Newcastle disease virus therapy of human tumor xenografts: antitumor effects of local or systemic administration.

Previously we showed that a single local injection of the avian paramyxovirus Newcastle disease virus (NDV) strain 73-T caused long-lasting, complete tumor regression of human neuroblastoma and fibrosarcoma xenografts in athymic mice. Here we report the antitumor effects of NDV administered by either the intratumoral (IT) route to treat a variety of human carcinoma xenografts or by the systemic (intraperitoneal, IP) route to treat neuroblastoma xenografts (6.5-12 mm in diameter). For IT treatments, mice were randomized into treatment groups and given a single IT injection of NDV 73-T, vehicle (phosphate buffered saline, PBS), or UV-inactivated NDV. For systemic therapy, mice (n=18) with subcutaneous IMR-32 human neuroblastoma xenografts received IP injections of NDV (5 x 10(9) PFU). Significant tumor growth inhibition (77-96%) was seen for epidermoid (KB8-5-11), colon (SW620 and HT29), large cell lung (NCIH460), breast (SKBR3), prostate (PC3), and low passage colon (MM17387) carcinoma xenografts treated IT with NDV. In all cases, tumors treated IT with PBS or replication-incompetent, UV-inactivated NDV displayed rapid tumor growth. After a single IP injection of NDV, complete regression of IMR-32 neuroblastomas was observed in 9 of 12 mice without recurrence for the 3-9 month follow-up period. Six mice with recurrent neuroblastomas after one IP injection received one to three additional IP treatments with NDV. Three of these six mice showed complete regression without recurrence. These data show that: (1) NDV administered either IT or IP is an effective antitumor therapy in this system, (2) replication competency is necessary for maximal effect, and (3) multiple NDV doses can be more effective than a single dose. These studies provide further rationale for the preclinical study of NDV as an oncolytic agent.

Use of porcine acellular dermal matrix as a dermal substitute in rats.

OBJECTIVE: To examine porcine acellular dermal matrix (ADM) as a xenogenic dermal substitute in a rat model. SUMMARY BACKGROUND DATA: Acellular dermal matrix has been used in the treatment of full-thickness skin injuries as an allogenic dermal substitute providing a stable wound base in human and animal studies. METHODS: Xenogenic and allogenic ADMs were produced by treating porcine or rat skin with Dispase and Triton X-100. Full-thickness skin defects (225 mm2) were created on the dorsum of rats (n = 29), porcine or rat ADMs were implanted in them, and these were overlain with ultrathin split-thickness skin grafts (STSGs). In two adjacent wounds, 0.005- or 0.017-inch-thick autografts were implanted. In other experiments, the antimicrobial agent used during ADM processing (azide or a mixture of antibiotics) and the orientation of the implanted ADM (papillary or reticular side of ADM facing the STSG) were studied. Grafts were evaluated grossly and histologically for 30 days after surgery. RESULTS: Significant wound contraction was seen at 14, 20, and 30 days after surgery in wounds receiving xenogenic ADM, allogenic ADM, and thin STSGs. Contraction of wounds containing xenogenic ADM was significantly greater than that of wounds containing allogenic ADM at 30 days after surgery. Graft take was poor in wounds containing xenogenic ADM and moderately good in those containing allogenic ADM. Wound healing was not significantly affected by the antimicrobial agent used during ADM preparation or by the ADM orientation. CONCLUSION: Dispase-Triton-treated allogenic ADM was useful as a dermal substitute in full-thickness skin defects, but healing with xenogenic ADM was poor.

Steroid sex hormones and macrophage function: regulation of chemiluminescence and phagocytosis.

PROBLEM: Female sex hormones modulate a variety of humoral and cell-mediated immunologic functions. In this study, the effects of estrogen, progesterone, and testosterone on the chemiluminescence (CL) response and phagocytic ability of male rat peritoneal macrophages (M luminal diameter) were examined. METHOD: In M luminal diameter pretreated with 10(-2) ng/ml of 17 beta-estradiol (E2) for 20 hours, the CL generated in response to phorbol myristate acetate (PMA), 1,2-dioctanoyl-rac-glycerol (C8:0), or opsonized zymosan (OZ) was significantly increased by 135%, 140%, and 136% of control values, respectively. In addition, M luminal diameter treated with 10(-5) ng/ml or 10 ng/ml of E2 exhibited a significantly greater PMA- or OZ-stimulated CL response than did untreated controls. RESULTS: At 10(-2) ng/ml, progesterone enhanced and testosterone reduced the CL response, but these changes were not statistically significant. In time course studies, the PMA-stimulated CL response of M luminal diameter treated with 10(-2) ng/ml of E2 or progesterone for 5 h was significantly less than that of the untreated group. In the presence of endotoxin (12 pg/ml), the CL response in M luminal diameter treated with E2 or testosterone was significantly depressed as compared to untreated controls. Phagocytosis of opsonized sheep erythrocytes also was significantly enhanced (140% to 190% of control) when M luminal diameter were pretreated with 10(-12) M to 10(-8) M of either E2 or progesterone. CONCLUSIONS: These findings suggest that, at physiological concentrations, E2 is capable of modulating both CL generation and phagocytic uptake by M luminal diameter in a manner not shared by other steroid hormones.

Complete regression of human fibrosarcoma xenografts after local Newcastle disease virus therapy.

We have recently demonstrated that a single local injection of the avian pathogen Newcastle disease virus (NDV; strain 73-T) causes complete regression of human neuroblastoma xenografts in athymic mice (R. M. Lorence, K. W. Reichard, B. B. Katubig, H. M. Reyes, A. Phuangsab, B. R. Mitchell, C. J. Cascino, R. J. Walter, and M. E. Peeples. J. Natl. Cancer Inst., 86: 1228-1233, 1994). In this report, we tried to determine if this in vivo antineoplastic effect of NDV extends to human sarcomas. Athymic mice with s.c. HT1080 fibrosarcoma xenografts (7-14 mm) were randomly divided into two groups and treated i.t. with a single injection of either 10(7) plaque-forming units of NDV or phosphate-buffered saline. Complete tumor regression occurred in 8 of 10 mice treated with NDV while unabated tumor growth occurred in all 9 mice treated with phosphate-buffered saline (P < 0.001). To determine if complete tumor regression was long lasting, the 8 mice were monitored for 1 year, during which time no tumor recurred. To test the antitumor effects of NDV on tumors derived from a fresh human sarcoma, a similar experiment was performed in athymic mice using TH15145 synovial sarcoma xenografts at their first and second passages. Of 9 mice with TH15145 xenografts, a single i.t. injection of NDV (10(7) plaque-forming units) caused complete regression of 3 tumors and > 80% regression in 3 more tumors. In contrast, tumors in all 5 mice treated with phosphate-buffered saline exhibited unabated growth (P < 0.03 for > 80% tumor regression). Since HT1080 fibrosarcoma cells express the N-ras oncogene, we explored the effects that transfection of this oncogene has on the sensitivity to NDV. Cultured human fibroblasts that were made tumorigenic following N-ras-transfection were found to be 1000-fold more sensitive to NDV than normal fibroblasts in a cytotoxicity assay. Oncogene expression by the HT1080 fibrosarcoma may therefore contribute to the long-lasting complete regression of this sarcoma following a single local injection of NDV.

Complete regression of human neuroblastoma xenografts in athymic mice after local Newcastle disease virus therapy.

BACKGROUND: Neuroblastoma is the most common pediatric extra-cranial solid cancer. Using conventional therapies, children older than 1 year of age with advanced neuroblastoma have a poor prognosis. The development of new approaches for treating such children with neuroblastoma continues to be one of the most important goals today in pediatric oncology. Despite numerous anecdotal reports of human tumor regression during viral infections, the use of viruses to directly lyse neuroblastoma cells has never been reported as a potential therapy. Newcastle disease virus (NDV) has been shown to replicate in and kill cultured human and rat neuroblastoma cells but not normal human fibroblasts. PURPOSE: Our purpose was to determine if this selective killing of human neuroblastoma (IMR-32) cells is maintained during the in vivo treatment of established tumors. METHODS: Two experiments were performed using NDV strain 73-T. Athymic mice with subcutaneous IMR-32 human neuroblastoma xenografts (6-12 mm) were treated intralesionally with live NDV, UV-inactivated NDV, or phosphate-buffered saline (PBS). To study virus replication in situ, mice were given intratumoral or intramuscular injections of NDV. These mice were then killed at various times, and the amount of infectious virus present in tumor or muscle was determined. RESULTS: After one injection of live NDV, 17 of 18 tumors regressed completely, whereas rapid tumor growth occurred in all 18 mice treated with PBS and in all nine mice treated with UV-inactivated NDV (P < .0001). The one tumor that showed only a partial response to a single injection regressed completely after a second NDV treatment. Six months following virus-induced regression, only one tumor had recurred. No significant acute or chronic side effects of live NDV were noted in athymic mice given doses up to 500 times that used in this study. Virus levels increased more than 80-fold between 5 and 24 hours in virus-injected tumors (P < .04), while no infectious virus was produced in NDV-injected muscle tissue. CONCLUSIONS: NDV 73-T appears to replicate selectively in human IMR-32 neuroblastoma xenografts, leading directly to a potent antitumor effect as demonstrated by long-lasting, complete tumor regression occurring after a single local injection of virus. IMPLICATION: These experiments may provide an important step in the development of new therapeutic approaches to challenging cancers such as neuroblastoma.

Intravenous fat emulsion acutely suppresses neutrophil chemiluminescence.

The immediate effect of intravenous fat emulsion on neutrophil oxidant release was studied. Opsonized nonencapsulated S. aureus was used to stimulate neutrophil activity. Luminol enhanced chemiluminescence was followed over 15 min and recorded as peak output (P; mV), integral under the curve (I; V-sec) and rate of increase (R; mV/sec). Eighteen chronically ill patients receiving glucose based total parenteral nutrition were studied before and after a 4- to 6-hr test infusion of 500 ml of 10% fat emulsion. P decreased from 719 +/- 46 to 461 +/- 42 mV (p less than 0.001), I decreased from 169 +/- 17 to 111 +/- 12 V-sec (p less than 0.001) and R decreased from 6.9 +/- 1.0 to 4.0 +/- 0.6 mV/sec (p less than 0.001). Preincubation of normal whole blood with fat emulsion in vitro did not adversely affect chemiluminescence (11 studies), nor did incubation of normal neutrophils with patient postinfusion plasma (10 studies). We conclude that fat emulsion infusion acutely suppresses neutrophil chemiluminescence. The suppression is not a direct effect of the fat emulsion per se and is not due to inhibitory substances in the plasma following infusion.

The effect of bacterial trapping by fibrin on the efficacy of systemic antibiotics in experimental peritonitis.

Sixty Sprague-Dawley rats were inoculated with 10(9) Escherichia coli either suspended in 2 milliliters of normal saline solution or incorporated in a 2 milliliter 0.4 per cent fibrin clot. One hour after inoculation, one-half of the rats in each group received gentamicin, 12.5 milligrams per kilogram, intramuscularly. Escherichia coli suspended in normal saline solution was uniformly lethal. Incorporation of the bacteria and fibrin resulted in a survival of 13 of 15 rats (p = less than 0.002), but in abscess formation in all survivors. Treatment with gentamicin nearly abolished the mortality of Escherichia coli suspended in normal saline solution (14 of 15 rats survived, p = less than 0.002) but did not prevent abscess formation when bacteria were incorporated into fibrin. The gentamicin level exceeded the minimal inhibitory concentration (0.98 microgram per milliliter) for the strain of Escherichia coli used in serum (15.84 micrograms per milliliter after one hour) and peritoneal fluid (14.75 micrograms per milliliter after one hour) but never reached inhibitory levels in the fibrin clot (0.75 microgram per milliliter after one hour). We conclude that entrapment of bacteria by fibrin abolishes systemic sepsis but also protects bacteria against the action of systemic antibiotics and favors abscess formation.

Irrigation of the peritoneal cavity and local antibiotics in the treatment of peritonitis.

Peritonitis was induced in the Sprague-Dawley rats by the implantation of gelatin capsules containing 0.3 milliliter of human fecal suspension into the peritoneal cavity. The rats were then treated with systemic antibiotics or peritoneal irrigation with normal saline solution alone or cephalotin solution, 2,000 micrograms per liter, or both. The results show that peritoneal irrigation with normal saline solution or with cephalotin alone was ineffective, even though the cephalotin concentration used inhibited the growth of all bacterial species isolated during the free stage of peritonitis in vitro. Systemic antibiotics alone as well as in combination with peritoneal irrigation significantly improved the survival rate, p less than or equal to 0.05 and p less than or equal to 0.02, respectively. However, only systemic antibiotics in combination with peritoneal irrigation resulted in a significant cure rate compared with the control group and the group treated with systemic antibiotics alone, p less than or equal to 0.002. There was no increase in the cure or survival rates when antibiotics were added to the peritoneal irrigation fluid. In the second experiment, seven of ten rats treated with systemic antibiotics survived, but only two rats had no intraperitoneal abscesses at autopsy. Intraperitoneal application of antibiotics did not improve these results: six rats survived, and one rat was cured. We conclude that peritoneal irrigation alone is ineffective in the treatment of peritonitis. The addition of peritoneal irrigation to systemic antibiotic therapy, however, resulted in a significantly increased cure rate. The addition of antibiotics to the irrigant does not further improve the results, and local antibiotics are not more effective than systemic antibiotics. We believe that benefit of peritoneal irrigation is due to its mechanical effect.

Chronic infections by herpes simplex viruses and by the horse and cat herpesviruses.

Rabbit eyes experimentally infected with either type 1 or type 2 herpes simplex viruses occasionally released virus spontaneously. Injection of adrenalin was not highly effective for stimulating virus release but did seem to have a slight and erratic activating capacity. No spontaneous virus release were detected from the eyes of six cats infected with cat herpesvirus, but, when adrenalin was administered, an episode of virus release did ensue in one animal. Rabbit spinal cords could be chronically infected with either herpes simplex virus type 2 or equine herpesvirus type 2. The viruses could be reisolated over subsequent months from about half the animals without prior stimulation; the interval between inoculation of trypsinized spinal tissue into tissue cultures and the development of cytopathic effect was often long-more than 4 weeks in some cases.

Type 1 and type 2 herpes simplex viruses: serological and biological differences.

Forty isolates of herpes simplex virus were compared by means of cross-neutralization curves. The 11 oral isolates were serotype 1, and all 29 genital/anal isolates were serotype 2. The cytopathic effects of the two serotypes were consistently different. Passage of strains of type 1 and type 2 in mice and in rabbits yielded two variants, although the majority of the strains remained unchanged serologically and in their cytopathic effects. The two variants were derived from type 1 strains and differed from the parent strains in their cytopathic effects, each of them producing syncytia and enlarged plaques. They had, however, retained the serotypic properties and the deoxyribonucleic acid (DNA) densities of their parent strains. The Roizman syncytial/macroplaque strain of herpes simplex virus was also included in the study; the density of its DNA (1.727 g/ml) was typical of type 1 strains, and serologically it seemed to be basically a type 1 strain, although it was neutralized by type 2 antiserum slightly better than were other type 1 strains. Growth curves were performed of the two serotypes in rabbit kidney, human fibroblast, and mouse embryo tissue cultures. The type 2 strains attained lower titers of infectivity in these three cell systems; the levels of infectivity of type 2 virus in the culture fluid decreased much more rapidly after the maximum had been attained than did the levels of infectivity of the type 1 strains, due to the greater instability of the type 2 virus. Parallel titrations of different strains in tissue cultures and intracerebrally in mice indicated that the latter assay system was usually more sensitive for type 2 strains than it was for type 1 strains. The paralytic sequelae and inflammatory changes of lumbar ganglia and spinal cord in young rabbits inoculated extraneurally with strains of the two serotypes also indicate that the type 2 virus is more virulent in laboratory animals than is type 1 virus.