Sustained intrathecal therapeutic protein delivery using genetically transduced tissue implants in a freely moving rat model.

Systemic delivery of therapeutic proteins to the central nervous system (CNS) is challenging because of the blood-brain barrier restrictions. Direct intrathecal delivery is possible but does not produce stable concentrations. We are proposing an alternative approach for localized delivery into the CNS based on the Transduced Autologous Restorative Gene Therapy (TARGT) system. This system was previously developed using a gene therapy approach with dermal tissue implants. Lewis rat dermal tissue was transduced to secrete human EPO (hEPO). TARGT viability and function were retained following cryopreservation. Upon implantation into the rat cisterna magna, a mild inflammatory response was observed at the TARGT-brain interface throughout 21-day implantation. hEPO expression was verified immunohistochemically and by secreted levels in cerebrospinal fluid (CSF), serum, and in vitro post explant. Detectable CSF hEPO levels were maintained during the study. Serum hEPO levels were similar to rat and human basal serum levels. In vitro, the highest hEPO concentration was observed on day 1 post-explant culture and then remained constant for over 21days. Prolonged incubation within the cisterna magna had no negative impact on TARGT hEPO secretion. These promising results suggest that TARGTs could be utilized for targeted delivery of therapeutic proteins to the CNS.

Helicobacter pylori eradication as a surrogate marker for the reduction of duodenal ulcer recurrence.

OBJECTIVES: An abundance of data exists documenting the association of H. pylori eradication with the reduction in duodenal ulcer recurrence. AIM: To evaluate the validity of using H. pylori eradication as a surrogate marker for the reduction in duodenal ulcer recurrence using rigorously controlled studies. METHODS: Three controlled clinical trials were conducted in patients with uncomplicated, active duodenal ulcers. Patients were treated with various combinations of omeprazole and amoxycillin. Ulcer healing and H. pylori eradication were assessed. For patients whose duodenal ulcer healed, duodenal ulcer recurrence was determined over a 6-month period in patients with H. pylori eradication and those remaining positive for H. pylori at least 4 weeks after treatment. To support the data obtained from these clinical trials, a search of the medical literature was conducted to identify additional human clinical trials in which duodenal ulcer recurrence rates were measured and categorized by H. pylori status at least 1 month post-treatment. RESULTS: In 11 controlled trials, the overall 6-18-month duodenal ulcer recurrence rate was 54% among patients remaining positive for H. pylori at least 4 weeks after treatment compared to 6% among patients with H. pylori eradication following treatment. This finding was corroborated by the uncontrolled trials, in which the duodenal ulcer recurrence rate was 64% among patients found to be H. pylori-positive and 6% for patients found to be H. pylori-negative at least 4 weeks after treatment. A time course of duodenal ulcer recurrence rates using pooled data from both controlled and uncontrolled studies demonstrated that duodenal ulcer recurrence rates for H. pylori-negative patients persisted for up to 4 years following treatment. Duodenal ulcer recurrence rates for H. pylori-positive patients increased for the first year, then levelled off. A comparison of the duodenal ulcer recurrence rates for different treatment regimens revealed that eradication regimens based on omeprazole plus antibiotics and bismuth plus antibiotics exhibited similar duodenal ulcer recurrence rates for H. pylori-positive and -negative patients. CONCLUSION: Regardless of treatment regimens, H. pylori eradication produced a consistent and significant reduction in duodenal ulcer recurrence. Therefore H. pylori eradication, 4 weeks post-therapy, can be used as a surrogate marker for reduced duodenal ulcer recurrence in investigational clinical trials.

A United States multicentre trial of dual and proton pump inhibitor-based triple therapies for Helicobacter pylori.

BACKGROUND: One-week proton pump inhibitor-based triple therapies are very popular in the US despite limited US data documenting efficacy. We assessed 1-week proton pump inhibitor triple therapies for Helicobacter pylori, and compared them to dual antibiotic therapies (to assess benefit of omeprazole) and to omeprazole-amoxycillin (to assess benefit of clarithromycin) in a large, randomized, US multicentre study. METHODS: Healthy subjects who were H. pylori-positive by rapid serological test and 13C-urea breath test were randomly assigned to (i) omeprazole (O) 20 mg b.d. + amoxycillin (A) 1 g t.d.s. for 14 days (OA); (ii) A 1 g b.d. + clarithromycin (C) 500 mg b.d. for 7 days (AC); (iii) C 250 mg b.d. + metronidazole (M) 500 mg b.d. for 7 days (CM); (iv) O 20 mg b.d. + C 250 mg b.d. + M 500 mg b.d. for 7 days (MOC); or (v) O 20 mg b.d. + C 500 mg b.d. + A 1 g b.d. for 7 days (OAC). Repeat breath tests were done at 6 weeks to assess H. pylori status. RESULTS: Three hundred and two H. pylori-positive subjects at 25 centres received medication. Intention-to-treat cure rate was significantly higher for OAC (82%) than for MOC (67%), CM (59%), AC (18%) or OA (58%), Per-protocol cure rates were 85% for OAC and 75% for MOC. Discontinuation of therapy due to a side-effect occurred in 0-3% of each study group. CONCLUSIONS: One-week twice-daily triple therapy with omeprazole, amoxycillin and clarithromycin provides the best rate of eradication of the five regimens studied. However, treatment in the US for 7 days may be unable to achieve eradication rates of > or = 90% with proton pump inhibitor-based triple therapy.

Do ulcers burn out or burn on? Managing duodenal ulcer diathesis in the Helicobacter pylori era. Ad Hoc Committee on FDA-Related Matters.

Now that the Food and Drug Administration is examining various treatment regimens for eradication of Helicobacter pylori (H. pylori) infection, the question of whom to treat has come to the forefront. Widespread attempts to eradicate the bacterium are not without risk, including the possibility of accelerating the emergence of resistant strains of the organism, in addition to possible adverse events of agents used to cure the infection. These risks are of concern to the Agency in the process of granting marketing approvals for various therapies. The association of H. pylori and duodenal ulcer (DU) is no longer disputed; however, data to date have been generated in studies of patients with active (acute) DU. In these patients, the benefits of eradication therapy are clear. Patients with a documented history of DU who do not have an ulcer crater at the time of presentation have not been well studied in controlled trials. If they are at similar risk for recurrence and complications, it follows that they, too, should be candidates for H. pylori testing and treatment. However, if, as some believe, duodenal ulcer disease becomes "inactive" or "burns out" with time, an argument could be made for expectant treatment for this subgroup of patients. The present review examines the available literature on the natural history of DU disease and explores the validity of the hypothesis of duodenal ulcer "burn out." Analysis of the data shows that there is little support for the phenomenon of duodenal ulcer disease "burn out," and that, in fact, DU disease resulting from H. pylori infection is a chronic, relapsing condition, lasting for decades, if not a lifetime. The literature is compatible with the view that, in the majority of patients, DU occurs on a background of an "ulcer diathesis" that is fueled by H. pylori infection, and "burns on, not out", until and unless the infection is extinguished. The true incidence of ulcer relapse is difficult to predict due to the common occurrence of asymptomatic ulcers and the poor correlation of symptoms with the presence of ulcer. Therefore, treatment of patients with a history of duodenal ulcer disease should be advocated, even if their ulcer disease is not "active," because the risk of recurrence and complications does not diminish unless the H. pylori bacterium is eradicated.

Time of Helicobacter pylori eradication assessment following treatment.

BACKGROUND: The most appropriate time to assess accurately Helicobacter pylori eradication following treatment has been debated, with recommendations ranging from 1 to 3 months. The purpose of this study was to validate the assessment of H. pylori eradication 1 month following treatment. MATERIALS AND METHODS: Three randomized, double-blind, active-controlled clinical trials were conducted in patients with endoscopically verified, active duodenal ulcers and H. pylori infection. Patients were treated with various treatment regimens of omeprazole plus amoxicillin. Ulcer healing, H. pylori eradication, and ulcer relapse were examined. Patients underwent repeat endoscopy and biopsy at 1 and 6 months following treatment (or sooner if symptoms returned) to determine the recurrence of ulcers and H. pylori status. To determine the accuracy of measuring H. pylori eradication at 1 month posttreatment, we compared the H. pylori status at 1 month and 6 months following treatment. RESULTS: In a combination of treatment groups and studies, a total of 384 evaluable patients represented data at both time points and were included in the analysis. Of those eradicated at 1 month posttreatment, 94% (141 of 150) remained eradicated at 6 months posttreatment. The proportion of patients with H. pylori eradicated at 1 month posttreatment did not differ significantly from that at 6 months posttreatment for each study. The overall efficiency of the two tests (agreement between tests) was 93% (359 of 384). Agreement between the 1-month and 6-month posttreatment H. pylori assessment was apparent, regardless of the treatment used. CONCLUSION: H. pylori eradication measured 1 month following cessation of treatment accurately reflects successful treatment of the infection.

Nerve growth factor synthesis by intestinal epithelial cells.

Nerve growth factor (NGF) exists in the gut of adult rats. The cells responsible for NGF synthesis in the gut remain unknown. IEC-6 and Caco-2 cells, established cell culture models of intestinal epithelial cells, were studied to determine whether intestinal epithelial cells, were studied to determine whether they synthesize and release NGF. Conditioned media from both IEC-6 and Caco-2 cells stimulated neurite outgrowth in both rat pheochromocytoma (PC-12) cells and sensory neurons derived from embryonic chick dorsal root ganglia (DRG). The addition of anti-NGF antibody blocked neurite outgrowth in PC-12 cells and partially blocked outgrowth in DRG cells. An NGF-enzyme-linked immunosorbant assay readily detected immunoreactive NGF in conditioned media from both cell lines, whereas cellular extracts from IEC-6, Caco-2, and isolated rat intestinal epithelial cells had low levels of immunoreactivity. Caco-2 monolayers primarily secreted NGF from the basolateral compartment, and interleukin-1 enhanced its secretion. IEC-6, Caco-2, and isolated rat intestinal epithelial cells expressed NGF mRNA as determined by reverse transcription polymerase chain reaction. These observations suggest that intestinal epithelial cells are capable of NGF synthesis.

Variability with omeprazole-amoxicillin combinations for treatment of Helicobacter pylori infection.

OBJECTIVE: Although omeprazole co-therapy enhances the effectiveness of some antimicrobials for the treatment of Helicobacter pylori infection, results have not been uniform. A meta-analysis suggested that 20 mg of omeprazole b.i.d. and 2 g or more of amoxicillin would yield a > 80% success rate (Gastroenterology 1994; 106: 142A). Our objective in this study was to test that hypothesis. METHODS: Volunteers with H. pylori infection were studied. Anti-H. pylori therapy was administered with meals for 14 days (omeprazole 20 mg b.i.d. plus amoxicillin 1 g t.i.d., or omeprazole 20 mg b.i.d. plus amoxicillin 0.5 g t.i.d.). Endoscopy was performed 4-6 wk after antimicrobial therapy ended, and the presence or absence of H. pylori was determined with biopsy specimens by Genta stain. RESULTS: Fifty-nine volunteers completed the study; 30 were studied twice. The overall success for initial treatment with either combination of amoxicillin and omeprazole was 18 of 59 [30.5%; 95% confidence interval (CI) = 19-44%]. The success rate with 500 mg amoxicillin t.i.d. was 7 of 29 (24%; 95% CI = 10-43%). With 1 g t.i.d. amoxicillin, the cure rate was higher (36.6%) (11 of 30; 95% CI = 20-56%), or intention-to-treat result was 11 of 31 (35.4%), which includes the early dropout. Compliance was > 95% for both therapies. Side effects were experienced by eight patients, two receiving 1.5 g amoxicillin and six receiving 3 g amoxicillin (p > 0.2). German trials suggest that better results might be achieved when amoxicillin is given as suspension while fasting. Thirty treatment failures were re-treated with 1 g amoxicillin suspension t.i.d., given fasting, and omeprazole 20 mg b.i.d. The cure rate was 16.6% (95% CI = 6-35%). CONCLUSION: Amoxicillin/omeprazole combinations for treatment of H. pylori infection do not yield consistent results. The reason is unknown, but the reported high rate of success with 40 mg of omeprazole and 750 mg t.i.d. suggests that almost complete inhibition of acid secretion is necessary to obtain consistent results with this combination.

Generation of a human monoclonal antibody to hepatitis C virus, JRA1 by activation of peripheral blood lymphocytes and hypo-osmolar electrofusion.

We have generated a human monoclonal antibody with binding specificity for hepatitis C virus (HCV)-specific peptides using peripheral blood lymphocytes isolated from a HCV antibody positive patient. The B-lymphocytes were stimulated with lipopolysaccharide (LPS) for 72 hours prior to the fusion. A recently described high efficiency hypo-osmolar electrofusion technique was employed, allowing generation of a large number of human hybridomas. The hybridomas were screened for human immunoglobulin and HCV-specific peptide binding by EIA. A single HCV-positive clone, JRA1, was detected and sub-cloned. Isotype analysis showed it to secrete an IgM lambda monoclonal antibody. The antibody was positive on both first and second generation HCV antibody analysis. This study confirms that viable pathogen-specific B-cells may be recovered from the peripheral blood. Although such cells are likely to be relatively uncommon in the circulating B-cell pool, they may be successfully immortalized by high efficiency electrofusion techniques. This technique might be valuable for the generation of human monoclonal antibodies with specificity for other human pathogens.

Caco-2 cells express type I interleukin-1 receptors: ligand binding enhances proliferation.

We examined the effect of interleukin-1 (IL-1) on the rate of proliferation of the human colon carcinoma Caco-2 and characterized the human intestinal epithelial cell IL-1 receptor (IL-1R). IL-1 dose dependently increased tritiated thymidine uptake in confluent Caco-2 monolayers fed complete growth medium. An anti-IL-1 beta completely blocked the increase in tritiated thymidine uptake, whereas an IL-1 receptor antagonist human recombinant blocked it partially. In long-term culture, IL-1 increased DNA content over control, an effect similar to that of epidermal growth factor (EGF). Unlike EGF, IL-1 did not enhance tritiated thymidine uptake in Caco-2 monolayers grown in serum-free medium, implying that IL-1 needs a cofactor(s) to elicit its proliferative effect. Cross-linking 125I-IL-1 beta to Caco-2 membranes revealed a binding protein of approximately 80 kDa with binding saturated at approximately 2.5 x 10(9) M-1 consistent with that for the type I IL-1R. cDNA transcribed from Caco-2 mRNA and amplified by polymerase chain reaction, using complementary oligonucleotides, resulted in a reaction product matching the sequence of the type I IL-1R. Our results demonstrate that IL-1 enhances proliferation of Caco-2 cells. This effect requires the presence of an unidentified cofactor(s). Also, Caco-2 cells express the type I IL-1R.

Induction of hepatic pathology in SCID-Hu mice engrafted with peripheral blood lymphocytes of patients with Schistosomiasis japonica.

SCID mice were engrafted with peripheral blood lymphocytes (PBL) derived from persons currently or previously infected with Schistosoma japonicum. After immunization with soluble worm antigenic preparation, the SCID-Hu mice were analyzed for a human immune response. ELISA revealed a low titer of human antibody recognizing soluble egg antigens in 2 of 10 mice. One mouse had detectable levels of interleukin (IL)-2 and gamma-interferon, TH1 phenotype cytokines. All mice had elevated levels of IL-4, a TH2 phenotype cytokine. The human cytokine profile of the mice paralleled the patient's serum profile at clinical examination. In addition, all mice had substantial hepatic pathology, including inflammatory cell infiltrates and macrovesicular fat deposition. The data indicate that activation of PBL from patients with a history of schistosomiasis japonica infection can result in focal hepatic pathology, which may be driven by specific cytokines.

Analysis of T-lymphocyte subpopulations in inflammatory bowel diseases by three-color flow cytometry.

In order to better define changes in the relative proportion of peripheral blood T-lymphocyte subpopulations in patients with inflammatory diseases of the bowel, we performed simultaneous three-color fluorescence-activated cytometric (FACS) analysis using fluorophore-conjugated monoclonal antibodies with specificity for CD4, CD8, Leu 8, and CD45RA on 22 normal control subjects, 28 patients with Crohn's disease (CD), 15 patients with ulcerative colitis (UC), and 11 patients with intestinal inflammation secondary to etiologies other than inflammatory bowel disease (NIBD). This staining combination allowed enumeration of distinct T-cell subpopulations as follows: virgin CD4+, recall antigen helper T cells, nonspecific B-cell helper T cell, virgin CD8+, cytotoxic effector and suppressor effector and recall antigen cytotoxic T cells based on a synthesis of published functional analyses. No differences in the proportion of CD4+ or CD8+ cells or in the CD4+/CD8+ ratios were evident when UC and NIBD patients were compared to normal subjects. A significant reduction in the proportion of CD4+ cells and an increase in CD8+ cells was observed, however, in the CD group. When two-color analysis was performed, several significant differences in the proportions of circulating lymphocytes were seen. Specifically, these included significant increases in the number of CD4+, Leu 8- (P < 0.01) cells in all disease groups and an increase in CD4+, CD45RA+ cells in the NIBD group. Conversely, significant decreases in the proportions of CD8+, Leu 8+ (P < 0.01) cells were evident in the Crohn's disease group.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification and characterization of the rat intestinal epithelial cell (IEC-18) interleukin-1 receptor.

Interleukin-1 (IL-1), an important mediator of inflammation, may act in chronic inflammatory disorders of the intestine such as idiopathic inflammatory bowel diseases. Although the IL-1 receptor (IL-1R) has been studied extensively in many cell lines, including T cells, B cells, and fibroblasts, it has not been demonstrated on intestinal epithelial cells. IL-1 affects intestinal epithelial cell proliferation in vitro, suggesting the presence of IL-1Rs on intestinal epithelium. This paper demonstrates and further characterizes an IL-1R in a rat intestinal epithelial cell. Cells from rat intestinal epithelial cell line IEC-18 were grown to confluence in six-well plates, and association studies with 125I-labeled IL-1 beta to determine specific binding and equilibrium conditions were performed. A competitive-inhibition curve verified the IL-1 concentration required to saturate the IL-1R, and a Scatchard plot revealed a dissociation constant (Kd) of 1.2 x 10(-10) M, with 1,000 receptors per epithelial cell. Binding studies using increasing concentrations of 125I IL-1 beta alone confirmed the receptor density. Cross-linking 125I-IL-1 beta to the IEC-18 IL-1R demonstrated an IL-1R of approximately 80.5 kDa. cDNA transcribed from IEC-18 mRNA was used as a template for amplification of a segment of the IL-1R using complementary oligonucleotides. The resulting sequence of the IL-1R demonstrated a high degree of homology with both the human and mouse type I IL-1R.(ABSTRACT TRUNCATED AT 250 WORDS)

Drainage of the gallbladder in patients with acute acalculous cholecystitis by transpapillary endoscopic cholecystotomy.

The mortality associated with acute acalculous cholecystitis approaches 50%. Removal or decompression of the gallbladder in these patients may prevent gallbladder rupture and may be lifesaving. This is usually accomplished by cholecystectomy, cholecystotomy, or percutaneous gallbladder drainage. We describe a novel transpapillary endoscopic approach to gallbladder drainage in patients at high surgical risk. A total of seven high surgical risk patients were treated with transpapillary endoscopic cholecystotomy. Cannulation of the cystic duct was accomplished by using standard hourglass-tipped catheters in two patients. A new "selector" catheter was developed for selective cannulation of the cystic duct and used in the other five patients. Five of the seven patients showed evidence of clinical, radiographic, and laboratory improvement after treatment. We conclude that transpapillary endoscopic cholecystotomy may be an effective treatment alternative for high surgical risk patients with acalculous cholecystitis.

Antineutrophil cytoplasmic antibody in inflammatory bowel and hepatobiliary diseases. High prevalence in ulcerative colitis, primary sclerosing cholangitis, and autoimmune hepatitis.

The prevalence of antineutrophil cytoplasmic antibodies was evaluated in patients with ulcerative colitis, primary sclerosing cholangitis, and various other gastrointestinal and hepatobiliary diseases to define the sensitivity and specificity of the test. The presence of antineutrophil cytoplasmic antibodies was detected in alcohol-fixed cytospin preparations of peripheral blood neutrophils with an indirect immunofluorescence technique. A perinuclear staining pattern was considered positive. Thirty-six of 50 patients (72%) with ulcerative colitis and/or primary sclerosing cholangitis had positive results. Twenty-two of 210 patients (10%) in the control group had positive findings, including a significant proportion of patients with autoimmune hepatitis (50%) and non-A, non-B and non-C hepatitis (27%). This test for antineutrophil cytoplasmic antibodies has a sensitivity of 72% and specificity of 90% for either ulcerative colitis or primary sclerosing cholangitis. It may be useful in the differential diagnosis of Crohn's disease and ulcerative colitis and in the early diagnosis of ulcerative colitis. It also may be employed to distinguish primary biliary cirrhosis from primary sclerosing cholangitis.

Immunization of SCID-Hu mice and generation of anti-hepatitis B surface antigen-specific hybridomas by electrofusion.

Human hybridomas with specificity for recombinant hepatitis B surface antigen (HBSAg) were produced by adoptive transfer of human peripheral blood mononuclear cells prepared from HBSAg-immune donors to CB.17 mice bearing the severe combined immunodeficiency (SCID) phenotype. A total of ten SCID-Hu mice were immunized with recombinant HBSAg. Eight SCID-Hu mice found to have human HBSAg antibody in their serum were sacrificed, and single-cell suspensions were made from their spleens. The SCID-Hu spleen cells were electrofused to the mouse-human fusion partner H7. HBSAg-specific hybridomas were recovered from all fusions. This method may provide the means for the production of other human hybridomas and may, in some cases, circumvent the need for in vitro immunization or Epstein-Barr virus transformation of human B lymphocytes.

Omeprazole heals mucosal ulcers associated with endoscopic injection sclerotherapy.

Endoscopic injection sclerotherapy (EIS) is a standard and definitive therapy for bleeding esophageal varices. While the overall complication rate of the procedure is low, a substantial minority of patients treated by EIS develop refractory mucosal ulceration and/or esophageal strictures. However, despite the prophylactic use of H2 blockers and sucralfate in our EIS protocol, we observed a number of patients who developed nonhealing esophageal and/or gastroduodenal ulceration. We conducted an open trial in which we enrolled nine patients who had refractory ulcer disease. Patients that enrolled in the trial exhibited complete healing of their mucosal ulcers. These results suggest that acid is an important contributory factor in the pathogenesis and perpetuation of EIS-associated mucosal ulceration. Patients with alcohol-associated liver disease may be at increased risk for the development of EIS-associated complications.

CD5+ B cells are decreased in peripheral blood of patients with Crohn's disease.

B cells bearing the CD5 surface marker comprise a substantial minority of the circulating lymphocyte population in healthy individuals. These recently described cells have been implicated in T-independent humoral responses, immunoregulation, and autoimmunity. We undertook to enumerate circulating CD5+ B cells by three-color fluorescence activated flow cytometry in 28 patients with Crohn's disease (CD). None of the CD patients were using immunosuppressive medication. The CD patients were subdivided into "inactive" and "active" groups based upon their Crohn's disease activity index (CDAI). Thirty-two normal subjects served as a control population. The percentage of CD19+ B cells was significantly reduced in both active and inactive CD patients as compared with normal controls (P less than or equal to 0.01). CD5+ B cells were likewise found to be significantly decreased in both inactive and active CD patients (P less than or equal to 0.01) as compared with normal controls. The proportion of CD5+ B cells was significantly lower in the peripheral blood of active as compared with inactive CD patients (P less than or equal to 0.05). The finding that CD5+ B cells are reduced in CD may provide an important clue to immunological dysfunction in inflammatory bowel disease and merits further study.

The effect of lowering faecal pH on the rate of proliferation of the normal colonic mucosa.

Epidemiological and animal studies suggest that faecal pH may be a risk factor for colorectal cancer with low faecal pH associated with a lower incidence of the disease. The aim of this study was to determine whether faecal pH (or dietary fibre) affects the short-term risk factors for colon cancer. Sixty-nine normal volunteers were randomized into three equal groups (A-C). They provided food records, faecal specimens and submitted to rectal biopsy for thymidine labelling studies before and after a 2-week intervention. Group A received a placebo of fruit juice. Group B, approximately 3.0 g d-1 sodium sulphate in juice. Group C, 30 g d-1 supplementary dietary fibre as wheat bran in bread. Age, sex, weight, height and intake of macronutrients and minerals were similar in the groups prior to intervention. Faecal pH was similar for the three groups before and was reduced in Group B after intervention (P = 0.001) with a relative reduction of 0.5 pH units. The labelling index for the three groups was similar prior to intervention; after, it was lowest in Group B with a relative reduction of 0.5% points, although this difference was not statistically significant. The results thus do not support the hypothesis that an acidification of faecal pH leads to a reduction in risk markers for colon cancer.