Genomic diversity and antimicrobial resistance in clinical Klebsiella pneumoniae isolates from tertiary hospitals in Southern Ghana.

OBJECTIVES: Comprehensive data on the genomic epidemiology of hospital-associated Klebsiella pneumoniae in Ghana are scarce. This study investigated the genomic diversity, antimicrobial resistance patterns, and clonal relationships of 103 clinical K. pneumoniae isolates from five tertiary hospitals in Southern Ghana-predominantly from paediatric patients aged under 5 years (67/103; 65%), with the majority collected from urine (32/103; 31%) and blood (25/103; 24%) cultures. METHODS: We generated hybrid Nanopore-Illumina assemblies and employed Pathogenwatch for genotyping via Kaptive [capsular (K) locus and lipopolysaccharide (O) antigens] and Kleborate (antimicrobial resistance and hypervirulence) and determined clonal relationships using core-genome MLST (cgMLST). RESULTS: Of 44 distinct STs detected, ST133 was the most common, comprising 23% of isolates (n = 23/103). KL116 (28/103; 27%) and O1 (66/103; 64%) were the most prevalent K-locus and O-antigen types. Single-linkage clustering highlighted the global spread of MDR clones such as ST15, ST307, ST17, ST11, ST101 and ST48, with minimal allele differences (1-5) from publicly available genomes worldwide. Conversely, 17 isolates constituted novel clonal groups and lacked close relatives among publicly available genomes, displaying unique genetic diversity within our study population. A significant proportion of isolates (88/103; 85%) carried resistance genes for ≥3 antibiotic classes, with the blaCTX-M-15 gene present in 78% (n = 80/103). Carbapenem resistance, predominantly due to blaOXA-181 and blaNDM-1 genes, was found in 10% (n = 10/103) of the isolates. CONCLUSIONS: Our findings reveal a complex genomic landscape of K. pneumoniae in Southern Ghana, underscoring the critical need for ongoing genomic surveillance to manage the substantial burden of antimicrobial resistance.

Heritable plant phenotypes track light and herbivory levels at fine spatial scales.

Organismal phenotypes often co-vary with environmental variables across broad geographic ranges. Less is known about the extent to which phenotypes match local conditions when multiple biotic and abiotic stressors vary at fine spatial scales. Bittercress (Brassicaceae: Cardamine cordifolia), a perennial forb, grows across a microgeographic mosaic of two contrasting herbivory regimes: high herbivory in meadows (sun habitats) and low herbivory in deeply shaded forest understories (shade habitats). We tested for local phenotypic differentiation in plant size, leaf morphology, and anti-herbivore defense (realized resistance and defensive chemicals, i.e., glucosinolates) across this habitat mosaic through reciprocal transplant-common garden experiments with clonally propagated rhizomes. We found habitat-specific divergence in morphological and defensive phenotypes that manifested as contrasting responses to growth in shade common gardens: weak petiole elongation and attenuated defenses in populations from shade habitats, and strong petiole elongation and elevated defenses in populations from sun habitats. These divergent phenotypes are generally consistent with reciprocal local adaptation: plants from shade habitats that naturally experience low herbivory show reduced investment in defense and an attenuated shade avoidance response, owing to its ineffectiveness within forest understories. By contrast, plants from sun habitats with high herbivory show shade-induced elongation, but no evidence of attenuated defenses canonically associated with elongation in shade-intolerant plant species. Finally, we observed differences in flowering phenology between habitat types that could potentially contribute to inter-habitat divergence by reducing gene flow. This study illuminates how clonally heritable plant phenotypes track a fine-grained mosaic of herbivore pressure and light availability in a native plant.

[The 2014 consensus conference of the ISUP on Gleason grading of prostatic carcinoma]. / Konsenskonferenz 2014 der ISUP zur Gleason-Graduierung des Prostatakarzinoms.

In 2005 the International Society of Urological Pathology (ISUP) held a concensus conference on Gleason grading in order to bring this grading system up to the current state of contemporary practice; however, it became clear that further modifications on the grading of prostatic carcinoma were necessary. The International Society of Urological Pathology therefore held a further consensus conference in 2014 to clarify these points. This article presents the essential results of the Chicago grading meeting.

Impaired prostate tumorigenesis in Egr1-deficient mice.

The transcription factor early growth response protein 1 (EGR1) is overexpressed in a majority of human prostate cancers and is implicated in the regulation of several genes important for prostate tumor progression. Here we have assessed the effect of Egr1 deficiency on tumor development in two transgenic mouse models of prostate cancer (CR2-T-Ag and TRAMP). Using a combination of high-resolution magnetic resonance imaging and histopathological and survival analyses, we show that tumor progression was significantly impaired in Egr1-/- mice. Tumor initiation and tumor growth rate were not affected by the lack of Egr1; however, Egr1 deficiency significantly delayed the progression from prostatic intra-epithelial neoplasia to invasive carcinoma. These results indicate a unique role for Egr1 in regulating the transition from localized, carcinoma in situ to invasive carcinoma.

Investigation into a new softening agent for use on formalin-fixed, paraffin wax-embedded tissue.

The use of tissue softening agents to improve microtomy of keratotic tissues is employed widely. Many of these softeners contain hazardous constituents such as phenol. In this study, the use of non-ionic surfactants or non-toxic ingredients are investigated with the aim of creating a new softening agent. The new agent should be more effective in facilitating the sectioning of hardened tissue while reducing toxicity and complications associated with sectioning hard tissue compared to a commercially available phenol-based formulation. Four formulations are compared against the commercial product for their capability to section routinely processed paraffin-embedded tissue under standard operating procedure parameters. The trial formulations were shown to be fast acting and enabled improved serial sectioning of hard keratotic tissue in nearly all the cases tested. There was no evidence of adverse staining using either tinctorial or immunohistochemical methods. The new formulations had advantages over the commercially available solutions, improving on the number and quality of sections attainable from the tissue blocks, as well as offering a composition less toxic than phenol-based products.

Musculoskeletal injuries in real tennis.

Introduction: Real tennis is a growing, unique, and well-established sport. To date, there has been no epidemiological data on real tennis injuries. The primary aim of this retrospective study is to record the incidence and document any trends in real tennis musculoskeletal injuries, so as to improve injury awareness of common and possibly preventable injuries. Methods: A surveillance questionnaire e-mailed to 2,036 Tennis & Rackets Association members to retrospectively capture injuries sustained by amateur and professional real tennis players over their playing careers. Results: A total of 485 (438 males and 47 females) questionnaires were fully completed over 4 weeks. A total of 662 musculoskeletal injuries were recorded with a mean of 1.4 injuries per player (range 0-7). The incidence of sustaining an acute real tennis musculoskeletal injury is 0.4/1000 hrs. The three main anatomical locations reported injured were elbow 15.6% (103/662), knee 11.6% (77/662), and face 10.0% (66/662). The most common structures reported injured were muscle 24% (161/661), tendon 23.4% (155/661), ligament 7.0% (46/661), soft tissue bruising 6.5% (43/661), and eye 6.2% (41/661). The majority of the upper limb injuries were gradual onset (64.7%, 143/221), and the lower limb injuries were sudden onset (72.0%, 188/261). Conclusion: This study uniquely provides valuable preliminary data on the incidence and patterns of musculoskeletal injuries in real tennis players. In addition, it highlights a number of reported eye injuries. The study is also a benchmark for future prospective studies on academy and professional real tennis players.

Prognostic value of various morphometric measurements of tumour extent in prostate needle core tissue.

AIMS: Predicting prostatic cancer patients' outcome is a major objective for clinicians and patients. Several nomograms are currently implemented prior to treatment to help predict clinical and pathological outcome. The aim of this study was to investigate the prognostic significance of morphometric measurements of cancer on the needle biopsy specimen in relation to the final pathological stage or the biochemical failure status following radical prostatectomy, and to determine which measurement of tumour length in cases with discontinuous foci of cancer (DFC) is most reliably reflective of the pathological stage. METHODS AND RESULTS: Of the 100 patients included in this study, 34% had high-stage disease (pT >or= 3 and/or pN1) and 16% experienced biochemical recurrence. The analysis showed that fraction of positive cores, total percentage of cancer and both total and greatest millimetric cancer lengths were the variables most closely associated with pathological stage and biochemical failure status. CONCLUSIONS: This study confirms the prognostic value of recording tumour extent in prostatic needle biopsy reporting. However, the results are inconclusive in determining the best method to record tumour length in cores with DFC and larger studies are needed to answer this question fully.

The in vitro pharmacological profile of TD-5108, a selective 5-HT(4) receptor agonist with high intrinsic activity.

The in vitro pharmacological profile of TD-5108, a novel, selective 5-HT(4) receptor agonist, was compared to that of clinically efficacious gastroprokinetic 5-HT(4) receptor agonists. TD-5108 produced an elevation of cyclic adenosine monophosphate in human embryonic kidney 293 cells expressing the human recombinant 5-HT(4(c)) (h5-HT(4(c))) receptor (pEC(50) = 8.3) and 5-HT(4) receptor-mediated relaxation of the rat esophagus (pEC(50) = 7.9) and contraction of the guinea pig colon (pEC(50) = 7.9). In all in vitro assays, TD-5108 was a high intrinsic activity agonist, unlike tegaserod, mosapride, and cisapride which, in the majority of test systems, had lower intrinsic activity. TD-5108 had high affinity (pK (i) = 7.7) and selectivity (> or =25-fold) for h5-HT(4(c)) receptors over other biogenic amine receptors. TD-5108 was >500-fold selective over other 5-HT receptors (including h5-HT(2B) and h5-HT(3A)) and, at 3 microM, had no effect on human ether-à-go-go-related gene K+ channels. In conclusion, TD-5108 is a selective 5-HT(4) receptor agonist in vitro. The high intrinsic activity and preferential binding of TD-5108 to 5-HT4 over other 5-HT receptors may result in an improved clinical profile for the treatment of gastrointestinal disorders of reduced motility.

The in vivo gastrointestinal activity of TD-5108, a selective 5-HT(4) receptor agonist with high intrinsic activity.

The in vivo preclinical pharmacodynamic profile of TD-5108, a selective 5-HT(4) receptor agonist with high intrinsic activity, was compared to that of the clinically studied gastrointestinal pro-kinetic agents, tegaserod, cisapride and mosapride. The activity of TD-5108 was evaluated in guinea pig colonic transit, rat oesophageal relaxation and dog gastrointestinal smooth muscle contractility models. Subcutaneous administration of TD-5108, tegaserod, cisapride and mosapride increased guinea pig colonic transit (rank order of potencies: TD-5108 > tegaserod > cisapride > mosapride). Following intravenous and intraduodenal dosing, TD-5108, tegaserod, cisapride and mosapride produced dose-dependent relaxation of the rat oesophagus. On a molar basis, TD-5108 was approximately twofold less potent than tegaserod following intravenous dosing but 6- or 86-fold more potent than cisapride or mosapride, respectively, and 9- or 18-fold more potent than tegaserod or cisapride, respectively, after intraduodenal administration. Orally dosed TD-5108 increased the contractility of the canine antrum, duodenum and jejunum with higher potency than tegaserod. The selective 5-HT(4) receptor agonist, TD-5108, demonstrates robust in vivo activity in the guinea pig, rat and dog gastrointestinal tracts.

The experiences of women receiving brachytherapy for cervical cancer: A systematic literature review.

OBJECTIVES: To determine women's experiences of brachytherapy for cervical cancer. KEY FINDINGS: Nineteen studies were included for data extraction/synthesis. Twelve studies focussed on psychological issues, seven on pharmacological aspects of women's experiences. Themes of anxiety, distress, pain, informational needs and non-pharmacological interventions were found. Nine out of ten psychological studies described brachytherapy as a distressing experience causing anxiety and distress for most women. Non-pharmacological interventions were found to be effective and inexpensive adjuncts. Peri and post-operative pharmacological management was variable, but duration of procedure was an important factor. CONCLUSION: Brachytherapy for gynaecological cancer causes varying levels of pain, anxiety and distress. To improve women's experiences there needs to be better pain management, patient information and the development of non-pharmacological interventions. Future recommendations are to develop clinical support guidelines, audit the quality of services and develop effective interventions to improve women's experiences of brachytherapy for locally advanced cervical cancer.

A comparison of the pharmacological properties of guinea-pig and human recombinant 5-HT4 receptors.

BACKGROUND AND PURPOSE: 5-HT(4) receptor agonists are used therapeutically to treat disorders of reduced gastrointestinal motility. Since such compounds are evaluated in guinea-pigs, we cloned, expressed and pharmacologically characterized the guinea-pig 5-HT(4) and human 5-HT(4(b)) splice variant, which share 95% homology. The functional properties of guinea-pig 5-HT(4(b)) receptors were compared with native receptors in guinea-pig colon. EXPERIMENTAL APPROACH: Membrane radioligand binding and whole cell cAMP accumulation assays were used to determine the affinities, potencies and intrinsic activities (IA). Contraction of the guinea-pig distal colon longitudinal muscle myenteric plexus preparation (LMMP) was monitored to evaluate functional activity. KEY RESULTS: pK(i) values for guinea-pig and human recombinant receptors, and guinea-pig striatum 5-HT(4) receptors, were in agreement, as were the potency and IA values for guinea-pig and human 5-HT(4) receptors expressed at a similar density ( approximately 0.2 pmol mg(-1) protein). Tegaserod was a potent (pEC(50)=8.4 and 8.7, respectively), full agonist at both guinea-pig and human 5-HT(4) receptors. In contrast, in the LMMP preparation, tegaserod was a potent, partial agonist (pEC(50)=8.2; IA=66%). CONCLUSIONS AND IMPLICATIONS: Close agreement between the pharmacological properties of guinea-pig and human 5-HT(4) receptors support the use of guinea-pig model systems for the identification of 5-HT(4) receptor therapeutics. However, the mechanisms underlying the different agonist properties of tegaserod in recombinant and isolated tissue preparations, and the extent to which these impact the clinical efficacy of tegaserod as a prokinetic agent, remain to be determined.

Diagnosis of adenocarcinoma in prostate needle biopsy tissue.

Prostate cancer is a major public health problem throughout the developed world. For patients with clinically localised prostate cancer, the diagnosis is typically established by histopathological examination of prostate needle biopsy samples. Major and minor criteria are used to establish the diagnosis, based on the microscopic appearance of slides stained using haematoxylin and eosin. Major criteria include an infiltrative glandular growth pattern, an absence of basal cells and nuclear atypia in the form of nucleomegaly and nucleolomegaly. In difficult cases, basal cell absence may be confirmed by immunohistochemical stains for high-molecular-weight cytokeratins (marked with antibody 34betaE12) or p63, which are basal cell markers. Minor criteria include intraluminal wispy blue mucin, pink amorphous secretions, mitotic figures, intraluminal crystalloids, adjacent high-grade prostatic intraepithelial neoplasia, amphophilic cytoplasm and nuclear hyperchromasia. Another useful diagnostic marker detectable by immunohistochemistry is alpha-methylacyl coenzyme A racemase (AMACR), an enzyme selectively expressed in neoplastic glandular epithelium. Cocktails of antibodies directed against basal cell markers and AMACR are particularly useful in evaluating small foci of atypical glands, and in substantiating a diagnosis of a minimal adenocarcinoma. Reporting of adenocarcinoma in needle biopsy specimens should always include the Gleason grade and measures of tumour extent in the needle core tissue. Measures of tumour extent are (1) number of cores positive for cancer in the number of cores examined, (2) percentage of needle core tissue affected by carcinoma and (3) linear millimetres of carcinoma present.

Receptor isoforms mediate opposing proliferative effects through gbetagamma-activated p38 or Akt pathways.

The opposing effects on proliferation mediated by G-protein-coupled receptor isoforms differing in their COOH termini could be correlated with the abilities of the receptors to differentially activate p38, implicated in apoptotic events, or phosphatidylinositol 3-kinase (PI 3-K), which provides a source of survival signals. These contrasting growth responses of the somatostatin sst(2) receptor isoforms, which couple to identical Galpha subunit pools (Galpha(i3) > Galpha(i2) >> Galpha(0)), were both inhibited following betagamma sequestration. The sst(2(a)) receptor-mediated ATF-2 activation and inhibition of proliferation induced by basic fibroblast growth factor (bFGF) were dependent on prolonged phosphorylation of p38. In contrast, cell proliferation and the associated transient phosphorylation of Akt and p70(rsk) induced by sst(2(b)) receptors were blocked by the PI 3-K inhibitor LY 294002. Stimulation with bFGF alone had no effect on the activity of either p38 or Akt but markedly enhanced p38 phosphorylation mediated by sst(2(a)) receptors, suggesting that a complex interplay exists between the transduction cascades activated by these distinct receptor types. In addition, although all receptors mediated a sustained activation of extracellular signal-regulated kinases (ERK1 and ERK2), induction of the tumor suppressor p21(cip1) was detected only following amplification of ERK and p38 phosphorylation by concomitant bFGF and sst(2(a)) receptor activation. Expression of constitutively active Akt in the presence of a p38 inhibitor enabled a proliferative response to be detected in sst(2(a)) receptor-expressing cells. These findings demonstrate that the duration of activation and a critical balance between the mitogen-activated protein kinase and PI 3-K pathways are important for controlling cell proliferation and that the COOH termini of the sst(2) receptor isoforms may determine the selection of appropriate betagamma-pairings necessary for interaction with distinct kinase cascades.

Safety of an attenuated West Nile virus vaccine, live Flavivirus chimera in horses.

REASON FOR PERFORMING STUDY: West Nile virus (WNV) infection is endemic and able to cause disease in naive hosts. It is necessary therefore to evaluate the safety of new vaccines. OBJECTIVES: To establish: 1) the safety of a modified live Flavivirus/West Nile virus (WN-FV) chimera by administration of an overdose and testing for shed of vaccine virus and spread to uninoculated sentinel horses; 2) that this vaccine did not become pathogenic once passaged in horses; and 3) vaccine safety under field conditions. METHODS: There were 3 protocols: 1) In the overdose/shed and spread study, horses were vaccinated with a 100x immunogenicity overdose of WN-FV chimera vaccine and housed with sentinel horses. 2) A reversion to virulence study, where horses were vaccinated with a 20x immunogenicity overdose of WN-FV chimera vaccine. Horses in both studies were evaluated for abnormal health conditions and samples obtained to detect virus, seroconversion and dissemination into tissues. 3) In a field safety test 919 healthy horses of various ages, breeds and sex were used. RESULTS: Vaccination did not result in site or systemic reactions in either experimental or field-injected horses. There was no shed of vaccine virus, no detection of vaccine virus into tissue and no reversion to virulence with passage. CONCLUSIONS: WN-FV chimera vaccine is safe to use in horses with no evidence of ill effects from very high doses of vaccine. There was no evidence of reversion to virulence. In addition, administration of this vaccine to several hundred horses that may have been previously exposed to WNV or WNV vaccine resulted in no untoward reactions. POTENTIAL RELEVANCE: These studies establish that this live attenuated Flavivirus chimera is safe to use for immunoprophylaxis against WNV disease in horses.

Efficacy, duration, and onset of immunogenicity of a West Nile virus vaccine, live Flavivirus chimera, in horses with a clinical disease challenge model.

REASON FOR PERFORMING STUDY: West Nile virus (WNF) is a Flavivirus responsible for a life-threatening neurological disease in man and horses. Development of improved vaccines against Flavivirus infections is therefore important. OBJECTIVES: To establish that a single immunogenicity dose of live Flavivirus chimera (WN-FV) vaccine protects horses from the disease and it induces a protective immune response, and to determine the duration of the protective immunity. METHODS: Clinical signs were compared between vaccinated (VACC) and control (CTRL) horses after an intrathecal WNV challenge given at 10 or 28 days, or 12 months post vaccination. RESULTS: Challenge of horses in the immunogenicity study at Day 28 post vaccination resulted in severe clinical signs of WNV infection in 10/10 control (CTRL) compared to 1/20 vaccinated (VACC) horses (P<0.01). None of the VACC horses developed viraemia and minimal histopathology was noted. Duration of immunity (DPI) was established at 12 months post vaccination. Eight of 10 CTRL exhibited severe clinical signs of infection compared to 1 of 9 VACC horses (P<0.05). There was a significant reduction in the occurrence of viraemia and histopathology lesion in VACC horses relative to CTRL horses. Horses challenged at Day 10 post vaccination experienced moderate or severe clinical signs of WNV infection in 3/3 CTRL compared to 5/6 VACC horses (P<0.05). CONCLUSIONS: This novel WN-FV chimera vaccine generates a protective immune response to WNV infection in horses that is demonstrated 10 days after a single vaccination and lasts for up to one year. POTENTIAL RELEVANCE: This is the first USDA licensed equine WNV vaccine to utilise a severe challenge model that produces the same WNV disease observed under field conditions to obtain a label claim for prevention of viraemia and aid in the prevention of WNV disease and encephalitis with a duration of immunity of 12 months.

Species and response dependent differences in the effects of MAPK inhibitors on P2X(7) receptor function.

BACKGROUND: Recent studies have implicated the mitogen activated protein kinase (MAPK) in cellular permeability changes following P2X(7) receptor activation in native tissues. In this study we have further studied the effect of MAPK inhibitors on recombinant and native P2X(7) receptors. EXPERIMENTAL APPROACH: The MAPK inhibitors SB-203580, SB-202190 and SB-242235 were examined in HEK293 cells expressing recombinant P2X(7) receptors and in THP-1 cells expressing native human P2X(7) receptors using a range of experimental approaches. KEY RESULTS: At human recombinant P2X(7) receptors, SB-203580 and SB-202190 were weak, non-competitive inhibitors (pIC(50)= 4.8 - 6.4) of ethidium accumulation stimulated by 2'- & 3'-O-(4benzoylbenzoyl)-ATP (BzATP) but SB-242235 (0.1-10 microM) had no effect. SB-203580 and SB-202190 had no effect on rat or mouse recombinant P2X(7) receptors and studies with chimeric P2X(7) receptors suggested that SB-203580 was only effective in chimeras containing the N-terminal 255aa of the human P2X(7) receptor. SB-203580 did not consistently affect BzATP-mediated increases in cell calcium levels and, in electrophysiological studies, it slightly decreased responses to 30 microM BzATP but potentiated responses to 100 microM BzATP. In THP1 cells, SB-203580 modestly inhibited BzATP-stimulated ethidium accumulation (pIC(50) 5.7 - < 5) but SB-202190 had no effect. Finally, SB-203580 did not block BzATP-stimulated interleukin-1beta release in THP-1 cells. CONCLUSIONS: This study confirms that high concentrations of SB-203580 and SB-202190 can block human P2X(7) receptor-mediated increases in cellular ethidium accumulation but suggest this is not related to MAPK inhibition. Overall, the data cast doubt on a general role of MAPK in mediating P2X(7) receptor mediated changes in cellular permeability.

Somatostatin receptors in the central nervous system.

Somatostatin was first identified chemically in 1973, since when much has been established about its synthesis, storage and release. It has important physiological actions, including a tonic inhibitory effect on growth hormone release from the pituitary. It has other central actions which are not well understood but recent cloning studies have identified at least five different types of cell membrane receptor for somatostatin. The identification of their genes has allowed studies on the distribution of the receptor transcripts in the central nervous system where they show distinct patterns of distribution, although there is evidence to indicate that more than one receptor type can co-exist in a single neuronal cell. Receptor selective radioligands and antibodies are being developed to further probe the exact location of the receptor proteins. This will lead to a better understanding of the functional role of these receptors in the brain and the prospect of determining the role, if any, of somatostatin in CNS disorders and the identification of potentially useful medicines.

Clonal origin of epithelial ovarian carcinoma: analysis by loss of heterozygosity, p53 mutation, and X-chromosome inactivation.

BACKGROUND: It has been suggested that multiple sites of epithelial ovarian carcinoma on the peritoneal surface reflect polyclonal disease arising from multiple primary tumors in the peritoneal mesothelium, rather than monoclonal disease spread by metastases from one primary ovarian cancer. PURPOSE: The purpose of this study was to investigate whether ovarian cancer has a monoclonal or polyclonal origin. METHODS: DNA specimens were obtained from peripheral blood lymphocytes (normal DNA) and from multiple tumor deposits of 17 women with epithelial ovarian carcinoma: primary tumors, metastatic deposits, and ascites. The clonal origin of each tumor was determined by performing (a) analysis to detect loss of heterozygosity at five loci on chromosomes 5, 11, 13, and 17; (b) sequencing of exons 5-8 of the p53 gene; and (c) X-chromosome inactivation analysis of the phosphoglycerate kinase (PGK) gene. RESULTS: In 15 of the 17 cases analyzed, there was clear evidence of monoclonal origin. The probability that the genetic events documented in these 15 cases occurred as independent events in each tumor deposit ranged from 2.5 x 10(-1) to 3.7 x 10(-16). In two cases, the pattern of allelic deletion and p53 gene mutation was compatible with either a monoclonal origin or origin from two primary ovarian tumors. CONCLUSIONS: The results did not support the hypothesis that ovarian cancer is a multifocal, polyclonal disease. Instead, the data suggest that sporadic epithelial ovarian carcinoma has either a monoclonal or a dual primary origin. IMPLICATIONS: These findings have important implications for understanding of the natural history of ovarian cancer and for clinical strategies aimed at prevention and early detection. Further studies will be required to determine the clonal origin of familial hereditary ovarian cancer.

Effect of canarypox virus (ALVAC)-mediated cytokine expression on murine prostate tumor growth.

BACKGROUND: Canarypox virus, ALVAC, does not replicate in infected mammalian cells and has potential as a vector for gene therapy in the treatment of cancer. PURPOSE: Recombinant viruses carrying DNA sequences encoding interleukin 2 (ALVAC-IL-2), interferon gamma (ALVAC-IFN gamma), tumor necrosis factor-alpha (ALVAC-TNF-alpha), or the co-stimulatory molecule B7-1 (ALVAC-B7-1) were investigated as agents for the treatment of a newly defined mouse prostate tumor model. METHODS: RM-1 mouse prostate cancer cells, which are syngeneic (i.e., same genetic background) to C57BL/6 mice, were used. The expression of foreign gene products in vitro in infected RM-1 cells was measured by immunoprecipitation, bioassay, or flow cytometry. The effects of foreign gene product expression on RM-1 tumor cell growth in C57BL/6 mice were measured after subcutaneous injection (in the back) of 5 x 10(5) uninfected or infected cells; measurements included determinations of time to a measurable tumor size, tumor size as a function of time, and survival. The induction of protective immunity by uninfected and infected RM-1 cells was tested by injection of lethally irradiated (70 Gy) cells and subsequent challenge with uninfected cells. The generation of cytotoxic T cells was monitored by use of a 51Cr release assay. Severe combined immunodeficient (SCID) mice were used to determine whether T or B lymphocytes were involved in ALVAC vector-mediated antitumor responses. Data were analyzed by use of Pearson's modification of the chi-squared test and Kaplan-Meier survival methods. Reported P values are two-sided. RESULTS: The level of foreign gene product expression in ALVAC-infected RM-1 cells was dependent on the multiplicity of virus infection used; a multiplicity of five viruses per infected cell was chosen for subsequent experiments. RM-1 tumor growth in C57BL/6 mice was not affected by tumor cell expression of IL-2 alone, IFN gamma alone, or B7-1 alone; however, expression of TNF-alpha alone significantly delayed tumor growth at early time points (compared with parental ALVAC-infected tumors, P = .0001 at day 21 and P = .037 at day 28). Tumor cell expression of both TNF-alpha and IL-2 completely inhibited tumor growth in 60%-100% of treated mice. No protection against subsequent tumor challenge was detected in mice previously exposed to RM-1 cells expressing both TNF-alpha and IL-2. Cytotoxic T-lymphocyte activity toward RM-1 cells was not observed in C57BL/6 mice that rejected tumors. Tumor cell expression of TNF-alpha and IL-2 also resulted in tumor growth inhibition in SCID mice. CONCLUSIONS: RM-1 mouse prostate cancer cells are readily infected by ALVAC vectors, and foreign gene products are efficiently expressed. Inhibition of RM-1 tumor growth by tumor cell expression of TNF-alpha and IL-2 appears to involve nonspecific antitumor activity.

Effect of epidermal growth factor on glioma cell growth, migration, and invasion in vitro.

Effects of epidermal growth factor (EGF) and an antibody (Ab-528) reactive against the binding site for EGF on human EGF receptors were studied on multicellular tumor spheroids obtained from three human glioma cell lines with high (D-37 MG), medium (D-247 MG), and low (D-263 MG) levels of EGF receptor expression. The D-247 MG and D-263 MG spheroids grew slowly or not at all in the absence of EGF, while in the presence of EGF they were growth stimulated. Tumor cell migration, as measured by the spread of cells from spheroids on a plastic substratum, was increased by the addition of EGF for all three cell lines. Stimulation of migration could be blocked by a subsequent addition of Ab-528 to the medium at a concentration of 50 micrograms/ml. Invasiveness of glioma cell spheroids into fetal rat brain aggregates was related to EGF receptor expression; the two lines with medium to high receptor expression (D-247 MG and D-37 MG) were invasive, while the line with low EGF receptor expression (D-263 MG) was noninvasive, as assessed by an in vitro coculture assay. In the D-247 MG cell line, morphometry revealed EGF-enhanced invasiveness of the tumor cells. The addition of the Ab-528 to EGF-treated cocultures reduced invasion in both D-247 MG and D-37 MG cell lines. Antibody Ab-528 alone did not affect glioma cell growth or migration but did inhibit invasiveness. The present study suggests that, in brain tumors with an increased number of normal-sized Mr 170,000 EGF receptors, EGF or an EGF-like ligand such as transforming growth factor-alpha may selectively facilitate expansive tumor growth and tumor cell invasion. This effect may in part be blocked or retarded by specific antibodies to the EGF receptor.