Theoretical Tools to Analyze Anorectal Mechanophysiological Data Generated by the Fecobionics Device.

A mechanical approach is needed for understanding anorectal function and defecation. Fecal continence is achieved by several interacting mechanisms including anatomical factors, anorectal sensation, rectal compliance, stool consistency, anal muscle strength, motility, and psychological factors. The balance is easily disturbed, resulting in symptoms such as fecal incontinence and constipation. Novel technologies have been developed in recent years for studying anorectal function. Especially, the Fecobionics device, a simulated feces, has gained attention recently. This facilitates new analysis of anorectal mechanical function. In this study, a theoretical model is developed to analyze anorectal mechanophysiological data generated by the Fecobionics device. Theoretical approaches can enhance future interdisciplinary research for unraveling defecatory function, sensory-motor disorders, and symptoms. This is a step in the direction of personalized treatment for gastrointestinal disorders based on optimized subtyping of anorectal disorders.

Tandem thiotepa with autologous hematopoietic cell rescue in patients with recurrent, refractory, or poor prognosis solid tumor malignancies.

BACKGROUND: The purpose of this study was to determine the feasibility and tolerability of tandem courses of high-dose thiotepa with autologous hematopoietic cell rescue (AHCR) in patients with recurrent, refractory solid tumors who were ineligible for a single course of high-dose therapy due to greater than minimal residual disease. Patients with decreased hearing or poor renal function were eligible. PROCEDURE: Thiotepa was administered intravenously at a dose of 200 mg/m2 /day (6.67 mg/kg/day) daily for 3 days followed by AHCR. A second course of thiotepa was given 4 weeks later provided blood counts recovered sufficiently without evidence of tumor progression. RESULTS: Fifty-eight patients received 96 courses. Thirty-eight (65%) patients received two courses of therapy. Twenty-seven courses (28%) were administered completely in the outpatient setting. A toxic mortality rate of 3.4% was observed. Five of 26 patients with medulloblastoma were alive at a median of 35 months, whereas 21 patients died at a median of 11.7 months. Four of five patients with central nervous system germ cell tumors (CNS GCT) were alive 68-103 months following AHCR. CONCLUSIONS: Two cycles of high-dose thiotepa with AHCR were well tolerated even in these heavily pretreated patients. This therapy may provide prolonged survival in patients with recurrent malignant brain tumors, particularly medulloblastoma and CNS GCT.

The impact of chlordane and dieldrin contamination of well water on cancer rates in North Stamford, Connecticut.

BACKGROUND: High levels of the carcinogenic organochlorine pesticides chlordane and dieldrin have been reported in the well water of homes in North Stamford. It is unclear if the contamination is associated with an increase in the cancer rate in North Stamford. METHODS: We reviewed the demographics of the towns surrounding North Stamford and chose New Canaan, Wilton, Weston, and Darien as towns with sufficiently similar demographics that would permit comparison of cancer incidence with North Stamford. Data were obtained from the Connecticut Tumor Registry regarding the number of different cancers diagnosed per year from 1998 to 2007 in North Stamford and the four nearby towns. We compared the annual cancer incidence of these communities in total and by cancer types. RESULTS: There was no statistically significant difference in the average annual cancer incidence from 1998 to 2007 between North Stamford and the four other communities. There was also no statistically significant difference seen in the incidence of the various cancer types. CONCLUSION: Chlordane and dieldrin contamination of the well water of homes in North Stamford may not be associated with a higher incidence of cancer.

Oligomerization-dependent regulation of motility and morphogenesis by the collagen XVIII NC1/endostatin domain.

Collagen XVIII (c18) is a triple helical endothelial/epithelial basement membrane protein whose noncollagenous (NC)1 region trimerizes a COOH-terminal endostatin (ES) domain conserved in vertebrates, Caenorhabditis elegans and Drosophila. Here, the c18 NC1 domain functioned as a motility-inducing factor regulating the extracellular matrix (ECM)-dependent morphogenesis of endothelial and other cell types. This motogenic activity required ES domain oligomerization, was dependent on rac, cdc42, and mitogen-activated protein kinase, and exhibited functional distinction from the archetypal motogenic scatter factors hepatocyte growth factor and macrophage stimulatory protein. The motility-inducing and mitogen-activated protein kinase-stimulating activities of c18 NC1 were blocked by its physiologic cleavage product ES monomer, consistent with a proteolysis-dependent negative feedback mechanism. These data indicate that the collagen XVIII NC1 region encodes a motogen strictly requiring ES domain oligomerization and suggest a previously unsuspected mechanism for ECM regulation of motility and morphogenesis.

Effects of angiogenesis inhibitors on multistage carcinogenesis in mice.

Solid tumors depend on angiogenesis for their growth. In a transgenic mouse model of pancreatic islet cell carcinogenesis (RIP1-Tag2), an angiogenic switch occurs in premalignant lesions, and angiogenesis persists during progression to expansive solid tumors and invasive carcinomas. RIP1-Tag2 mice were treated so as to compare the effects of four angiogenesis inhibitors at three distinct stages of disease progression. AGM-1470, angiostatin, BB-94, and endostatin each produced distinct efficacy profiles in trials aimed at preventing the angiogenic switch in premalignant lesions, intervening in the rapid expansion of small tumors, or inducing the regression of large end-stage cancers. Thus, anti-angiogenic drugs may prove most efficacious when they are targeted to specific stages of cancer.

Activation of Ha-ras p21 by substitution, deletion, and insertion mutations.

The transforming activity of naturally arising ras oncogenes results from point mutations that affect residue 12 or 61 of the encoded 21-kilodalton protein (p21). By use of site-directed mutagenesis, we showed that deletions and insertions of amino acid residues in the region of residue 12 are also effective in conferring oncogenic activity on p21. Common to these various alterations is the disruption that they create in this domain of the protein, which we propose results in the inactivation of a normal function of the protein.

FP-21399 blocks HIV envelope protein-mediated membrane fusion and concentrates in lymph nodes.

The identification of fusin and other chemokine receptors as coreceptors for HIV-1 has renewed the interest in agents that may prevent viral entry. Polyanionic compounds such as dextran sulfate, curdian sulfate, and suramin act on the V3 loop of the viral envelope and may prevent its interaction with fusin. These agents show activity against a wide range of HIV-1 strains, but have undesirable circulating half-life, bioavailability, and toxicity. We have developed a small molecule inhibitor of HIV-1 that has several advantages over these other agents. FP-21399 is a novel compound of the bis(disulfonaphthalene) dimethoxybenzene class that blocks entry of HIV into CD4+ cells and blocks fusion of infected and noninfected CD4+ cells. This compound only weakly inhibits binding of CD4 and gp120, at concentrations much greater than are required to block viral entry. Furthermore, FP-21399 can block the interaction between gp120 and antibodies directed against the V3 loop, but does not block binding of antibodies directed against the V4 loop. Animal studies demonstrate that FP-21399 is concentrated in lymph nodes, making it a promising compound for anti-HIV therapy. In SCID mice reconstituted with human immune cells, maintenance of HIV-1 infection was blocked by a 5-day treatment with low doses of FP-21399, suggesting that lymph node accumulation may contribute to antiviral activity. Finally, attempts to generate drug-resistant virus in cell culture resulted in only weakly resistant variants with IC90 values that are much lower than concentrations of FP-21399 found in lymph nodes.

Ablation of paternal accessory sex glands imparts physical and behavioural abnormalities to the progeny: an in vivo study in the golden hamster.

The functional significance of male accessory sex glands (ASG) remains unclear. This study explored their importance in reproduction. In previous investigations, embryos sired by males with ASG either totally or partially removed had a shift in the cell cycle and delayed cleavage during preimplantation development, higher incidence of apoptosis, early oviductal-uterine transit, higher proportion of embryo degeneration, lower implantation rate, and ultimately reduced fertility and fecundity. Some pups were born alive; but would they be normal? We hypothesized that the first generation offspring (F1) could also bear undesirable traits. To test our hypothesis, we raised and studied these F1 pups from birth to 8 weeks. We monitored physical growth and assessed behaviour such as nest patch odor preference, acoustic startle response (ASR) and exploratory activity. We detected deviations from the norm in physical growth, a premature cessation of nest patch odor preferences, accelerated acoustic startle habituation and more frequent rearing when exposed to a novel environment. In terms of structure, we found one incidence of diphallus with duplicated urethra. We concluded that sperm lacking contact with ASG secretions gave rise to progeny with abnormal traits.

Endostatin therapy reveals a U-shaped curve for antitumor activity.

Developing continuous systemic delivery of endostatin has been a goal of many laboratories. We have employed a method of gene therapy utilizing different viral constructs. Here, we report that a new serotype of adeno-associated viruses, which incorporates canine endostatin, provides dose-dependent transgene expression in the circulation after intramuscular injection in mice. Elevated levels of endostatin remained stable in the circulation for at least 4 months. In vitro assays determined that the protein expressed was biologically active. Antitumor activities of the above construct demonstrated a U-shape curve, where the maximum activity was observed within a certain critical concentration range. These data suggest that an optimum dose range may be required to achieve therapeutic efficacy in large animal models.

Effect of antiangiogenic therapy on slowly growing, poorly vascularized tumors in mice.

BACKGROUND: Angiogenesis is essential for tumor growth and progression. Therefore, inhibition of angiogenesis is being studied as a new anticancer therapy. Because cytotoxic chemotherapy is more effective on rapidly growing tumors than on slowly growing tumors, it has been assumed that antiangiogenic therapy will also be effective only on rapidly growing, highly vascularized tumors. We compared the effects of two angiogenesis inhibitors, TNP-470 and angiostatin, on slowly growing, poorly vascularized and rapidly growing, highly vascularized human tumors in mice. METHODS: Slowly growing (RT-4) and rapidly growing (MGH-U1) human bladder carcinoma cell lines were grown in severe combined immunodeficiency mice. Established tumors were treated with one of the two angiogenesis inhibitors. Tumor volumes, vascularity, and proliferation indices were determined. The in vitro effects of TNP-470 and of angiostatin on the proliferation of RT-4 and MGH-U1 cells were also investigated. All statistical tests were two-sided. RESULTS: RT-4 and MGH-U1 tumor growth was statistically significantly inhibited by both angiogenesis inhibitors (P<.001). Both inhibitors decreased the blood vessel density in both tumor types but did not alter the in vivo proliferation indices of the tumors. TNP-470, but not angiostatin, marginally decreased the in vitro proliferation of MGH-U1 cells. CONCLUSION: Slowly growing, poorly vascularized tumors in animal models respond as well as rapidly growing, highly vascularized tumors to therapy with the angiogenesis inhibitors TNP-470 and angiostatin.

Improving the efficacy of antibody-interleukin 2 fusion proteins by reducing their interaction with Fc receptors.

Fusion proteins between whole antibodies (Abs) and cytokines (immunocytokines) such as interleukin 2 have shown efficacy in several mouse tumor models despite a circulating half-life that is significantly shorter than that of the original Ab. We have examined the potential mechanisms responsible for clearance and shown that an important factor is enhanced binding to Fc receptor (FcR). Improvements in the half-lives of two different immunocytokines were made by changing the isotype of the human heavy chain C region from IgG1 or IgG3 to those with reduced binding to FcR, e.g., IgG4. The same effect could also be achieved through site-directed mutagenesis of the FcR binding site in the IgG1 H chain. In vitro studies using mouse J774 FcR-expressing cells showed increased binding of interleukin 2-based immunocytokines, relative to their corresponding Abs, and that this was reversed in those fusion proteins made with IgG4 or mutated IgG1 H chains. All of the fusion proteins showing reduced FcR binding also had reduced Ab-dependent cellular cytotoxicity activity, as measured in 4-h chromium release assays. A complete loss of complement-dependent cytotoxicity activity was seen with an IgG4-based immunocytokine derived from an IgG1 Ab with potent activity. Despite these reduced effector functions, the IgG4-based immunocytokines with extended circulating half-lives showed equivalent (in the case of severe combined immunodeficiency mouse xenograft models) or better (in the case of syngeneic models) efficacy in mouse tumor models than the original IgG1-based molecules. These novel immunocytokines may show improved efficacy in therapeutic situations where T cell- rather than natural killer- or complement-mediated antitumor mechanisms are involved.

Elimination of established murine colon carcinoma metastases by antibody-interleukin 2 fusion protein therapy.

A recombinant humanized antibody-interleukin 2 fusion protein (huKS1/4-IL-2) was used to direct IL-2 to the tumor microenvironment and elicit a T cell-mediated eradication of established pulmonary and hepatic CT26-KSA colon carcinoma metastases in syngeneic BALB/c mice. This antitumor effect was specific because a fusion protein, which was nonreactive with these tumor cells, failed to exert any such effect. The efficacy of the huKS1/4-IL-2 fusion protein in eliminating metastases was documented because mixtures of monoclonal antibody huKS1/4 with recombinant human IL-2 were ineffective and, at best, only partially reduced tumor load. Two lines of evidence indicated the eradication of metastases and the absence of minimal residual disease in animals treated with the fusion protein: first, the lack of detection of CT26-KSA cells by reverse transcription-PCR, which can detect one tumor cell in 10(6) liver cells; and second, the tripling of life span. The effector mechanism involved in this tumor eradication is dependent on T cells because the IL-2-directed therapy is ineffective in T cell-deficient SCID mice. The essential effector cells were further characterized as CD8+ T cells by in vivo depletion studies. Such T cells, isolated from tumor-bearing mice after fusion protein therapy, elicited MHC class I-restricted cytotoxicity in vitro against colon carcinoma target cells. Taken together, these data indicate that fusion protein-directed IL-2 therapy induces a T cell-dependent host immune response capable of eradicating established colon cancer metastases in an animal tumor model.

HRad17, a human homologue of the Schizosaccharomyces pombe checkpoint gene rad17, is overexpressed in colon carcinoma.

Using the palindromic PCR-cDNA display method, we have cloned a novel gene overexpressed by human colon carcinoma relative to normal colon. Among normal tissues examined, only testis expresses it at a high level. Sequence analysis revealed its extensive homology with checkpoint genes rad17 of Schizosaccharomyces pombe and RAD24 of Saccharomyces cerevisiae. This novel gene designated as hRad17 is localized to chromosome 5q12,13.1, a region known to be deleted in a variety of human cancers. Promoter region and one pseudogene of hRad17 have been identified. Whereas the increased expression of hRad17 by human colon carcinomas may be related to the known resistance of these cells to DNA-damaging agents during therapy, the deletion of hRad17 in a variety of cancers may predispose them to increased rate of mutation and heightened sensitivity to DNA-damaging agents, including radiation and anticancer drugs.

High level expression of human proteins in murine hybridoma cells: induction by methotrexate in the absence of gene amplification.

We have developed an inducible system for high level expression of heterologous genes in murine hybridoma cells. The rapid induction by methotrexate (MTX) does not involve gene amplification and is controlled at the level of mRNA accumulation. Transfection was achieved by protoplast fusion with an expression vector containing the cDNA of interest and a marker gene encoding dihydrofolate reductase. The initial clones, selected at 100 nM MTX, produced high levels of the protein of interest and contained about 100-400 copies of the integrated plasmid DNA. They could adapt to a 100- to 1000-fold stepwise increase in MTX concentration in a few weeks, during which the expression of the gene of interest but not its copy number, increased several-fold. Furthermore, the induction is freely reversible. If cells were propagated in MTX-free media, the expression level decreased, but the cells could be reinduced to their original high level of expression by adding MTX back to the media. A several-fold increase in the mRNA levels of the dihydrofolate reductase and the gene of interest could be detected after induction for 18 h.

Randomized trial of black cohosh for the treatment of hot flashes among women with a history of breast cancer.

PURPOSE: Most breast cancer survivors experience hot flashes; many use complementary or alternative remedies for these symptoms. We undertook a randomized clinical trial of black cohosh, a widely used herbal remedy for menopausal symptoms, among breast cancer patients. PATIENTS AND METHODS: Patients diagnosed with breast cancer who had completed their primary treatment were randomly assigned to black cohosh or placebo, stratified on tamoxifen use. At enrollment, patients completed a questionnaire about demographic factors and menopausal symptoms. Before starting to take the pills and at 30 and 60 days, they completed a 4-day hot flash diary. At the final visit, they completed another menopausal symptom questionnaire. Follicle-stimulating hormone (FSH) and luteinizing hormone (LH) levels were measured in a subset of patients at the first and final visits. RESULTS: Of 85 patients (59 on tamoxifen, 26 not on tamoxifen) enrolled in the study, 42 were assigned to treatment and 43 were assigned to placebo; 69 completed all three hot flash diaries. Both treatment and placebo groups reported declines in number and intensity of hot flashes; the differences between the groups were not statistically significant. Both groups also reported improvements in menopausal symptoms that were, for the most part, not significantly different. Changes in blood levels of FSH and LH also did not differ in the two groups. CONCLUSION: Black cohosh was not significantly more efficacious than placebo against most menopausal symptoms, including number and intensity of hot flashes. Our study illustrates the feasibility and value of standard clinical trial methodology in assessing the efficacy and safety of herbal agents.

Pilot study of tandem high-dose chemotherapy and autologous stem cell transplantation with a novel combination of regimens in patients with poor risk lymphoma.

In an effort to improve the outcome of poor-risk lymphoma patients, we evaluated a novel regimen of tandem high-dose chemotherapy (THDC) with autologous stem cell transplantation. A total of 41 patients (median age 40 years, range 15-68 years) with poor-risk non-Hodgkin's lymphoma and Hodgkin's disease were enrolled. THDC consisted of melphalan (180 mg/m2) and escalating dose mitoxantrone (30-50 mg/m2) (MMt) for the first conditioning regimen, and thiotepa (500 mg/m2), carboplatin (800 mg/m2), and escalating dose etoposide phosphate (400-850 mg/m2), (ETCb) as the second regimen. In all, 31 patients (76%) completed both transplants, with a median time between transplants of 55 days (range 26-120). The maximum tolerated dose was determined as 40 mg/m2 for mitoxantrone and 550 mg/m2 for etoposide phosphate. The overall toxic death rate was 12%. Following high-dose chemotherapy, 10 of 24 evaluable patients (42%) were in CR. The two-year overall survival and event-free survival is 67% (95% CI, 52-81%) and 45%, (95% CI, 29-61%) for the 41 patients enrolled; and 69% (95% CI, 525-586%) and 48% (95% CI, 30-67%) for the 31 patients completing both transplants. This THDC regimen is feasible but with notable toxicity in heavily pretreated patients; its role in the current treatment of high-risk lymphoma remains to be determined.

Fractures after stroke: frequency, types, and associations.

BACKGROUND AND PURPOSE: Stroke patients may have an increased risk of fractures because of weak bones or an increased risk of falling. Our goal was to estimate the frequency of fracture after stroke and to identify those at greatest risk. METHODS: This study incorporated 2 complementary strategies: a prospective, single-center, cohort study and an analysis of Scottish routine hospital discharge data. RESULTS: Eighty-eight fractures (30% hip) occurred in 2696 hospital-referred stroke patients. The proportions sustaining any fracture or hip fracture within 2 years were 4% and 1.1%, respectively, 1.4 (95% CI, 0.92 to 2.07) times the rate of hip fracture in the general population (ie, observed number divided by expected number or standardized morbidity ratio). Female sex, older age, low abbreviated mental test score, and prestroke dependence were associated with an increased hip fracture rate. Routine data identified 129 935 acute stroke patients admitted to Scottish hospitals. During 363 447 patient-years, 4528 patients had hip fractures, 2.0% had fractures by 1 year, and 10.6% had fractures by 10 years. This is 1.7 times the rate of hip fracture in the general population and 2.3 times that in patients with myocardial infarction. Older patients predictably had the highest rate of poststroke hip fractures but a lower standardized morbidity ratio than younger patients. CONCLUSIONS: Fractures after stroke are probably frequent and serious enough to justify the development of preventive strategies, but the modest event rate would mean that randomized, controlled trials to test these strategies specifically in stroke patients would need to enroll thousands of patients.

The use of a wild-type dihydrofolate reductase-encoding cDNA as a dominant selectable marker and induction of expression by methotrexate.

The cDNA (DHFR) encoding the wild-type (wt) dihydrofolate reductase (DHFR) was used as a dominant selectable marker in the transfection of murine hybridoma Sp2/0-Ag14 cells by protoplast fusion. The initial clones contained 100-400 copies of integrated plasmid DNA, and the high level of wt DHFR protein produced enabled the cells to survive the drug selection at 100 nM methotrexate (MTX). The expression of the gene of interest was several fold higher than when the mutant DHFR with decreased MTX binding was used as the selection marker, presumably because the clones were more sensitive to the stress induced by MTX. When the clones were propagated at higher concentrations of MTX, expression of both DHFR and the gene of interest increased. This induction is freely reversible, and we have shown that it is controlled at the transcriptional level, by nuclear run-off transcription assays.

High-level expression of chimeric antibodies using adapted cDNA variable region cassettes.

A rapid and generally applicable method for the modification of immunoglobulin cDNAs was developed so that the variable (V) regions could be expressed as cassettes, together with a variety of constant regions. Murine cDNAs were isolated, sequenced and the V regions joined to short oligonucleotides providing both splice donor sites and unique restriction sites for insertion into an expression vector. Using this strategy we have expressed the V regions of several murine antibodies, together with the human gamma 1 constant region. Although most of these chimeric antibodies were readily expressed, one murine light-chain cDNA sequence could not be expressed in transfected hybridoma cells. Reconstruction experiments indicate that the sequence created by the fusion of the murine leader and variable region blocked expression at the level of RNA accumulation. The methods described, as well as the potential problems of expression, are applicable to both traditional cDNA fragments and those obtained by in vitro amplification techniques.