretion. Testosterone secretion is reduced in NPC mouse testes incubated with 8-bromo-cAMP, 20 alpha-hydroxycholesterol, and pregnenolone compared to testosterone release by normal mouse testes under identical conditions. Ultrastructural examination of testes revealed a paucity of lipid droplets, extensive accumulation of inclusion bodies, and distorted endoplasmic reticulum in Leydig cells of adult NPC mice. The hypoandrogenemia caused systemic deficiencies in NPC mice. Seminal vesicles, a testosterone-responsive tissue, were underdeveloped in NPC male mice. The testosterone-responsive kidney beta-glucuronidase activity was also underexpressed. Seminal vesicle mass and beta-glucuronidase activity were increased by testosterone treatment of NPC mice. Many hepatic proteins, identified by microsequencing, were also deficient in NPC male mice. Levels of alpha 2-mu-globulin, glutathione S-transferase-pi, carbonic anhydrase-III, and selenium-binding protein increased in normal male mice during puberty, but did not increase in the NPC male mice. Based on the increases in protein expression during puberty, differential expression in males and females, and the reported involvement of androgens in regulating expression of some of these proteins, deficient expression of most of these proteins in male NPC mice appears to result from low testosterone levels. We conclude that a defect in testicular testosterone production in NPC male mice causes a pleiotropic deficiency in androgen-sensitive expression of proteins in various organs.

The neurogenetics of mucolipidosis type IV.

BACKGROUND: Mucolipidosis type IV (MLIV) is an autosomal recessive disease caused by mutations in the MCOLN1 gene that codes for mucolipin, a member of the transient receptor potential (TRP) gene family. OBJECTIVE: To comprehensively characterize the clinical and genetic abnormalities of MLIV. METHODS: Twenty-eight patients with MLIV, aged 2 to 25 years, were studied. Ten returned for follow-up every 1 to 2 years for up to 5 years. Standard clinical, neuroimaging, neurophysiologic, and genetic techniques were used. RESULTS: All patients had varying degrees of corneal clouding, with progressive optic atrophy and retinal dystrophy. Twenty-three patients had severe motor and mental impairment. Motor function deteriorated in three patients and remained stable in the rest. All had a constitutive achlorhydria with elevated plasma gastrin level, and 12 had iron deficiency or anemia. Head MRI showed consistent characteristic findings of a thin corpus callosum and remained unchanged during the follow-up period. Prominent abnormalities of speech, hand usage, and swallowing were also noted. Mutations in the MCOLN1 gene were present in all patients. Correlation of the genotype with the neurologic handicap and corpus callosum dysplasia was found. CONCLUSIONS: MLIV is both a developmental and a degenerative disorder. The presentation as a cerebral palsy-like encephalopathy may delay diagnosis.

Separation of ferritin labeled antibody from free antibody.

A procedure for the separation of ferritin-antibody conjugates from free antibody has been developed using isokinetic sucrose density gradient sedimentation. The gradient (5% w/w sucrose at the top to about 35% at the tube bottom) provides optimal separation of 7S free immunoglobulin from the ferritin (65S) and ferritin-antibody conjugate. Recovery of the conjugate band is simple and avoids problems of aggregation found by centrifugal pelleting and resuspension procedures. The method was tested by labeling human globulin and fractionating the gradient. Free antibody was detected at the top one-fifth of the gradient by immunodiffusion. Ferritin and the labeled antibody were found one-third to one-half of the way down the gradient. Immunoelectrophoresis was also used to demonstrate the separation of unconjugated ferritinand ferritin labeled antibody from free antibody.

Lysosomal inclusions in gastric parietal cells in mucolipidosis type IV: a novel cause of achlorhydria and hypergastrinemia.

Mucolipidosis type IV (ML-IV) is an autosomal recessive lysosomal storage disease that causes severe neurologic abnormalities. The brain disease is characterized by pigmented cytoplasmic granules in neurons and accumulation of lamellated membrane structures in lysosomes. The gastrointestinal disease in ML-IV was not previously recognized. Clinical examination of 20 patients with ML-IV (age range, 2-23 years) at the National Institutes of Health showed hypergastrinemia and constitutive achlorhydria. Endoscopic biopsy specimens from the gastric fundus, body, and antrum and from the duodenum of four such patients (ages 4, 6, 7, and 22 years) were evaluated histologically and by electron microscopy. Histologically, all gastric fundus and body biopsy specimens showed parietal cells in normal numbers. However, a striking cytoplasmic vacuolization of parietal cells was seen on hematoxylin and eosin stain. Electron microscopy showed the parietal cells to be markedly distended by large lysosomes containing lamellar, concentric, and cystic membranous inclusions. Additionally, chronic atrophic gastritis and enterochromaffin-like (ECL) cell hyperplasia were observed. Foveolar and chief cells in stomach and duodenum biopsy specimens were normal. We conclude that the cytoplasmic lysosomal inclusions in gastric parietal cells is a unique histologic feature of gastric biopsy in ML-IV.

The effect of ethanol on the expression of HLA class I genes in human colon adenocarcinoma cell lines.

The loss of HLA antigens by neoplastic cells may allow tumors to escape immune surveillance. We observed reduced expression of HLA antigens during human colon carcinogenesis. Since ethanol, which is associated with human colonic carcinogenesis, modulates the expression of HLA genes, we examined whether it affects the expression of HLA class I genes in human colon adenocarcinoma cell lines. Ethanol (1.7 x 10(-10) M to 1.7 x 10(-1) M), had no effect on the expression of HLA class I antigens on these colonocytes, the corresponding mRNA levels, or the expression of HLA constructs. Our findings do not support the hypothesis that ethanol may modulate the expression of HLA class I genes in human colon cancer cells.

Niemann-Pick type-C disease: deficient intracellular transport of exogenously derived cholesterol.

NPC disease is an autosomal recessive neurovisceral storage disorder. A pleiotropic array of secondary enzymatic and storage abnormalities has in the past obscured a cohesive understanding of the underlying metabolic basis of this disorder. Recent findings, reviewed in this report, demonstrate that NPC disease is a cholesterol lipidosis resulting from defective intracellular cholesterol transport. The sequence of cellular events characteristic of NPC is 1) deficient intracellular transport of exogenously derived cholesterol resulting in retarded induction of cellular cholesterol homeostatic regulation; 2) accumulation of cholesterol in lysosomes; and 3) secondary cellular effects. Retarded esterification of exogenous cholesterol and accumulation of unesterified cholesterol in lysosomes is tightly coupled to the primary defect and serves as the basis for biochemical diagnosis of NPC.

Aspirin and aspirin-like drugs induce HLA-DR expression in HT29 colon cancer cells.

Nonsteroidal anti-inflammatory drugs (NSAIDs) decrease the incidence of colon cancer. The underexpression of HLA antigens during colon cancer development is suspected to be a mechanism by which malignant cells escape immune surveillance. We examined whether NSAIDs affect the expression of HLA-DR in HT29 human colon adenocarcinoma cells, which do not express HLA-DR. Aspirin, indomethacin and sulindac induced several-fold the expression of HLA-DR in these cells in a concentration- and time-dependent manner. Aspirin increased HLA-DR alpha steady-state mRNA levels and HLA-DR alpha gene transcription rate. These findings raise the possibility that such a mechanism may be operative in vivo.

Efficacy of misoprostol and ranitidine in the prevention of duodenal ulcer relapse and its correlation with endogenous gastric prostanoid synthesis.

We determined endogenous gastric prostaglandin synthesis and its correlation with the prevention of duodenal ulcer relapse by misoprostol and ranitidine. Sixty-one patients with recent endoscopically healed duodenal ulcer were randomly allocated in a double-blind fashion for one year of treatment with misoprostol 400 micrograms nocte, ranitidine 150 mg nocte or placebo. Patients were followed every two months. Endoscopy was repeated at six and 12 months or beforehand, if relapse was suspected. Antral and fundic biopsies, 3-4 from each region, were obtained at each endoscopy for determination of prostaglandin synthesis. During the one year of treatment, 11 out of the 12 placebo treated patients flared up, as opposed to 10 out of 25 and four out of 24 misoprostol and ranitidine treated patients, respectively. The difference between all treatment groups was significant (P less than 0.0001). In all subjects who flared up, pretrial endogenous antral and fundic prostaglandin E2 synthesis were not different from their respective synthesis in those who did not relapse.

No correlation between indomethacin-induced gastroduodenal damage and inhibition of gastric prostanoid synthesis.

The effect of 1 week of treatment with indomethacin 150 mg/day on human gastric prostanoid synthesis was correlated with its effect on gastric and duodenal mucosa. Before and following 1 week of treatment, endoscopic appearance of the mucosa was evaluated and scored. Following 1 week of treatment with indomethacin, antral PGE2 and 6-keto-PGF1 alpha were significantly lower than in normal subjects, but similar in patients with significant or with no mucosal damage. Co-treatment with ranitidine 150 mg b.d. or with cimetidine 400 mg b.d. reduced the mean mucosal damage score but did not affect gastric prostanoid synthesis, which was similar irrespective of the presence or absence of mucosal damage. It is therefore suggested that there is no correlation between indomethacin-induced inhibition of gastric prostanoid synthesis and its induction of mucosal damage.

Ischemia stress and arachidonic acid metabolites in the fetal brain.

Hypoxic-ischemic insults caused by placental insufficiency in perinatal life are today considered a major cause for neuronal injury and impaired postnatal development. A major consequence of placental insufficiency and ischemia is the change in metabolism of arachidonic acid and its oxidation products. A burst of postischemic production of prostaglandins, unequivocally shown in many systems, is documented in the fetal rabbit brain as well as in placenta tissue soon after vascular restriction. PGE2, a most abundant prostaglandin of the fetal brain, is particularly elevated. Similarly, thromboxane B2 and 6-keto PGF1 alpha, the stable metabolites of thromboxane A2 and prostacyclin, are both increased over the control values. However, after 48 h of restriction, the levels of these eicosanoids are restored to near-normal values. The metabolic machinery responsible for the conversion of arachidonic acid into eicosanoids in brain and placenta tissues appears to be impaired following a period of placental insufficiency. This inhibition can be accounted for by excessive production of eicosanoids and also by formation of an endogenous inhibitor or free radicals. Studies are in progress to test these possibilities.

Endoscopic insertion of endoprostheses in the treatment of postoperative cutaneous biliary fistula.

We report a case in which a large, postoperative, biliary cutaneous fistula was successfully treated by means of endoscopic insertion of two endoprostheses into the common bile duct. Twenty-four hours after the procedure, bile flow from the cutaneous fistula ceased completely. The endoprostheses were removed after 6 weeks, and no evidence of leak from the biliary tract was seen on a retrograde cholangiogram. For the treatment of postoperative biliary leaks, insertion of endoprostheses is a good and safe alternative to surgery.

Decreased substance P content in the rectal mucosa of diabetics with diarrhea and constipation.

Substance P (SP), vasoactive intestinal polypeptide (VIP), and somatostatin content in rectal mucosa were determined by radioimmunoassay (RIA) in 38 diabetic patients (12 with normal bowel function, 13 with diabetic diarrhea, and 13 with constipation) and in 10 nondiabetic controls with normal bowel function. SP content (picograms per milligram) in the rectal mucosa of diabetics with normal bowel function was significantly higher than that of nondiabetic controls (P < .05). SP content in the rectal mucosa of diabetics with diabetic diarrhea and constipation was significantly lower than in diabetics with normal bowel habits and nondiabetic controls (P < .05). No differences were found in the rectal mucosa content of VIP and somatostatin between the different groups of diabetics and controls. Diabetic diarrhea is a condition with an intermittent nature and frequently alternates with constipation. Our findings showing low levels of rectal mucosa SP in both conditions suggest a possible common role of SP in the pathogenesis of diabetic diarrhea and constipation.