Characteristics Associated With Clinically Important Treatment Responses in Women Undergoing Nonsurgical Therapy for Fecal Incontinence.

OBJECTIVE: To identify baseline clinical and demographic characteristics associated with clinically important treatment responses in a randomized trial of nonsurgical therapies for fecal incontinence (FI). METHODS: Women (N = 296) with FI were randomized to loperamide or placebo- and manometry-assisted biofeedback exercises or educational pamphlet in a 2 × 2 factorial design. Treatment response was defined in 3 ways from baseline to 24 weeks: minimal clinically important difference (MID) of -5 points in St. Mark's score, ≥50% reduction in FI episodes, and combined St. Mark's MID and ≥50% reduction FI episodes. Multivariable logistic regression models included baseline characteristics and treatment groups with and without controlling for drug and exercise adherence. RESULTS: Treatment response defined by St. Mark's MID was associated with higher symptom severity (adjusted odds ratio [aOR] 1.20, 95% confidence interval [CI] 1.11-1.28) and being overweight vs normal/underweight (aOR 2.15, 95% CI 1.07-4.34); these predictors remained controlling for adherence. Fifty percent reduction in FI episodes was associated with the combined loperamide/biofeedback group compared with placebo/pamphlet (aOR 4.04, 95% CI 1.36-11.98), St. Mark's score in the placebo/pamphlet group (aOR 1.29, 95% CI 1.01-1.65), FI subtype of urge vs urge plus passive FI (aOR 2.39, 95% CI 1.09-5.25), and passive vs urge plus passive FI (aOR 3.26, 95% CI 1.48-7.17). Controlling for adherence, associations remained, except St. Mark's score. DISCUSSION: Higher severity of FI symptoms, being overweight, drug adherence, FI subtype, and combined biofeedback and medication treatment were associated with clinically important treatment responses. This information may assist in counseling patients, regarding efficacy and expectations of nonsurgical treatments of FI.

Formation of exoplanetary satellites by pull-down capture.

The large size and wide orbit of the recently announced exomoon candidate Kepler-1625b-i are hard to explain within traditional theories of satellite formation. We show that these properties can be reproduced if the satellite began as a circumstellar co-orbital body with the original core of the giant planet Kepler-1625b. This body was then drawn down into a circumplanetary orbit during the rapid accretion of the giant planet gaseous envelope, a process termed "pull-down capture." Our numerical integrations demonstrate the stability of the original configuration and the capture process. In this model, the exomoon Kepler-1625b-i is the protocore of a giant planet that never accreted a substantial gas envelope. Different initial conditions can give rise to capture into other co-orbital configurations, motivating the search for Trojan-like companions to this and other giant planets.

Flow cytometric analysis of human bone marrow perfusion cultures: erythroid development and relationship with burst-forming units-erythroid.

Flow cytometry has been used in recent years to study antigenic and physical changes accompanying hematopoietic cell differentiation. Such studies have provided the basis for rapid and objective assays that are potential alternatives to the colony assays currently in widespread use. In this report, erythropoiesis was examined in growth factor-supplemented perfused cultures of bone marrow mononuclear cells (BMMNC) using flow cytometric analysis of the transferrin receptor (CD71), glycophorin antigen expression occurred with time, Initially, fewer than 10% of the cells were (CD71++, but by day 8, 19-34% of the cells were CD71++gly A-. This was followed by the appearance of gly A on 10-60% of the CD71++ cells. After day 10, CD71 expression on many gly A+ cells decreased so that a population of CD71-gly A+ cells (11-54 accumulated by day 14. Each phenotype was sorted for morphologic identification and colony assay analysis. CD71++gly A- cells were 85% blasts, one-half of which were erythroblasts, and were significantly enriched for burst-forming units-erythroid (BFU-E). The time-varying number of CD71++gly A- cells in these cultures was found to correlate with the number of BF.78-0.97). Three-color analysis was next used to examine CD33 expression on BFU-E, and in fresh BM, most were found to be CD33-. During culture, however, the number of BFU-E recovered from CD33+ populations first increased and then decreased. Therefore, CD33 was not particularly useful for identifying BFU-E. In contrast, CFU-GM were mostly found to be in the CD71+CD33+ population throughout the culture period. When erythropoietin (Epo) was not added to these cultures, the percentage of gly A+ cells was reduced from 33 to 3.3%. Further, the omission of Epo caused an 80% decrease in the number of BFU-E and a corresponding 94% decrease in the number of CD71++gly A- cells, thereby maintaining the relationship between CD71++gly A- cells and BFU-E. Therefore, flow cytometric analysis was found to be useful in assessing erythroid development, and this approach may be used to develop flow cytometric assays for other populations of interest in hematopoietic cell cultures.

Growth factor consumption and production in perfusion cultures of human bone marrow correlate with specific cell production.

Perfusion cultures of human bone marrow mononuclear cells (BMMNC) provide a unique in vitro model of hematopoiesis, supporting growth of both accessory and hematopoietic elements. In this study, bioreactors were used to analyze the consumption and production of growth factors (GFs) in relation to each other and to the cells produced. The exogenously added GFs interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), stem cell factor (SCF), and erythropoietin (Epo) each exhibited different, but reproducible, consumption kinetics. Epo and IL-3 were consumed slowly for the first 5-7 days, and then the consumption rate of both increased. Epo consumption reached a plateau by day 10, whereas IL-3 consumption continued to increase. Consumption of SCF was similar to that of Epo, but began 2-3 days earlier. GM-CSF was consumed throughout the culture period in an accelerating manner. Consumption of SCF and Epo were related, because omission of Epo from the growth medium reduced SCF consumption by 53% and omission of SCF reduced Epo consumption by 82%. A reproducible relationship between cumulative GF consumption and total cell production was observed. Epo was most potent, with 5900 molecules consumed per cell produced, whereas 69,400 molecules of SCF were consumed per cell generated. More specifically, Epo consumption was correlated (r = 0.92 and 0.96) with the number of glycophorin A-positive (glyA+) cells produced, and the rate of Epo consumption varied with the progression of cells through the erythroid lineage. Consequently, measurement of GF consumption rates may be useful for quantifying the types of cells present in a culture. Endogenous GF production was also examined. G-CSF and MIP-1 alpha were present at high levels during the first 4 days but then declined rapidly. LIF first appeared in the second week and steadily increased thereafter. Omission of SCF from the medium allowed the detection of endogenous SCF production, and the kinetics was similar to that of LIF. IL-6 production was biphasic, with a peak and decline in week 1 and an increase during week 2. TGF-beta was below the level of detection in these cultures. The results suggest that perfusion supports accessory and hematopoietic elements which interact and therefore represent a partially functional tissue ex vivo. This system provides a useful model for studying relationships within GF networks and for elucidating the conditions that result in primitive cell expansion ex vivo.

Functional properties of a synthetic chicken parathyroid hormone-related protein 1-36 fragment.

The biologic activities of human parathyroid hormone-related protein [hPTHrP(1-34] and bovine PTH [bPTH(1-34)] are remarkably similar despite marked sequence divergence in their primary binding domain, residues 25-34. Chicken PTHrP (cPTHrP) is identical to hPTHrP through residue 21. However, in the 25-34 region, cPTHrP displays three fewer basic residues than hPTHrP and contains five residues not present in any other member of the PTH/PTHrP family. To assess the biologic consequences of these structural differences, we compared the activities of synthetic [36Tyr]cPTHrP(1-36)NH2 and hPTHrP(1-34)NH2 with those of bPTH(1-34) in avian systems (chicken renal plasma membranes and 19 day chick embryonic bone cells) and mammalian systems [canine renal plasma membranes and rat osteosarcoma cells (UMR-106-H5)]. In both avian and mammalian systems the binding affinity of [36Tyr]cPTHrP(1-36)NH2 (0.8-3.4 nM) was approximately one-half that of hPTHrP(1-34)NH2 (0.4-1.1 nM). The potencies of [36Tyr]cPTHrP(1-36)NH2 and hPTHrP(1-34)NH2 for activation of adenylate cyclase were similar in canine renal membranes (5.2 and 6.7 nM) and chick bone cells (1.0 nM). In UMR-106 cells and chicken renal membranes the potency of [36Tyr[cPTHrP(1-36)NH2 for activation of adenylate cyclase was about one-half that of [36Tyr]hPTHrP(1-36)NH2. Binding of 125I-[36Tyr]cPTHrP(1-36)NH2 to chick bone cells and chicken renal membranes was completely displaced by bPTH(1-34) and hPTHrP(1-34)NH2: thus there was no evidence for a distinct chicken PTHrP receptor. In general, [36Tyr]cPTHrP(1-36)NH2 and hPTHrP(1-34)NH2 activated adenylate cyclase similarly despite their sequence differences in the 25-32 region.(ABSTRACT TRUNCATED AT 250 WORDS)

Two homologs of the Drosophila polarity gene frizzled (fz) are widely expressed in mammalian tissues.

The frizzled (fz) locus of Drosophila encodes a protein (Fz) with a seven-transmembrane-domain profile characteristic of G-protein-coupled receptors. In Drosophila, genetic evidence suggests that Fz functions to transmit and transduce polarity signals in epidermal cells during hair and bristle development. We have isolated from a UMR 106 rat osteosarcoma cell library a cDNA (fz-1) encoding a predicted 641-residue protein (Fz-1) with 46% homology with Drosophila Fz. We also identified a second cDNA (fz-2) encoding a protein (Fz-2) of 570 amino acids that is 80% homologous with Fz-1, with divergence most evident in the extracellular domains. Southern blots of rat genomic DNA indicated that fz-1 and fz-2 represent distinct genes. Northern analysis revealed the presence of a single fz-1 mRNA (4.7 kilobases) and two fz-2 mRNAs (2.5 and 4.5 kilobases) in rat tissues. The fz-1 and fz-2 genes are widely expressed in rat tissues with the highest steady-state levels of mRNA in kidney, liver, heart, uterus, and ovary. fz-1 and -2 mRNA levels were greater in neonatal than in corresponding adult tissues. Treatment of UMR 106 cells with bone resorbing agents including parathyroid hormone, epidermal growth factor, and 1,25-dihydroxyvitamin D3 produced increases in fz-1 and -2 mRNA levels. We suggest that hormonal induction of Fz proteins in osteoblasts serves to promote intercellular signaling required for functional responses such as increased bone resorption. Fz-1 and Fz-2 may represent products of a gene family whose members serve as transducers or intercellular transmitters of signals required for normal morphogenesis and/or differentiated function in diverse tissues.

Analogues of parathyroid hormone modified at positions 3 and 6. Effects on receptor binding and activation of adenylyl cyclase in kidney and bone.

Predictive and spectroscopic methods were used to develop a model of the structures of the 1-34 peptides of parathyroid hormone (PTH) and the PTH-related protein (PTHrP). Circular dichroism (CD) studies of bovine PTH-(1-34) and human PTHrP-(1-34)amide in the presence of trifluoroethanol suggest the presence of 24-26 alpha-helical residues. For both peptides, interactions between amino- and carboxyl-region alpha-helices are predicted to result in a hydrophobic core with externally facing hydrophilic residues that include probable determinants of receptor binding and activation. Two such residues, Ser3 and Gln6, are conserved in all known members of the PTH/PTHrP family. We have synthesized 13 novel analogues of bovine PTH-(1-34) monosubstituted at positions 3 and 6 and have determined their biological activities in renal and bone cell radioreceptor and adenylyl cyclase assays. Position 3 analogues displayed biological activity that was reduced in direct proportion to the volume of the substituent side-chain. Position 6 analogues also displayed reduced biological activity, but no simple correlation with side-chain volume or hydrophobicity was evident. The analogues fully displaced labeled PTH from binding sites in renal membranes and bone cells, but [Phe3]bPTH-(1-34), [Tyr3]bPTH-(1-34), [Phe6] bPTH-(1-34), and [Ser6]bPTH-(1-34) were only partial agonists in one or both adenylyl cyclase assays. Of these, [Phe3]bPTH-(1-34) and [Phe6]bPTH-(1-34) were tested for antagonist activity and were found to inhibit the activation of adenylyl cyclase in response to bPTH-(1-34) or hPTHrP-(1-34)amide. These results indicate that positions 3 and 6 contribute important determinants of PTH receptor binding and activation. Modification at these positions represents a novel approach to the development of antagonists of PTH action.

An unexpected response to vaccination with a purified major membrane tachyzoite antigen (P30) of Toxoplasma gondii.

A purified Toxoplasma gondii tachyzoite membrane protein (P30) and a monoclonal antibody directed against this antigen were used to immunize mice. The P30 protein has an apparent m.w. of 30,000 and is the major radioiodinated tachyzoite membrane antigen identified by human and mouse antitoxoplasma antisera. Polyclonal and monoclonal antibody to the P30 antigen are parasiticidal in the presence of human serum. A series of mice were immunized with affinity column-purified P30 protein. This produced a dose-dependent, antigen-specific IgG and IgM response. The mice were challenged with the less virulent C strain tachyzoite. Immunized mice showed a statistically significant increase in mortality over nonimmunized control mice. In addition, vaccinated mice had an increased number of intracerebral tissue cysts when compared with the control group. Similar results were obtained with passive transfer immunization by using monoclonal antibody directed against the P30 antigen. Immunofluorescence assay of brain tissue cyst bradyzoites revealed a total absence of P30 antigen. Bradyzoites were also deficient in another major tachyzoite antigen of approximate m.w. 22,000 (P22). Mouse antibradyzoite serum absorbed with tachyzoites recognized bradyzoites but failed to identify tachyzoites. This suggests that there are stage-specific bradyzoite antigens of Toxoplasma gondii.

Identification of stage-specific sporozoite antigens of Toxoplasma gondii by monoclonal antibodies.

Stage-specific antigens of Toxoplasma gondii have not been reported. We developed a panel of monoclonal antibodies that identified unique surface membrane antigens on the sporozoite and oocyst wall of T. gondii. These monoclonal antibodies failed to react with T. gondii tachyzoite surface membrane antigens in several immunologic assays. A comparison of surface membrane radioiodinated tachyzoites and sporozoites by one- and two-dimensional electrophoresis showed that T. gondii sporozoites have two major membrane proteins of approximate m.w. 67,000 and 25,000, not present in the tachyzoite stage. Two of the stage-specific monoclonal antibodies (2E4 and 5A4) were directed against the sporozoite 67,000 m.w. protein. In addition, T. gondii sporozoites are deficient in the major radioiodinated tachyzoite protein P30, which is identified by both human and mouse convalescent antitoxoplasma antisera. A convalescent antitoxoplasma serum failed to recognize sporozoite antigens by both immunoprecipitation and reaction with nitrocellulose transfer blots.