Demonstration of differences in colonic volumes, transit, chyme consistency, and response to psyllium between healthy and constipated subjects using magnetic resonance imaging.

BACKGROUND: In functional gastrointestinal disorders a lack of objective biomarkers limits evaluation of underlying mechanisms. We aimed to demonstrate the utility of magnetic resonance imaging for this task using psyllium, an effective constipation treatment, in patients and controls. METHODS: Two crossover studies: (i) adults without constipation (controls, n = 9) took three treatments in randomized order for 6 days - maltodextrin (placebo), psyllium 3.5 g t.d.s and 7 g t.d.s., (ii) adults with chronic constipation (patients, n = 20) took placebo and psyllium 7 g t.d.s. for 6 days. MRI was performed fasting and postprandially on day 6. Measurements included small bowel and ascending colon water content, colonic volume, transit time, and MR relaxometry (T1, T2) to assess colonic chyme. Stool water percentage was measured. RESULTS: 7 g psyllium t.d.s. increased fasting colonic volumes in controls from median 372 mL (IQR 284-601) to 578 mL (IQR 510-882), and in patients from median 831 mL (IQR 745-934) to 1104 mL (847-1316), P < .05. Mean postprandial small bowel water was higher in controls and patients after 7 g psyllium t.d.s. vs placebo. Whole gut transit was slower in patients than controls (P < .05). T1 of the descending colon chyme (fasting) was lower in patients (213 ms, 176-420) than controls (440 ms, 352-884, P < .05) on placebo, but increased by 7 g psyllium t.d.s. (590 ms, 446-1338), P < .001. Descending colon T1 correlated with baseline stool water content and stool frequency on treatment. CONCLUSIONS AND INFERENCES: MRI measurements can objectively demonstrate the mode of action of therapy targeting intestinal fluid content in constipation.

The impact of abdominal pain on global measures in patients with chronic idiopathic constipation, before and after treatment with linaclotide: a pooled analysis of two randomised, double-blind, placebo-controlled, phase 3 trials.

BACKGROUND: Few clinical trials in chronic idiopathic constipation (CIC) patients have evaluated abdominal symptom severity and whether CIC patients with abdominal symptoms respond similarly to patients with limited abdominal symptoms. AIMS: To examine abdominal symptom severity and relationships between symptoms and global measures at baseline; compare linaclotide's effect on symptoms in subpopulations with more or less abdominal pain; and assess relationships between symptom improvement and global measures in these two subpopulations. METHODS: In two phase 3 trials, patients meeting modified Rome II CIC criteria were assigned to linaclotide 145 µg, 290 µg, or placebo once daily. Patients rated abdominal and bowel symptoms daily during 2-week pre-treatment and 12-week treatment periods. Linaclotide's effect on symptoms and global measures [constipation severity, health-related quality of life (HRQOL), treatment satisfaction] and their inter-relationships were assessed in post hoc analyses of abdominal pain subpopulations. RESULTS: Of 1271 CIC patients, 23%, 32%, and 43% reported moderate-to-severe abdominal pain, discomfort, and bloating, respectively, during baseline. In more-severe abdominal pain patients, abdominal symptoms were more strongly correlated than bowel symptoms with global measures, but in less-severe abdominal pain patients, abdominal and bowel symptoms were similarly correlated with global measures, at baseline and post-treatment. Linaclotide significantly improved all symptoms and global measures in both subpopulations. CONCLUSIONS: When abdominal pain is present in CIC, abdominal and not bowel symptoms may drive patient assessments of constipation severity, HRQOL, and treatment satisfaction. Linaclotide (145 µg and 290 µg) is an effective treatment for both abdominal and bowel symptoms, even in CIC patients with more severe abdominal pain at baseline. (Clinicaltrials.gov: NCT00765882, NCT00730015).

Responders vs clinical response: a critical analysis of data from linaclotide phase 3 clinical trials in IBS-C.

BACKGROUND: US Food and Drug Administration (FDA) set a rigorous standard for defining patient responders in irritable bowel syndrome-C (IBS-C; i.e., FDA's Responder Endpoint) for regulatory approval. However, this endpoint's utility for health-care practitioners to assess clinical response has not been determined. We analyzed pooled IBS-C linaclotide trial data to evaluate clinically significant responses in linaclotide-treated patients who did not meet the FDA responder definition. METHODS: Percentages of FDA non-responders reporting improvement in abdominal pain, bowel function and/or global relief measures were determined using pooled data from two linaclotide Phase 3 IBS-C trials. KEY RESULTS: 1602 IBS-C patients enrolled; 34% of linaclotide-treated and 17% of placebo-treated patients met the FDA Responder Endpoint (p < 0.0001). Among FDA non-responders at week 12, 63% of linaclotide-treated patients reported their abdominal pain was at least somewhat relieved, compared with 48% of placebo-treated patients. For stool frequency, 62% of linaclotide-treated patients reported that they were at least somewhat improved at week 12, compared with 46% of placebo-treated patients. For global IBS symptoms, 65% of linaclotide-treated patients reported at least some IBS-symptom relief, 43% reported adequate relief of IBS symptoms, and 57% reported being satisfied with linaclotide treatment, vs placebo rates of 48%, 34%, and 41% respectively. CONCLUSIONS & INFERENCES: Most linaclotide-treated IBS-C patients who were FDA non-responders reported some improvement in abdominal pain and stool frequency, and global relief/satisfaction. In addition to the FDA Responder Endpoint, differing response thresholds and symptom-specific change from baseline should be considered by clinicians for a complete understanding of clinical response to linaclotide and other IBS-C therapies.

An evaluation of the FDA responder endpoint for IBS-C clinical trials: analysis of data from linaclotide Phase 3 clinical trials.

BACKGROUND: Our objective was to evaluate the performance of the Food and Drug Administration (FDA) Responder Endpoint for clinical trials in IBS-C, using data from two large Phase 3 clinical trials of linaclotide. The FDA interim endpoint requires that, for 50% of trial weeks, patients report ≥30% decrease in Abdominal Pain at its worst and (in the same week) an increase in Complete Spontaneous Bowel Movements (CSBMs) of ≥1 from baseline. METHODS: Anchor-based methodology was used to estimate thresholds of clinically meaningful change using symptom-specific patient rating of change questions (PRCQs) and symptom severity questions. The diagnostic accuracy of the FDA Responder Endpoint was assessed using sensitivity/specificity-based methods. KEY RESULTS: Using anchor-based methods, the estimates of the clinically meaningful improvement thresholds for Abdominal Pain ranged from 25.9% to 32.4% and thresholds for increase in weekly CSBM rate ranged from 1.4 to 1.6 CSBMs per week. Compared with the symptom-specific PRCQs for patient rating of relief, the FDA Responder Endpoint has a sensitivity of 60.7%, a specificity of 93.5%, and an accuracy of 82.0%. Changing the number of weeks required to be a responder or the percentage improvement in the Abdominal Pain criteria did not result in notable improvement in the accuracy of the FDA Responder Endpoint. CONCLUSIONS & INFERENCES: The FDA Responder Endpoint for IBS-C clinical trials represents clinically meaningful improvements in IBS-C symptoms for patients with excellent specificity and reasonable sensitivity.

GT160-246, a toxin binding polymer for treatment of Clostridium difficile colitis.

GT160-246, a high-molecular-weight soluble anionic polymer, was tested in vitro and in vivo for neutralization of Clostridium difficile toxin A and B activities. Five milligrams of GT160-246 per ml neutralized toxin-mediated inhibition of protein synthesis in Vero cells induced by 5 ng of toxin A per ml or 1.25 ng of toxin B per ml. In ligated rat ileal loops, 1 mg of GT160-246 neutralized fluid accumulation caused by 5 microg of toxin A. At doses as high as 80 mg/loop, cholestyramine provided incomplete neutralization of fluid accumulation caused by 5 microg of toxin A. GT160-246 protected 80% of the hamsters from mortality caused by infection with C. difficile, whereas cholestyramine protected only 10% of animals. Treatment of C. difficile-infected hamsters with metronidazole initially protected 100% of the hamsters from mortality, but upon removal of treatment, 80% of the hamsters had relapses and died. In contrast, removal of GT160-246 treatment did not result in disease relapse in the hamsters. GT160-246 showed no antimicrobial activity in tests with a panel of 16 aerobic bacteria and yeast and 22 anaerobic bacteria and did not interfere with the in vitro activities of most antibiotics. GT160-246 offers a novel, nonantimicrobial treatment of C. difficile disease in humans.

Murine complement receptor gene expression: Cr2 gene transcripts are depressed during a high dose microbial challenge.

The murine Cr2 gene encodes mRNA that produce two protein products predicted to be approximately 145,000 M(r) (Cr2-145) and 190,000 M(r) (Cr2-190). All cells examined which express the Cr2 gene produce transcripts encoding both the Cr2-145 and Cr2-190 proteins: both transcripts are constitutively expressed by mature B cells. To determine if Cr2 expression could be altered by activating splenic B cells, splenic cultures were incubated with lipopolysaccharide (LPS) and cell surface Ig chains were cross-linked with anti-mu. In the presence of LPS and anti-mu both Cr2 and Oct-2 transcripts were diminished while the control beta-actin transcript levels remained unchanged. However, when LPS alone was added, only the Cr2 transcript levels were diminished. To test if these findings could be reproduced in vivo, animals were provided with a peritoneal injection of either Escherichia coli or Listeria monocytogenes and transcript levels analysed. The quantities of both Cr2 transcripts, as well as those encoding Oct-2, were substantially reduced in splenocytes and peripheral lymphatic tissues obtained from these infected mice while those encoding the mouse Crry protein, the B-cell marker CD19 and beta-actin remained unchanged. These data suggest that when confronted with a major bacterial infection, murine B cells respond by shutting down synthesis of transcripts encoding the Cr2 and Oct-2 gene products.

Loss of human CR1- and murine Crry-like exons in human CR2 transcripts due to CR2 gene mutations.

Analysis of the coding sequences of the murine Cr2 gene indicated that it contains two distinct regions of homology to the murine Crry, and human CR1 and CR2 genes. The last 15 short consensus repeats (SCR) of Cr2 are very similar to the 15 reported SCR of human CR2. These 15 SCR of Cr2, plus the transmembrane and cytoplasmic domains, make up a protein very similar in organization and sequence to the human CR2 gene product. Another Cr2 transcript contains the sequences encoding the previously described 15 SCR plus those encoding another six amino terminal SCR. These amino terminal SCR are very similar to those of Crry and the first six SCR seen within the CR1 long homologous repeats. Amino acid sequence similarity analysis, however, indicated that the sequences encoding the six amino terminal SCR of Cr2 evolved from a separate lineage of SCR than Crry and CR1, suggesting that the human counterparts to these additional Cr2 SCR had not been identified. Using the cDNA sequences specific for the amino terminal SCR of murine Cr2, the human counterparts were isolated and localized within the human CR2 gene between those nucleotides encoding the signal sequence and those encoding the first SCR of the mature human CR2 protein. Unlike the murine Cr2 gene products, these Crry/CR1-like sequences of the CR2 gene are not maintained in mature CR2 mRNA, and thus represent pseudoexons. DNA sequence analysis of the pseudoexon homologous to that encoding the first SCR of the murine Cr2 gene indicated that a stop codon has been introduced within the human coding sequence but that the 5' and 3' splice recognition sequences appear to be functional. Although the stop codon would block the translation of a transcript with this exon, it would not inhibit the incorporation of the exon within the mature CR2 transcript. Therefore, another mutation must have been introduced within the human CR2 gene such that both this exon and the other CR1/Crry-like exons are removed from mature CR2 transcripts.

The murine complement receptor gene family. IV. Alternative splicing of Cr2 gene transcripts predicts two distinct gene products that share homologous domains with both human CR2 and CR1.

The murine Cr2 gene encodes at least two related proteins. The first of these is predicted to include 1408 amino acids from a transcript including 4224 coding nucleotides. This protein is predicted to contain 21 60-amino acid repeats plus those residues encoding transmembrane and cytoplasmic regions for a total peptide m.w. of 155,307. The first six of these repeats are similar to human CR1 in sequence and organization. The second protein is encoded by an alternatively spliced Cr2 transcript that is lacking those sequences which encode the first six 60-amino acid repeats of the larger protein. This second, smaller protein, encoded by a transcript of 3096 coding nucleotides, is predicted to include 15 60-amino acid repeats, plus the transmembrane and cytoplasmic regions. This smaller protein contains 1032 amino acids for a peptide m.w. of 113,328. This second protein is extremely homologous in size and sequence to human CR2. Both proteins share the same signal sequence for membrane insertion. DNA sequence analysis, RNA protection studies and genomic phage mapping indicate the transcripts which encode these proteins are derived from the Cr2 gene via alternative splicing.

Murine complement receptor gene family. II. Identification and characterization of the murine homolog (Cr2) to human CR2 and its molecular linkage to Crry.

CR2, a 145,000 to 150,000 Mr protein which binds specific breakdown products of C3, has been identified on the surface of both human and murine B cells. In order to understand the evolutionary relatedness of the human and murine proteins, we have used the coding sequences from the human CR2 gene to investigate those homologous sequences of murine Cr2. Human CR2 cDNA sequences were used as probes on a cDNA library derived from BALB/c spleen mRNA to identify cross-reacting cDNA sequences. A number of putative cDNA clones encoding murine Cr2 have been isolated and examined. DNA sequence analysis of these Cr2 cDNA clones indicates that they represent the murine homolog to human CR2. mRNA analysis with these Cr2 cDNA clones has revealed a transcription pattern similar to, but distinct from that seen for CR2. Whereas human CR2 coding sequences identify a single mRNA species of approximately 5 kb from human tonsillar mRNA, the murine counterpart identifies four transcripts from murine spleen of approximately 3, 5, 9 and 11 kb in size. The Cr2 cDNA clones which detect the four forms of spleen mRNA overlap in coding sequences and contain exons mapping to three colinear fragments as defined by EcoRI digestion. This suggests that the 3- 5-, 9-, and 11-kb mRNA forms arise by alternative splicing from a single gene. Use of these murine Cr2-specific cDNA clones to isolate their respective genomic sequences has allowed for the linkage of the 3' end of the Cr2 gene to the 5' end of the Crry gene, the evolutionary homolog to human CR1.

Genetic organization of complement receptor-related genes in the mouse.

Using an interspecific cross, gene linkage relationships among members of the murine complement receptor-related genes, C4bp, Cfh, Mcry, and Mcr2, were analyzed by segregation of RFLP in 200 mice. The human homologues of these genes are tightly linked, composing the RCA locus, which maps to human chromosome (Chr.)1q32, within a large linkage group conserved between human Chr.1q21-32 and mouse Chr.1. RFLP associated with C4bp and Cfh map within this conserved linkage group; Cfh is located 9 cM telomeric to C4bp, which is consistent with linkage data for their human homologues. Mcry and Mcr2, while tightly linked, are located outside the conserved group, 40 cM telomeric to C4bp. These data suggest that a translocation or inversion occurred within the RCA family during the evolution of the mouse, defining a breakpoint of this large conserved linkage group.

The murine complement receptor gene family. Analysis of mCRY gene products and their homology to human CR1.

The mouse genome contains two sets of gene sequences which are highly homologous to the gene encoding the human C3b/C4b receptor (CR1). These genes, termed murine CRY (mCRY) and murine CRX (mCRX) reside on murine chromosomes 1 and 8, respectively. Analysis of cDNA isolated by using these sequences as probes indicates that there are two related but distinct mRNA which are expressed in a wide variety of murine tissues including spleen, liver, lung, and brain. Both of these transcripts encode proteins which should contain a signal sequence for membrane insertion, a transmembrane/cytoplasmic tail region for membrane anchoring, and five extracellular domains made up of 60 amino acid consensus repeat sequences. The difference between the two is the presence of an additional exon of 129 bp immediately 3' of the signal sequence. This additional exon does not encode a 60 amino acid repeat. The sizes of the mature proteins predicted from the cDNA sequences are 43,998 Mr and 48,680 Mr; however, antisera raised against carboxy-terminal sequences detects a 70,000 Mr protein from murine fibroblasts suggesting a high degree of post-translational modification of the mature protein. A comparison of these murine gene sequences with a partial human CR1 sequence suggests that the human CR1 gene evolved by direct duplication of the ancestral coding sequences contained within these murine genes including those sequences important for membrane anchoring and cytoplasmic protein attachment.

Effects of products of activated leukocytes (lymphokines and monokines) on the growth of malignant trophoblast cells in vitro.

Supernatants from activated leukocyte cultures and individual lymphokines and monokines were added to cultures of JEG-3 human gestational choriocarcinoma cells in vitro, and effects on cell proliferation were measured. Activated leukocyte culture supernatants, recombinant gamma-interferon, tumor necrosis factor, and colony-stimulating factor significantly inhibited JEG-3 proliferation. In contrast, high doses of both interleukin 1 and 2 stimulated JEG-3 proliferation. Low doses of B cell growth factor stimulated JEG-3 proliferation, whereas the highest dose was inhibitory. Further understanding of the effects of lymphokines and monokines on trophoblastic growth may provide important insights into immunologic mechanisms affecting early pregnancy development and tumor-host interactions in gestational trophoblastic neoplasia.