Infectivity enhancement for adenoviral transduction of canine osteosarcoma cells.

The full realization of conditionally replicative adenoviruses (CRAds) for cancer therapy has been hampered by the limited knowledge of CRAd function in vivo and particularly in an immunocompetent host. To address this issue, we previously proposed a canine adenovirus type 2 (CAV2)-based CRAd for clinical evaluation in canine patients with osteosarcoma (OS). In this study, we evaluated infectivity-enhancement strategies to establish the foundation for designing a potent CAV2 CRAd with effective transduction capacity in dog osteosarcoma cells. The results indicate that the native CAV2 fiber-knob can mediate increased binding, and consequently gene transfer, in both canine osteosarcoma immortalized and primary cell lines relative to previously reported Ad5 infectivity-enhancement strategies. Gene delivery was further enhanced by incorporating a polylysine polypeptide onto the carboxy terminus of the CAV2 knob. This vector demonstrated improved gene delivery in osteosarcoma xenograft tumors. These data provide the rationale for generation of infectivity-enhanced syngeneic CAV2 CRAds for clinical evaluation in a dog osteosarcoma model.

Utilization of umbilical cords to assess in utero exposure to persistent pesticides and polychlorinated biphenyls.

In support of a study to relate developmental and cognitive effects with prenatal exposure to selected environmental toxicants, we developed and applied an analytical method to determine the concentration of two persistent pesticides, hexachlorobenzene (HCB) and p,p'-dichlorodiphenyldichloroethylene (DDE), and 32 specific polychlorinated biphenyl (PCB) congeners in 316 umbilical cords taken in 1986-1987 from women of the Faroe Islands. The analytical method consisted of homogenization of the cords, partitioning, microsilica gel column chromatography for clean-up, and dual-column capillary gas chromatography (DB-5 and DB-1701) with electron capture detection. Several quality control parameters were followed to monitor the performance of the method. Important criteria used before reporting unknown data were the recovery of in vitro-spiked analytes from a bovine umbilical cord (BUC) and the percentage lipid obtained for a Certified Reference Material (CRM)-350 of mackerel oil (MO). Recoveries of analytes that had been spiked at two concentration ranges (0.26-0.95 ng/g whole weight; 0.35-2.42 ng/g whole weight) into bovine cords ranged from 38.5% to 158% and from 50.4% to 145%, respectively, with a median recovery of 77.7%. Measurement of the percentage lipid for CRM-350 ranged from 73.8% to 107% with a median lipid value of 96.0%. The most prevalent analytes detected (%) in unknown umbilical cords were HCB (100), DDE (100), Ballschmiter/Zell PCBs 153 (100), 138 (98), 180 (98), 170 (93), 118 (88), 187 (86), and 146 (83), with corresponding median concentrations (ng/g whole weight) of 0.17, 1.19, 0.38, 0.30, 0.17, 0.11, 0.12, 0.09, and 0.07, respectively. Total PCB--sum of all measurable PCB congeners--had a median concentration of 1.37 ng/g whole weight. The analytes, which were very low in lipid content were also quantified on a lipid-adjusted basis, which provided an analytical challenge in these umbilical cord samples. The gravimetrically measured lipids in the human specimens ranged from 0.01% to 1.43% (median of 0.18%). In the pooled BUCs, our lipid measurements varied from 0.05% to 0.33% with a median value of 0.13%. The utility of using the umbilical cord as a matrix to assess in utero exposure to persistent environmental pollutants, compared with the use of umbilical cord blood or mother's blood, is worthy of debate.

A case-control assessment of risk factors for gallbladder carcinoma.

Gallbladder carcinoma is an uncommon, but highly fatal disease. Its symptoms frequently mirror those of gallstone disease, and in most instances, diagnosis is an incidental finding at surgery. While risk factors have been suggested for this cancer, many may in reality simply be a consequence of the older age of the population. This study is one of the few to approach this question by using a case-control study design comparing gallbladder carcinoma patients with a gallstone population, coupled with multivariate analysis to determine age-independent risk factors. Univariate analyses showed gallbladder carcinoma patients to be older than gallstone patients and to have many age-associated diseases. Following multiple regression adjustment for age, this disease was associated with female gender and with a previous history of gallstone symptoms. Carcinoma patients were less likely to have cholesterol gallstones in their gallbladders at surgery. A previous history of smoking was a substantial risk but of borderline statistical significance. Previous studies report associations that may be due to the older age of the gallbladder carcinoma patient. Our results show that after adjusting for age with multivariate analysis, gallbladder cancer subjects were predominantly female, more likely to report previous gallstone symptomology, and to smoke. While gallstones were not universally isolated from carcinoma patients at cholecystectomy, when present, they were less frequently classified as cholesterol gallstones based on visual inspection. Further cohort studies which target these populations will allow us to gain a more solid consensus on the risk factors for this disease.

Flow microsphere immunoassay-based method of virus quantitation.

A sensitive assay for adenovirus quantitation in vitro was developed using the flow microsphere immunoassay (FMIA) approach. Polystyrene microspheres were covalently coated with purified anti-adenoviral antibodies and incubated with virus-containing samples. After incubation, the samples were stained with DNA-specific fluorescent dyes, and microsphere-associated fluorescence was quantitated with a flow cytometer. The adsorption of virus to microspheres was examined under different experimental conditions. The flow cytometric assay was determined to be as accurate in detecting adenovirus as titering on 293 cells. The proposed method can be used to quantify virus in viral stocks and in biological samples.

Humoral and cellular immune responses of dogs immunized with a nucleic acid vaccine encoding human carcinoembryonic antigen.

A nucleic acid vaccine encoding human carcinoembryonic antigen (CEA) was administered to 10 juvenile dogs, 10-15 weeks of age, by four parenteral routes. The routes tested were intramuscular injection using a needle and syringe, intramuscular injection using the Biojector needleless injection device, intradermal injection or intravenous injection. All groups received 150 micrograms of plasmid DNA on weeks 0, 4, 7 and 13. All dogs were bled weekly for 17 weeks and tested for antibody against human CEA. Dogs given plasmid intramuscularly either by needle and syringe or Biojector showed significant antibody responses by week 9 which peaked by week 15. Dogs receiving plasmid intravenously showed slight, unsustained increases in antibody titers while dogs receiving plasmid intradermally had significant titers, but at levels approximately one log less than those induced by intramuscular injection. The five dogs immunized by intramuscular delivery of plasmid DNA were examined for cellular immune responses to human CEA by lymphoblast transformation (LBT) assay. All five showed significant CEA-specific lymphoproliferation when compared with unvaccinated dogs. Physical examination, clinical chemistry, hematology and histopathology examinations revealed no abnormalities associated with nucleic acid immunization.

Variables associated with anticipatory nausea and vomiting in pediatric cancer patients receiving ondansetron antiemetic therapy.

Investigated the prevalence of anticipatory nausea and vomiting (ANV) among 59 pediatric cancer patients who had routinely received ondansetron (Zofran) antiemetic therapy and determined patient- and treatment-related factors associated with ANV. Of the sample, 59% indicated at least mild ANV symptoms, suggesting that a significant number of patients report ANV and are bothered by it, despite the use of Zofran. These children were compared to those reporting no ANV symptoms. Most ANV symptomatology was consistent with a traditional classical conditioning model although cognitive processes may also play a role. Children with greater expectations of severe postchemotherapy vomiting and those who were more distressed by nausea and vomiting were more likely to experience ANV symptoms. Implications for psychological and pharmacological treatments of ANV are discussed.

Nutritional and treatment-related characteristics of pediatric oncology patients referred or not referred for nutritional support.

Nutritional problems often result from malignancies and aggressive multimodal treatment. Early identification of reliable risk factors associated with malnutrition and need for nutritional support is necessary for development of preventative approaches. Nutritional and treatment-related characteristics were examined for 173 pediatric oncology patients referred for nutritional support and a comparison sample of 43 patients matched on treatment protocol and/or diagnosis who had never been referred for nutritional support. Abnormally low serum albumin levels, poor oral intake, mucositis, prior radiation therapy, and increased gastrointestinal toxicity were significantly more frequent among referred than non-referred patients. A discriminant function analysis indicated that poor oral intake was the single best predictor of need for nutritional support. Patients with solid tumors were more nutritionally depleted at the time of referral; all bone marrow transplant patients received nutritional support. Patients with central nervous system (CNS) tumors required nutritional support for longer time periods. We conclude that routine documentation of poor oral intake (i.e., observation of change in a child's eating patterns) is the most reliable indicator of children who eventually require nutritional support and who may benefit from interventions that could delay or prevent nutritional problems. Prophylactic interventions should be tailored to meet the specific needs of individual diagnostic groups.

Squamous metaplasia of extrahepatic biliary system in an AIDS patient with cryptosporidia and cholangitis.

AIDS-related cholangiopathy is an increasingly recognized syndrome associated with significant morbidity and mortality. The mechanism of cholangiopathy is unknown but is assumed to be related to infectious pathogens such as CMV and cryptosporidia. The case of a Haitian with HIV and long-standing intestinal cryptosporidiosis who presented with cholangitis and protuberant intrabiliary filling defects is reported. Histopathological examination of biliary biopsies revealed previously unreported extensive squamous metaplasia of the bile duct epithelium, and the histogenesis of this condition is discussed. Chronic cryptosporidial infestation may be directly pathogenic resulting in squamous metaplasia that mimics biliary malignancy.

Nutritional outcome and pneumonia in critical care patients randomized to gastric versus jejunal tube feedings. The Critical Care Research Team.

OBJECTIVE: To compare nutritional status, gastric colonization, and rates of nosocomial pneumonia in ICU patients randomized to gastric tube feeding vs. patients fed by an endoscopically placed jejunal tube. DESIGN: Randomized, prospective study. SETTING: Medical and surgical ICUs at Boston City Hospital; surgical ICU at University Hospital. PATIENTS: Of the 38 study patients, 19 were randomized to gastric tube feeding and 19 were randomized to an endoscopically placed jejunal tube. The two groups were similar in age, sex, race, underlying disease, and type of surgery. RESULTS: The two patient groups were similar in number of days fed, duration of ICU stay, duration of mechanical ventilation, days of antibiotic therapy, and days with fever. Compared with the gastric group, the jejunal group had more patients with circulatory shock on admission (79% vs. 68.4%), higher admission Acute Physiology Score (24.0 vs. 21.7), and fewer patients with pneumonia at randomization (26.3% vs. 31.6%). The jejunal group received a significantly higher percentage of their daily goal caloric intake (p = .05), and had greater increases in serum prealbumin concentrations (p < .05) than the patients with gastric tube feeding. Although the jejunal tube group had more days of diarrhea (3.3 +/- 6.6 vs. 1.8 +/- 2.9), this difference was not statistically significant. Nosocomial pneumonia was diagnosed clinically in two (10.5%) patients in the gastric tube group and in no patients in the jejunal tube group. CONCLUSIONS: Patients fed by jejunal tube received a significantly higher proportion of their daily goal caloric intake, had a significantly greater increase in serum prealbumin concentrations, and had a lower rate of pneumonia than patients fed by continuous gastric tube feeding.

Characterization of bovine gallbladder mucin. Amino acid sequences of tryptic peptides from the glycosylated domain of the protein core.

Gallbladder mucin is a densely glycosylated macro-molecule that promotes cholesterol gallstone formation in experimental animals and in humans. Bovine gallbladder mucin structure was studied after chemical deglycosylation by treatment with anhydrous hydrogen fluoride at 23 degrees C for 3 hours. Deglycosylated mucin contained less than 5% of the amino sugar and neutral hexose content of native mucin. Electrophoretic and molecular sieve chromatographic analyses indicated that significant cleavage of the mucin polypeptide core had occurred during deglycosylation. Deglycosylated mucin was separated into three major fractions by reverse-phase chromatography, one of which was enriched with respect to threonine and proline. Tryptic peptides prepared from this fraction were purified by molecular sieve and reverse-phase chromatography, and the amino acid sequences (8-20 residues) of the four principal tryptic peptides were determined. These peptides contained 65%-75% threonine and proline residues and demonstrated 80%-100% sequence similarity. These data provide the first information on the primary structure of gallbladder mucin and suggest that repeating amino acid sequences occur in this protein. Comparison of gallbladder mucin peptide structure with the consensus repeat sequence of human intestinal mucin showed approximately 60% sequence similarity. It was concluded that mammalian gastrointestinal mucins may be derived from a common ancestral gene.

Isolation and characterization of peptides from the protein core of bovine gallbladder mucin.

Gallbladder mucin may promote cholesterol gallstone formation by accelerating cholesterol monohydrate crystal nucleation in supersaturated bile. In this study, peptides were isolated from the mucin protein core by protease digestion and molecular-sieve high-performance liquid chromatography. Tryptic peptides were purified by anion exchange or reverse-phase high-performance liquid chromatography, and amino acid compositions were determined. Tryptic peptides were (a) nonglycosylated, (b) selectively enriched in serine, glutamic acid plus glutamine, and glycine, and (c) depleted in threonine and proline compared with native gallbladder mucin. Bilirubin derivatized with Woodward's reagent K covalently bound to purified mucin. Tryptic digestion of the mucin-bilirubin complex yielded low-molecular-weight nonglycosylated peptides with covalently bound bilirubin. These data indicate that the mucin protein core contains at least two distinct domains. One domain is rich in threonine and proline and contains the majority of covalently bound carbohydrate. A second domain, possibly internally located, is nonglycosylated, enriched in serine, glutamic acid plus glutamine, and glycine, and binds hydrophobic ligands such as bilirubin and 1-anilino-8-naphthalene sulfonate. Hydrophobic domains on the mucin protein core may contribute to the pathogenesis of cholesterol cholelithiasis.

Genes transferred by retroviral vectors into normal and mutant myoblasts in primary cultures are expressed in myotubes.

Retroviral vectors were used to transfer genes efficiently into rat and dog myoblasts in primary cultures under conditions which permitted the transduced myoblasts to differentiate into myotubes expressing the transferred genes. The transduced myotubes expressed normal markers of differentiation and were morphologically indistinguishable from uninfected myotubes. Retroviral vector-mediated gene transfer was also used to correct a genetic enzyme deficiency in mutant canine muscle cells.

Effect of laser fragmentation of cholesterol and mixed gallstones on in vitro dissolution in methyl tert-butyl ether.

This study examined cholesterol and mixed gallstone dissolution in vitro by methyl-tert-butyl ether (MTBE) after gallstone fragmentation. Three morphologically identical gallstones were obtained from 42 patients. One stone from each patient was fragmented with laser energy at a wavelength of 504 nm delivered to the stone surface with a 320-microns quartz fiber. Intact and fragmented stones from the same patient were incubated without stirring in MTBE and dissolution was expressed as the percent of initial stone weight remaining after 2 hr. Stone composition did not correlate with the amount of laser energy required for stone fragmentation. Fragmented stones dissolved faster than intact stones in MTBE with 13.97% +/- 0.37% vs 31.0% +/- 0.51% respectively (mean +/- SEM) of initial stone weight remaining at 2 hr (P less than 0.0001). Initial stone weight and stone matrix content significantly predicted dissolution of intact (P = 0.0033 and P = 0.0483, respectively) and fragmented stones (P = 0.003 and P = 0.0001, respectively) in MTBE. These data suggest that the gallstone matrix may inhibit stone dissolution even after stone fragmentation.

Addition of N-acetylcysteine to aqueous model bile systems accelerates dissolution of cholesterol gallstones.

The organic matrix of cholesterol gallstones contains a macromolecular complex of mucin and bilirubin that may inhibit stone dissolution by limiting contact of desaturated bile with crystalline cholesterol. The goal of this study was to determine if the mucolytic agent N-acetylcysteine could accelerate gallstone dissolution in vitro. Paired gallstones were dissolved in either pure taurocholate (140 mM) or ursodeoxycholate (100 mM), or in bovine bile supplemented with either taurocholate or ursodeoxycholate to achieve the same respective bile-salt concentrations. N-acetylcysteine was added to 1 stone from each pair at a concentration of 500 mM in pure bile salts and 100 mM in supplemented bile. Gallstones dissolved significantly faster in bovine bile supplemented with taurocholate or ursodeoxycholate than in pure solutions of the respective bile salts (n = 30, p less than 0.001). N-acetylcysteine significantly accelerated gallstone dissolution in pure solutions of bile acids (n = 30, p less than 0.001 for each) and in supplemented bovine biles (n = 30, p less than 0.001). N-acetylcysteine also significantly increased the frequency of complete gallstone dissolution in taurocholate-supplemented (66.6% vs. 40.0%) and ursodeoxycholate-supplemented (76.6% vs. 50.0%) bile. These results indicate that the mucolytic agent N-acetylcysteine significantly accelerates in vitro gallstone dissolution. We speculate that adjuvant therapy with an appropriate mucolytic agent may potentially increase the efficacy of clinical gallstone dissolution.

Differentiated astrocytoma with osteoblastic skeletal metastases in a child.

A 13-year-old boy developed widespread osteoblastic bone metastases, with dissemination throughout the neuraxis, 1 year following treatment of a differentiated (Kernohan and Sayre grade II) astrocytoma of the left parietal cortex. This unusual clinical picture and the possible route of tumour dissemination are discussed.

Identification of gallbladder mucin-bilirubin complex in human cholesterol gallstone matrix. Effects of reducing agents on in vitro dissolution of matrix and intact gallstones.

The goals of this study were to isolate and characterize the nonlipid matrix of human cholesterol gallstones. The lipid portion of gallstones was dissolved in ethanol/ether, leaving an insoluble, granular, brown-black matrix that constituted 12.5% of solitary large stones and 3.5% of multiple small stones. The matrix was partially solubilized by sonication and studied by exclusion gel chromatography and density gradient ultracentrifugation. On Sepharose 2B column chromatography, bile pigment eluted with glycoprotein in the void volume, suggesting the presence of a high molecular weight complex (Mr greater than 2 X 10(6)). The identity of mucin in this complex was confirmed by its typical buoyant density during ultracentrifugation. The major bile pigments in the matrix were identified as bilirubin (84%) and bilirubin monoglucuronide (15%) by thin-layer chromatography. Because of their ability to solubilize mucin-type glycoproteins, we tested the ability of the reducing agents 2-mercaptoethanol (2ME) and N-acetylcysteine (NAcCys) to solubilize gallstone matrix. Both reducing agents caused a two- to threefold enhancement of matrix dissolution after 4 d compared to aqueous buffer alone (P less than 0.01). Sepharose 2B chromatography revealed that 2ME released a high molecular weight mucin-bilirubin complex as well as unbound pigment from the insoluble matrix. We also tested the effect of reducing agents on dissolution of matched cholesterol gallstones by monooctanoin, a cholesterol solvent. Both 2ME and NAcCys significantly accelerated gallstone dissolution in monooctanoin. Matched human cholesterol stones (n = 10) incubated for 4 d in monooctanoin plus either 2ME or NAcCys (1 M final concentration) weighed approximately half as much (P less than 0.01 for each) as stones incubated in monooctanoin alone. This study describes, for the first time, the isolation of a bilirubin-mucin complex in the insoluble matrix of human cholesterol gallstones. The ability of reducing agents to dissolve the matrix and thereby accelerate gallstone dissolution by monooctanoin in vitro may be relevant to gallstone dissolution in humans.

Role of gallbladder mucin in pathophysiology of gallstones.

A critical step in the formation of cholesterol gallstones in nucleation (i.e., the formation of cholesterol monohydrate crystals from supersaturated bile). The rate of nucleation of cholesterol depends upon a critical balance between pronucleating and antinucleating factors in bile. Mucin, a high molecular weight glycoprotein secreted by the gallbladder and biliary duct epithelium, is a pronucleating agent in experimental and human gallstone disease. Gallbladder mucin shares with other epithelial mucins the ability to bind lipids and bile pigment. The hydrophobic binding sites in the polypeptide core of mucin may provide a favorable environment for nucleation of cholesterol monohydrate from supersaturated bile. In nearly all animal models of cholelithiasis, mucin hypersecretion is prominent. The stimulus for gallbladder mucin hypersecretion appears to be a component of lithogenic bile. Prostaglandins regulate mucin release in gallbladder epithelium in vitro and probably in vivo. In the cholesterol-fed prairie dog, blockage of mucin release with aspirin inhibits gallstone formation. These findings suggest that inhibition of mucin release may prevent cholesterol stone formation during high-risk periods or after dissolution therapy with bile salts.

Maximal conservation and minimal usage of blood products in open heart surgery.

Open heart surgery has previously been associated with the use of large volumes of blood products. This paper describes methods of blood conservation and a simple method of intraoperative autotransfusion that together have resulted in minimal blood product usage in elective open heart surgery cases. This has reduced our dependence on blood bank supplies for the performance of elective open heart surgery.