Polyethylene glycol (molecular weight 400 DA) vehicle improves gene expression of adenovirus mediated gene therapy.

PURPOSE: A significant limitation of adenoviral mediated suicide gene therapy is poor gene distribution in vivo. The choice of vehicle has been demonstrated to affect the level of adenoviral delivered gene transduction. We examined the hypotheses that 1) adenovirus suspended in PEG400 improves gene expression in the naïve canine prostate model, 2) improved transgene expression with PEG400 results in improved tumor control and 3) vehicle affects the initial adenoviral spread from a single intratumor injection. MATERIALS AND METHODS: The magnitude and volume of gene expression were measured 24 hours following intraprostatic injection of adenovirus suspended in PEG400 (12.5% weight per volume) or saline as vehicle. Tumor growth delay was measured in mice bearing human tumor xenografts following the injection of adenovirus in PEG400 and saline. The initial spread of adenovirus was measured by confocal microscopy following a single injection of fluorescently labeled adenoviral particles in human tumor xenografts using each vehicle. RESULTS: Adenovirus suspended in PEG400 provided an average of twice the level of gene expression in the canine prostate and significantly better tumor control relative to saline in preclinical tumor models (p = 0.046 and 0.036, respectively). The initial spread of adenovirus with PEG400 was superior to that of adenovirus in saline and the latter was largely limited to the needle tract. CONCLUSIONS: Adenoviral gene therapy vectors suspended in PEG400 results in improved tumor control because of greater initial adenoviral spread, and the increased volume and magnitude of gene expression in vivo.

Epidemiology and characterization of leptospirosis at an urban and provincial site in Thailand.

Patients with FUOs at the Children's Hospital in Bangkok and the Chao Phya Abhai Bhu Bejhr Hospital in Prachinburi were screened for leptospirosis by blood and urine culture in addition to microagglutination testing of their serum. Animal populations in urban and periurban areas of Bangkok were surveyed for evidence of leptospira infection. Three rural sites near the Prachinburi Provincial Hospital were also surveyed. The rodents' and domestic animals' blood, urine, and/or kidney cell samples were cultured for leptospira. Sera from these animals were also tested for leptospira antibody. The bataviae serovar was the most commonly detected leptospiral agent in both man and animals. Presenting symptoms varied with age with children showing primarily fever, vomiting, headache, abdominal and generalized muscle pain and diarrhea whereas adults had fever, headache, anorexia, muscle pain and constipation. Blood samples from patients suspected of having leptospirosis were tested for antibody by the MAT and cultured in EMJH media. The following serogroups were identified: bataviae, autumanalis, javanica, hebdomadis, and pyrogens. Leptospirosis incidence in humans was much higher in the rainy/flooding year of 1983 compared to the relatively dry year of 1984. Results of our animal surveillance studies indicate that in addition to rats, which have previously been mentioned, dogs, bandicoots, cattle and pigs could be the source of human leptospirosis infection in both urban and provincial locations in Thailand.

Wild Malaysian cynomolgus monkeys are exposed to hepatitis A virus.

Serum samples were obtained within 3 days of capture from 106 cynomolgus monkeys (Macaca fascicularis) in peninsular Malaysia. Fifty-two monkeys were trapped on the fringes of palm oil estates and 54 in dense primary jungle. Sera were tested for antibodies to hepatitis A virus (HAV) with a commercial radioimmunoassay. Twenty-four animals had detectable serum anti-HAV activity (6 of 52 from palm oil estate sites and 18 of 54 from primary jungle sites). Among monkeys at both sites, antibody prevalence was strongly correlated with animal weight: overall only four of 69 monkeys (6%) weighing less than 2.0 kg had serum anti-HAV antibodies, while 14 of 29 (48%) weighing 2.0 to 3.9 kg, and 6 of 8 (75%) weighing 4.0 kg or more, had serum anti-HAV antibodies. These data suggest that wild cynomolgus monkeys in Malaysian jungles become infected with HAV or an HAV-like virus at a rate comparable to that of humans in the same region, and raise the possibility of a sylvatic cycle for HAV.

The effect of mouse age on the determination of Rickettsia tsutsugamushi virulence.

The effect of age on the susceptibility of ICR mice to lethal intraperitoneal (ip), Rickettsia tsutsugamushi infections was tested with five virulent strains--Karp, Kato, Gilliam, TA763, and TH1817--and three strains of reduced virulence--TA678, TA686, and TA716. Susceptibility differences were noted only in the ICR mice inoculated with two of the strains of reduced virulence, TA716 and TA678. With both strains, mice in the 12-weeks and younger age groups had lower death rates than did mice in the 21-weeks and older age groups. Also, CBA/CaJ mice of varying ages were inoculated intravenously with large doses of the Gilliam strain to determine the effect of age on susceptibility to acute death syndrome (ADS). A progressive increase in ADS resistance was seen in the 4-, 8-, 12-, and 16-week-old age groups. This study indicates that the age of mice used to test the virulence of R. tsutsugamushi strains may be an important consideration, especially when testing the ip lethality of strains of reduced virulence.

A technique for collection and cultivation of macrophages from Cynomolgus monkeys (Macaca fascicularis).

Several techniques were compared for collection of peritoneal mononuclear phagocytes from cynomolgus monkeys. A multiple-holed cannula proved superior to a hypodermic needle for the collection of peritoneal washings because it was not occluded by the omentum. With the cannula technique 55--70% of the harvest medium could be recovered from the peritoneal cavity with a yield of 2.5--5 x 10(5) macrophages per ml. A closed system for injecting and collecting harvest medium was better than a syringe in preventing bacterial contamination of peritoneal washings. After collection, cells were resuspended in growth medium to a concentration of 5 x 10(5) macrophages per ml, distributed into Leighton tubes, and incubated at 36 degrees C. After 12 hours, cultures were washed to remove non-adherent cells and refed with growth medium. This technique provided a sterile, reliable method for the collection and cultivation of macrophages from monkeys.

Scrub Typhus antibody in cynomolgus monkeys (Macaca fascicularis) in Malaysia.

Using an indirect immunofluorescence technique, sera from 113 cynomolgus monkeys (Macaca fascicularis), trapped in Peninsular Malaysia, were screened for the presence of antibody to six prototype strains of Rickettsia tsutsugamushi combined into three polyvalent groupings: I--Karp, TA716, and TA763; II--Gilliam; and III--TA678 and TH1817. Fifteen percent (17/113) of the monkeys had antibody titers greater than or equal to 1:50 to one or more of the antigenic groups. Although a titer greater than or equal to 1:150 is generally considered indicative or prior Rickettsia tsutsugamushi infection, we selected a less than 1:25 titer as a conservative standard to insure non-infected animals. Using this criterion, 62 (55%) of the 113 monkeys were accepted for use in scrub typhus studies. The high prevalence of antibody to scrub typhus in the semi-arboreal cynomolgus monkey is in marked contrast to the low prevalence reported in the strictly arboreal silvered leaf monkeys (Presbytis cristatus). The results of this study indicate that cynomolgus monkeys should be rigorously screened for evidence of prior infection before they are included in experimental scrub typhus studies.

Evaluation of vascular clearance as a marker for virulence of alphaviruses: disassociation of rapid clearance with low virulence of venezuelan encephalitis virus strains in guinea pigs.

The concept that relates low virulence of certain alphaviruses to low viremia and efficient vascular clearance of virus was tested in guinea pigs. Previously published studies with hamsters suggested that virulent strains maintain high viremias primarily because they are cleared inefficiently from the blood. In the present study, with guinea pigs, six of six virulent strains of Venezuelan encephalitis virus were cleared inefficiently, whereas three of six nonlethal or benign virus strains were cleared rapidly. However, three other guinea pig-benign Venezuelan encephalitis virus strains cleared slowly, to produce a high viremia was correlated with inefficient growth in primary viral replication sites. Thus, the potential of some alphaviruses to produce destructive lesions may be restricted by efficient clearance of virus from the blood, whereas the growth of other benign alphavirus strains may be restricted after the virus is presented to target cells.