Comparison of outpatient visits and hospitalisations, in patients with chronic obstructive pulmonary disease, before and after influenza vaccination.

OBJECTIVE: To determine the effectiveness of influenza vaccination on acute respiratory illness (ARI) and on acute exacerbation of chronic obstructive pulmonary disease (AECOPD) during a 2-year study conducted prior to and after influenza vaccination in patients with chronic obstructive pulmonary disease (COPD). MATERIALS AND METHODS: Eighty-seven male patients with COPD were stratified on the basis of their forced expiratory volume in 1 s (FEV(1)) as having mild, moderate and severe COPD. These patients were evaluated for a total duration of 2 years; 1 year prior to vaccination and for a period of 1 year following influenza vaccination. The vaccine (split virion, inactivated) composed of A/New Caledonia/20/99 (H1N1); A/California/7/2004(H3N2) and B/Shanghai/361/2002 strains all with 15 mug of haemagglutinin in each 0.5 ml dose. MEASUREMENTS: The number of episodes and severity of ARI and AECOPD, classified as outpatient treatment, hospitalisation and requirement of mechanical ventilation, for a period of 1 year before and 1 year after influenza vaccination were recorded. RESULTS: The incidence of ARI and AECOPD was 28.6 per 100 person-years prior to vaccination and 9.7 per 100 person-years postvaccination [relative risk (RR) 0.33; p = 0.005). Among the exacerbations because of natural infections prior to vaccination the incidences were 16.12, 42.1 and 33.14 per 100 person-years in the patients with mild, moderate and severe COPD respectively. These were significantly lower following vaccination with the incidences being 6.5, 18.5 and 8.42 per 100 person-years in the same subgroup of patients. Vaccine effectiveness in patients with mild COPD was 60% RR, 0.4 (p = 0.26); in patients with moderate COPD was 60% RR, 0.4 (p = 0.56); and in patients with severe COPD was 75% RR, 0.25 (p = 0.02). The total number of outpatient visits and hospitalisations before vaccination was eight and 14, respectively for a duration of 1 year in the total 87 patients with COPD being studied which decreased significantly to four outpatient visits and four hospitalisations postvaccination (p = 0.02). The overall effectiveness of influenza vaccination was 67%. CONCLUSIONS: Influenza vaccination is highly effective in the prevention of ARI. Maximum protection was found to be in patients with severe COPD. Influenza vaccination in patients is associated with fewer outpatient visits and fewer hospitalisations.

Evaluation of the wap-ras transgenic mouse as a model system for testing anticancer drugs.

Transgenic mouse models have provided many valuable insights into the molecular mechanisms of tumorigenesis; unfortunately, there is a paucity of published information on the utility of these models for evaluating potential anticancer therapeutics. Line 69 wap-ras transgenic mice have an activated, human c-Ha-ras gene on their Y chromosome. Adult males develop salivary and/or mammary adenocarcinomas. Both tumor types express high levels of human ras oncoprotein. Two new sublines, designated wap-ras/F, were created by selective breeding. Subline 69-2 wap-ras/F males developed multiple mammary tumors at puberty. Tumor onset was delayed by cyclophosphamide treatment prior to puberty. Mammary tumors from cyclophosphamide-treated mice weighed 0.57 +/- 0.09 g/mouse (SD +/- SEM; n = 8), while tumors from control mice weighed significantly more at 2.36 +/- 0.25 g/mouse (n = 8; P less than or equal to 0.001; SD +/- SEM). These results suggest that subline 69-2F mice will be valuable for testing therapeutic regimes designed to interfere with processes occurring early in tumorigenesis, before palpable tumor presentation. Tumor sensitivity to several clinically relevant cytotoxins was also tested in adult wap-ras males with palpable tumors. Both salivary and mammary tumors were sensitive to cyclophosphamide and 5-fluorouracil, but not methotrexate. This suggests that wap-ras transgenic mice will indeed be useful in the discovery of novel therapeutics against neoplasia.

Histopathology of salivary and mammary gland tumors in transgenic mice expressing a human Ha-ras oncogene.

Mutated ras genes are powerful transforming agents in vitro and are found in a wide variety of human tumors in vivo. We characterized the histopathology and p21 protein expression associated with tumorigenesis in line 69 transgenic mice carrying an activated, human c-Ha-ras gene on the Y-chromosome (A. C. Andres, C. A. Schonenberger, B. Groner, L. Hennighausen, M. LeMeur, and P. Gerlinger, Proc. Natl. Acad. Sci. USA, 84: 1299-1303, 1987). Male mice developed salivary and/or mammary gland tumors. The salivary tumors were adenosquamous carcinomas arising from serous areas of the submandibular gland. They characteristically exhibited densely packed cords and sheets of moderately anaplastic cells. Tumorigenic tissue had a high mitotic index, and all tumor-bearing animals had an ongoing inflammatory response as evidenced by extensive immune cell infiltration of affected tissue. Half of the mammary gland tumors were adenosquamous carcinomas with multiple foci of squamous metaplasia, while the rest were adenocarcinomas containing glandular tissue. Most tumors had a high mitotic index, and abnormal mitotic figures were common. All tumors produced p21 ras, as confirmed by immunohistochemistry and Western blots. Both tumor types expressed elevated levels of p21 protein. Microscopic lung metastases were present in 5 of 35 animals (14%). Our results suggest that this transgenic mouse will provide a useful model for testing therapies directed against ras-associated tumorigenesis.

Derivation and initial characterization of a mouse mammary tumor cell line carrying the polyomavirus middle T antigen: utility in the development of novel cancer therapeutics.

Here we describe the derivation of novel cell lines from spontaneous mammary tumors that arose in mouse mammary tumor virus-polyomavirus (MMTV-PyV) Middle T (MidT) transgenic mice. Clonal cell lines from four mixed cell populations were tested for adenovirus transducibility and sensitivity to p53 tumor suppressor gene therapy mediated by SCH58500, a replication-deficient adenovirus that expresses human p53. The MidT2-1 cell line was selected for further characterization in vitro and in vivo. This cell line carried the PyV MidT antigen, had wild-type p53 DNA, and was sensitive to suppression of proliferation by MMAC/PTEN tumor suppressor gene therapy. MidT2-1 cells gave rise to highly aggressive tumors in syngeneic FVB mice in both the mammary fat pad and the peritoneal cavity. The histopathology of MidT2-1 tumors closely resembled the histopathology of the primary transgenic tumors. Tumor growth in vivo was inhibited by p53 gene therapy or by MMAC gene therapy. In addition, combination therapy with a number of anticancer agents had synergistic or additive efficacy in vitro. In particular, MMAC gene therapy synergized with SCH58500 or paclitaxel. In the i.p. MidT2-1 tumor model p53 gene therapy enhanced the survival benefits of paclitaxel/cisplatin chemotherapy. Combination therapy has become a mainstay in cancer treatment. In this report, we use a novel transgenic mouse tumor cell line to suggest new combinations that might be explored in clinical cancer care. These include gene therapy using the tumor suppressors MMAC and p53, chemotherapy using farnesyl transferase inhibitors, the microtubule stabilizing taxanes, and the DNA synthesis disruptors gemcitabine and cisplatin. The precise biological mechanisms by which these therapies induce their antitumor effects are not fully elucidated. However, the work presented here suggests that many of these therapeutic approaches have synergistic antitumor activity when used in combination.

Combination therapy with the farnesyl protein transferase inhibitor SCH66336 and SCH58500 (p53 adenovirus) in preclinical cancer models.

SCH66336 is a p.o.-active, farnesyl protein transferase inhibitor. SCH66336 inhibits farnesylation of RAS and other proteins in tumor cells and suppresses tumor growth in human xenograft and transgenic mouse cancer models in vivo. SCH58500 is a replication-deficient, recombinant adenovirus, which expresses the human p53 tumor suppressor. In preclinical models, SCH58500 has therapeutic efficacy against a wide range of human tumor types containing nonfunctional p53 and enhanced activity in combination with many chemotherapeutic drugs. Here we report that combination therapy with SCH66336 and SCH58500 has synergistic or additive antiproliferative effects on a panel of tumor cells lines in vitro. The efficacy of the three-drug combination of SCH66336, SCH58500, and paclitaxel was also examined in vitro. Each two-drug interaction displayed such marked synergy, the addition of a third drug to the statistical model could only yield additivity. Greater combined efficacy for SCH66336 and SCH58500 was also observed in vivo in the DU-145 human prostate and wap-ras/F transgenic mouse cancer models.

Adenovirus-mediated p53 gene therapy and paclitaxel have synergistic efficacy in models of human head and neck, ovarian, prostate, and breast cancer.

Synergy (or antagonism) between two chemical agents is an in vitro empirical phenomenon, in which the observed effect of the combination is more (or less) than what would be predicted from the effects of each agent working alone. Although mathematical synergy is not directly provable in the clinical setting, it does predict a favorable outcome when the two therapeutics are combined in vivo and strongly suggests the presence of in vivo synergy. In contrast, overt antagonism warns of future problems. Sophisticated three-dimensional statistical modeling was used to evaluate the presence of synergistic, additive, or antagonistic efficacy between adenovirus (Ad)-mediated p53 gene therapy (p53 Ad) and paclitaxel (Taxol) in a panel of human tumor cell lines. Cells were either pretreated with paclitaxel 24 h before p53 Ad or treated with both agents simultaneously. Cell proliferation was measured 3 days later. Paclitaxel had synergistic or additive efficacy with p53 gene therapy. In no case was the interaction antagonistic. Cell cycle analysis demonstrated that p53 Ad arrested cells in G0/G1 prior to apoptotic cell death, whereas paclitaxel arrested cells in G2-M prior to apoptotic cell death. When combined, the relative concentration of each agent determined the dominant cellular response. These results are consistent with the previously reported cell cycle effects of p53 or paclitaxel, respectively; however, these data fail to explain the observed drug synergy. We found that low concentrations of paclitaxel (1-14 nM) increased the number of cells transduced by recombinant Ad 3-35% in a dose-dependent manner, which is one possible mechanism for the observed synergy. Of particular note, the concentrations of paclitaxel responsible for increased Ad transduction were lower than the concentrations required for microtubule condensation. The efficacy of combination therapy was also evaluated in vivo. In the p53null SK-OV-3 xenograft model of ovarian cancer, a dosing schedule of p53 Ad that, by itself, had a relatively minimal effect on tumor burden (16%) caused a much greater decrease in tumor burden (55%) when combined with paclitaxel. Greater combined efficacy was also observed in the p53mut DU-145 prostate, p53mut MDA-MB-468 breast, and p53mut MDA-MB-231 breast cancer xenograft models in vivo. In summary, p53 Ad for cancer shows enhanced efficacy when combined with paclitaxel. This combination is recommended for clinical cancer trials.

Recombinant E1-deleted adenovirus-mediated gene therapy for cancer: efficacy studies with p53 tumor suppressor gene and liver histology in tumor xenograft models.

Type 5 adenoviral (Ad) vectors have been the "vector-of-choice" for preclinical studies on p53 tumor suppressor gene therapy of cancer. Previous studies have examined the in vivo efficacy of p53 Ad when given intratumorally. However published information does little to guide clinicians in the design of intraperitoneal (i.p.) dosing trials for i.p. tumors, e.g., ovarian, or clinical trials using regional organ perfusion, e.g., for lung tumors. Therefore, we examined several parameters with special significance for these routes of administration. Lung metastases from p53mut MDA-MB-231 mammary xenografts were treated with therapeutic levels of intravenous buffer, beta-galactosidase (beta-Gal) Ad, or p53 Ad. Treatment with intravenous p53 Ad significantly reduced the number of metastases per lung and there was a dramatic reduction in the surface area occupied by these tumors as compared to control groups. Two types of i.p. tumor xenografts were used for preclinical modeling of i.p. gene therapy, the p53null SK-OV-3 ovarian and the p53mut DU-145 prostate human cancers. In a study examining the effect of different vehicle volumes on the efficacy of a constant drug dose, all mice treated with p53 Ad had reduced tumor burden compared to controls. Dosing volumes between 0.2 and 1 ml were equally effective and all were more effective than a dosing volume of 0.1 ml. However, reduced efficacy was observed when a volume of 1.5 ml was used. When the effect of dosing frequency on antitumor efficacy was examined, fractionated doses of p53 Ad had somewhat greater efficacy than fewer, bolus injections. One of the significant elements in the emerging toxicology associated with recombinant adenoviruses is the hepatocyte pathology caused by high systemic concentrations of adenovirus. For recombinant Ad used in this study, there was a pronounced dose-dependence for the liver response, with very high, repeated doses causing significant hepatocellular insult. Expression of cytoplasmic beta-Gal protein coincided with areas of greatest damage in mice treated with high doses of beta-Gal Ad. Ultrastructural examination of hepatocyte intranuclear inclusions revealed moderately electron-dense, tightly packed granular material interspersed with more electron-dense nuclear material. Human tumor xenografts, but not mouse tissues, expressed viral hexon protein. In summary, hepatic toxicity caused by high concentrations of recombinant adenovirus was observed in murine cancer models. However, therapeutic levels of p53 Ad could be achieved which had dramatic efficacy without significant pathology.

Development and validation of sensitive assays to quantitate gene expression after p53 gene therapy and paclitaxel chemotherapy using in vivo dosing in tumor xenograft models.

SCH58500 (ACN53) is a replication-deficient, type 5 adenovirus (Ad) expressing human wild-type p53 tumor suppressor. It is currently undergoing clinical trials as a cancer therapeutic. Many SCH58500 clinical trials incorporate an arm comparing traditional chemotherapy against chemotherapy combined with SCH58500. Paclitaxel was chosen for combination therapy in the preclinical study reported here due to its extensive use as a first-line therapy in ovarian cancer, its synergy with SCH58500 in preclinical cancer models, and its activation of p53-independent apoptosis, which might result in a "lowered threshold" for tumor cell death. SCID mice bearing human tumor xenografts were dosed with intratumoral vehicle, control Ad vector, or SCH58500, with or without paclitaxel. Real-time quantitative reverse transcriptase polymerase chain reaction assays were developed and validated to quantitate expression of p53, the p53 downstream effector gene p21, and the apoptosis-related genes, bax, bcl-2, and survivin. Protein expression was confirmed using immunohistochemical assays for p53 and p21. Only tumors injected with SCH58500 had detectable levels of exogenous p53 DNA and mRNA. After SCH58500 treatment, 3-11-fold elevations of p21 expression were observed in tumor xenografts containing nonfunctional p53 (MDA-MB-468, MDA-MB-231, MIAPaCa2, DU-145, and SK-OV-3), but no change in p21 mRNA in wild-type p53 PA-1 tumors. Immunohistochemical assays confirmed induction of p21 protein in MDAMB-468 and SK-OV-3 cells, but not in PA-1 cells. Ad vector alone or paclitaxel alone had no effect on p21 mRNA levels in most tumors. However, paclitaxel suppressed p21 expression induced by SCH58500 4-fold in DU-145 and SK-OV-3 tumors. Paclitaxel also affected expression of the housekeeping gene gapdh. There was no consistent pattern to the changes in bax, bcl-2, or survivin after SCH58500 treatment with or without paclitaxel between tumor types, although there were consistent responses within individual tumor lines. The mRNA ratios for bax/bcl-2 and bax/survivin were also not informative across tumor types. Of the genes examined, only p21 gave a predictable response 24 hours after p53 gene therapy and therefore, p21 expression may be useful for confirming SCH58500 activity in human tumor biopsies.

Adenovirus-mediated p53 gene therapy has greater efficacy when combined with chemotherapy against human head and neck, ovarian, prostate, and breast cancer.

PURPOSE: Adenovirus-mediated p53 gene therapy for cancer is currently undergoing phase I/II clinical trials. The drug used in our clinical trials (p53 Ad; ACN53; SCH58500) consists of a replication-deficient, type 5 adenovirus vector expressing human wildtype p53 tumor suppressor under the control of the cytomegalovirus promoter. In preclinical models, p53 Ad has therapeutic efficacy against a wide range of human tumor types containing nonfunctional p53, both in vitro and in vivo. Results from early clinical trials using p53 gene therapy by itself support optimism for the future of this therapeutic approach. However, it is likely that many phase II/III trials will incorporate an arm comparing traditional chemotherapy against chemotherapy combined with p53 gene therapy. Therefore, it is important to study possible interactions between p53 Ad and chemotherapeutic drugs in preclinical models before starting the clinical trials. METHODS: Proliferation of tumor cells was quantitated after incubation with various combinations of p53 Ad and chemotherapeutic drugs. Human tumor xenografts in scid mice were dosed with intraperitoneal or intratumoral p53 Ad with or without chemotherapeutic drugs and the tumor burden after therapy monitored. RESULTS: p53 Ad combined with cisplatin, doxorubicin, 5-fluorouracil, methotrexate, or etoposide inhibited cell proliferation more effectively than chemotherapy alone in SCC-9 head and neck, SCC-15 head and neck, SCC-25 head and neck, SK-OV-3 ovarian, DU-145 prostate, MDA-MB-468 breast, and MDA-MB-231 breast tumor cells. No obvious dependence on dosing schedule was observed. Greater anticancer efficacy was also demonstrated in four human tumor xenograft models in vivo. Of particular significance, there was enhanced efficacy using the three drug combination of p53 Ad, cisplatin, and paclitaxel in an ovarian cancer model. CONCLUSION: These results support the combination of p53 gene therapy with chemotherapy in clinical trials.

Somatotype, physical growth, and sexual maturation in young male smokers.

One thousand school boys aged 8 to 16 were examined for their somatotype, physical growth, sexual maturation, and smoking habits. Fifty-two boys were found to be smokers, of whom 30 were regularly smoking between two and 20 bidis or cigarettes a day for a mean duration of 2.5 years. The mean height and weight of the smokers was significantly lower than that of the non-smokers at all ages, more so in regular than occasional smokers. Sixty-nine per cent of the smokers had mesomorphic type of body build; about 65% of the non-smokers had ectomorphic somatotype (P less than 0.001). Onset of puberty occurred significantly earlier among smokers compared with non-smokers, as was evident from the early appearance of genital stage 2, and an early and rapid increase in testicular size. Genital stage 2 appeared at a mean age of 11 years in smokers and 11.6 years in non-smokers. However, the appearance of pubic, axillary, and facial hair was delayed. The possible significance of this is discussed.

In wap-ras transgenic mice, tumor phenotype but not cyclophosphamide-sensitivity is affected by genetic background.

Male wap-ras transgenic mice develop adenocarcinomas in salivary and/or mammary tissue by age 1 year. When the wap-ras transgene was bred into the FVB/N strain, males developed multiple mammary tumors between 1.5 and 3 mo. of age, but no salivary tumors. Crosses between ras/FVB mice and other strains produced moderate changes in mammary tumor onset and severity, but no salivary tumors. Histopathological analysis of 62 adenocarcinomas from 18 mice yielded: 14 tumors with areas of squamous metaplasia, many tumors with epithelium-lined cysts, few immune cells in tumors, and no lung metastases. Cyclophosphamide delayed tumor onset and inhibited the growth of established tumors. Our results suggest that wap-ras mice will be useful for studying ras-mediated tumor genetics and should be a good assay system for both preventative and curative anticancer therapies.

Development of a nude mouse model of ras-mediated neoplasia using WR21 cells from a transgenic mouse salivary tumor.

A novel cell line (WR21) was derived from a salivary tumor in a male wap-ras transgenic mouse. Salivary tumors in wap-ras transgenic mice are extremely aggressive and express high levels of oncogenic ras protein from the activated, human Ha-ras transgene. WR21 cells also expressed high levels of oncogenic ras protein in vitro and in vivo. They gave rise to aggressive, highly anaplastic solid tumors when injected subcutaneously into athymic nude mice and approximately 90% of the mice had lung metastases by the fifth week of tumor growth. WR21 tumors were inhibited by cyclophosphamide, 5-fluorouracil, adriamycin, mitomycin C and actinomycin D, but not methotrexate. Our results suggest that the WR21/nude mice model will be useful for testing the efficacy of drug therapies against ras-mediated neoplasias.

Environmental and genetic variation in milk yield of native cattle and crosses with Brown Swiss in India.

Effects of year, season, parity, age, their two-way interactions, lactation length and calving interval on milk yield were investigated utilizing 9,086 lactation records collected from 1930 to 1975 from six breed groups kept in one herd at Karnal, India. The breed groups involved three native breeds (Sahiwal, Red Sindhi and Tharpakar) and three crossbreds with Brown Swiss (F1 crosses between Brown Swiss and three native breeds, inter se crosses, and 3/4 Brown Swiss). Breed, year, season, parity, age and all of the two-way interactions with the exception of breed X season and parity X season were important. Tharpakar produced more milk than Sahiwal and Red Sindhi by 232 and 204 kg. The milk production difference between Sahiwal and Red Sindhi was only 28 kg. The three crossbreds outproduced the purebreds by an average of 766 kg; however, differences in management could have favored crossbreds. Among the crossbreds, F1 crosses were superior. The linear and quadratic regressions on lactation length accounted for 28% of the variation in milk yield after year, season, parity, age and their interactions were absorbed. Calving interval and lactation length together accounted for 29%. Estimates of heritability, from paternal half-sib analyses, and repeatability of milk yield for Red Sindhi, Sahiwal and Tharpakar ranged from .10 to .30. Differences among heritability estimates from different parities were small when more than 500 records were involved.

The use of laser scanning cytometry to assess depth of penetration of adenovirus p53 gene therapy in human xenograft biopsies.

SCH58500 is an agent for gene therapy of cancer, consisting of a replication-deficient type 5 adenovirus (Ad5) expressing the human p53 tumor suppressor gene (Ad5/p53). An important question about the use of Ad5/p53 gene therapy is how to achieve the therapeutically effective delivery of an Ad5/p53 vector to the tumor. We wanted to determine the effective depth of penetration of an Ad5/p53 vector by dosing the vector in an experimental human xenograft/SCID model. To assess depth of penetration, we developed a novel methodology for scanning tissue sections by laser scanning cytometry (LSC). SCID mice were given intraperitoneal injections of either p53(null) SK-OV-3 human ovarian tumor cells or p53(mut) DU-145 human prostate tumor cells to establish xenograft solid tumors. Mice were then dosed once or twice at 24-hour intervals by intraperitoneal injection with SCH58500 (Ad5/p53), an adenovirus construct expressing beta-galactosidase (Ad5/beta-gal), or a buffer control. Additional groups of mice received a single intraperitoneal dose of 10 mg/kg paclitaxel either alone or coadministered with Ad5/p53. Twenty-four hours after each last dose, the human solid tumor xenograft and relevant mouse tissue were removed from each mouse for the analysis of Ad5/p53 penetration. Immunohistochemistry (IHC) for beta-galactosidase protein revealed a depth of penetration of between 1 and 10 cells from the tumor surface. In some mice, hepatocytes in the periportal regions of liver lobules were also positive, indicating systemic absorption of adenovirus from the peritoneal cavity. IHC staining for p53 and p21 proteins in SK-OV-3 solid tumor xenografts revealed similar Ad/p53 penetration. LSC was used to map and quantitate apoptosis in both tumor and liver tissue biopsies, with over 450,000 nuclei from liver tissue and 150,000 nuclei from tumor tissue being evaluated. LSC analysis demonstrated a high level of apoptosis in the tumors that had been removed from Ad5/p53-dosed mice (12.7-19.7%). This level of apoptosis was significantly higher (P < 0.05) than was observed for liver tissues taken from Ad5/p53-dosed mice (2.7-8.0%) or tumor tissues taken from either Ad5/beta-gal-dosed mice (3.0-6.4%) or buffer control-dosed mice (3.0-5.3%). Scan bit maps from the extensive LSC analyses confirmed that apoptosis was present to about the same depth (1-10 cells) as had been identified by IHC for beta-galactosidase, p53, and p21 proteins. Paclitaxel coadministered with Ad5/p53 had no effect on Ad5 penetration into solid tumors in vivo as measured by IHC for p53 or p21 protein. However, the combination therapy did cause an elevation in the number of tumor cells undergoing apoptosis.