Encephalitozoon cuniculi causes focal anterior cataract and uveitis in dogs.

Three mongrel dogs, aged 10 months (case 1), 14 months (case 2) and 7.5 years (case 3), were presented because of ophthalmologic disorders of 4 months, 6 months and 7 years duration, respectively. All three dogs were offspring of stray dogs from Hungary and Serbia and had positive serum antibody titres against Encephalitozoon (E.) cuniculi. The two young dogs showed unilateral, the older dog bilateral chronic anterior uveitis with posterior synechia and focal anterior cortical cataract. The fundi that could be evaluated developed focal tapetal hyporeflective lesions in the course of the disease. Dogs 1 and 2 underwent removal of the lens via phacoemulsification. PCR of the lens material was positive for E. cuniculi strains IV and II, respectively. In dog 2 findings suggestive of microsporidia were detected underneath the anterior lens capsule by immunohistochemical staining. In all cases medical treatment consisted of systemic fenbendazole, prednisolone, and topical anti-inflammatory drugs, and additional brinzolamid/timolol for dog 3. For the time being all cases (follow up 23 months, 6 months and 3 months, respectively) are still on topical anti-inflammatory therapy. It is concluded that E. cuniculi infections can cause cataract and chorioretinal lesions in dogs.

Urogenital inflammation: changes of leucocytes and ROS.

The presence of excess leucocytes in the semen has been associated with male infertility. According to the WHO, concentrations of more than 106 leucocytes ml-1 are considered as leucocytospermia, indicating genital tract infections. Up to now, no consensus has been achieved on how leucocytes should be quantified in semen. Using the peroxidase staining and monoclonal antibodies to CD15, CD45 and CD68, we found significant differences between the detection methods. Only 47.4% of the semen samples that were assessed as leucocytospermic by CD45 were identified as such by peroxidase staining. The concentration of peroxidase-positive cells was significantly correlated with polymorphonuclear granulocyte (PMN) elastase (P < 0.0001). However, a negative correlation of peroxidase-positive cells with the sperm concentration was only found in oligozoospermic patients (P < 0.0001). Moreover, the slightly positive correlation with normal sperm morphology seems to be applicable only in cases of oligozoospermia. Significant negative correlation of the number of peroxidase-positive cells were found for both maximal inducible acrosome reaction (P = 0.0219) and the inducibility of acrosome reaction (P = 0.0370), indicating a rather deleterious effect of leucocytes on this important sperm function. Concerning the result in the in vitro fertilization programme, none of the examined parameters (PMN elastase, concentration of round cells and peroxidase-positive cells) showed a correlation with either fertilization or pregnancy. This result seems to be reasonable as severely damaged spermatozoa and leucocytes are eliminated from the ejaculate by different sperm separation methods. Interestingly, a significant negative correlation of the TUNEL assay as a measure of sperm DNA fragmentation was found only with pregnancy (P = 0.006) but not with fertilization. As DNA fragmentation can also be caused by ROS that are generated by leucocytes, this causality should not be neglected.

Computerizing incident reporting at a community hospital.

BACKGROUND: A community hospital in Seattle, Northwest Hospital (NWH), reengineered its paper-based incident reporting system into a computerized reporting, notification, and tracking tool. In July 1996 a small interdisciplinary working group was formed to improve the incident reporting system so that it would decrease the time needed to complete an incident report, collect more precise data about the incident, allow department managers instant access to all open memos involving or generated by their departments, allow ad hoc reporting by managers and administration, and allow only involved parties access to memos. IMPLEMENTATION AND EVALUATION: After a pilot study was conducted in the Childbirth Center and the pharmacy department began using the computerized incident reporting system, other units began using the system according to a weekly roll-out schedule during the first two quarters of 1998. In the third quarter of 1998, NWH began using the system as its primary quality assurance and incident reporting tool. As soon as an incident is documented, it is in the database and available for reporting. Data collected from January 1998 through December 1999 indicated that turnaround time for the life cycle of an incident report decreased from 53 to 12 days. At least 20 hours a month were saved in transcription and data entry time using the new system. DISCUSSION: Although incident reporting is now more streamlined and efficient, a few issues have emerged that need to be addressed, some relating to users' incorrect entering of information. Improvements are still being made to the system on an ongoing basis.

Recombinant adeno-associated virus for the generation of autologous, gene-modified tumor vaccines: evidence for a high transduction efficiency into primary epithelial cancer cells.

To explore the potential of recombinant vectors based on recombinant adeno-associated virus (rAAV) for cancer vaccination, we investigated the transduction efficiency of rAAV into cancer cells ex vivo. Infection of human epithelial cancer cell lines with rAAV carrying reporter genes encoding beta-galactosidase (rAAV/LacZ) or luciferase (rAAV/Luc) resulted in high levels of reporter gene expression (>90% positive cells). In marked contrast, rAAV poorly transduced all murine tumor cell lines, as well as human hematopoietic cell lines. Either irradiation or adenovirus infection of tumor cells prior to rAAV infection induced a 10- to 100-fold increase of reporter gene expression. To determine the transduction efficiency of rAAV into primary cancer cells, freshly isolated, irradiated tumor cells from malignant melanoma and ovarian carcinoma patients were infected with rAAV/Luc, resulting in up to 6.9-fold higher levels of gene expression than in a HeLa tumor cell line. Time course experiments with freshly isolated tumor cells infected with rAAV/Luc showed maximal levels of luciferase expression between days 3 and 9 posttransduction. Simultaneous infection of primary tumor cells with up to three rAAV vectors containing genes encoding the immunostimulatory proteins B7-2 (CD86), p35 subunit of IL-12, and p40 subunit of IL-12 resulted in high expression of B7-2 in more than 90% of the tumor cells and in the secretion of high levels of IL-12. Taken together, our results demonstrate that rAAV efficiently transduces freshly isolated human, epithelial tumor cells and might therefore be a potent tool to produce improved, gene-modified cancer vaccines.

Cloning of prokaryotic genomes in yeast artificial chromosomes: application to the population genetics of Pseudomonas aeruginosa.

Yeast artificial chromosomes (YACs) can accommodate large inserts and hence should be attractive tools for intra- and interspecies comparisons of bacterial genomes. YAC libraries were constructed from size-selected partial digests of human and Pseudomonas aeruginosa PAO DNA and SpeI-restricted PAO DNA. Whereas YACs from human DNA had an average size of 350 kilobase pairs (kbp), a P. aeruginosa sequence larger than 120 kbp was absent or truncated in the eukaryotic host. Coligation occurred for YACs smaller than 40 kbp, but stable YACs with 40-120 kbp large inserts of P. aeruginosa DNA were obtained in high yield. SpeI-restricted chromosomes from 97 P. aeruginosa strains representing 47 genotypes were hybridized with stable YACs from three equidistant regions of the PAO genome. The low complexity of hybridizing bands demonstrated that the analyzed 100 kbp sequence contigs were stably maintained in most P. aeruginosa isolates from both disease and environmental habitats.

Gene transfer of the costimulatory molecules B7-1 and B7-2 into human multiple myeloma cells by recombinant adeno-associated virus enhances the cytolytic T cell response.

Gene transfer of the costimulatory molecules B7-1 and B7-2 induces a potent antitumor immune response in a variety of tumor models. B cell neoplasms including multiple myeloma (MM) often show little or no expression of B7 antigens; they are therefore a potential target for this approach. To increase the expression of human B7 genes in MM cells, both genes and the neomycin phosphotransferase gene were packaged into recombinant adeno-associated virus vectors (rAAV). The resulting recombinant viruses rAAV/B7-1, rAAV/B7-2 and rAAV/Neo were used to transduce the MM cell lines LP-1 and RPMI 8226. This allowed the transduction of up to 80% of LP-1 cells 4 days after infection with purified rAAV particles. The response of human allogeneic T cells to rAAV/B7-1 and rAAV/B7-2 transduced, gamma-irradiated LP-1 cells was assessed by [3H]thymidine incorporation, by RT-PCR-based detection of immunostimulatory cytokine transcripts and by ELISA quantification of cytokines in the supernatant. Stimulation of T cells with rAAV/B7-1 or rAAV/B7-2 transduced LP-1 cells resulted in an up to 10-fold increase of T cell proliferation when compared with LP-1 cells transduced with rAAV/Neo. Similar results were obtained with RPMI 8226 cells. Both rAAV/B7-1 and rAAV/B7-2 transduced LP-1 cells stimulated the T cell secretion of IL-2 and IFN-gamma. Furthermore, [51Cr] release assays showed that rAAV/B7-1 or rAAV/B7-2 transduced LP-1 cells induced a cytolytic T cell (CTL) response, in contrast to LP-1 cells transduced with rAAV/Neo. In all assays, the effects of rAAV/B7-1 and rAAV/B7-2 were similar. Taken together, the results show that rAAV-mediated transfer of B7 genes into MM cell lines is able to enhance the antitumor T cell response and to elicit a cytolytic T cell response.

Generation of effective cancer vaccines genetically engineered to secrete cytokines using adenovirus-enhanced transferrinfection (AVET).

Cancer vaccines are based on the concept that tumors express novel antigens and thus differ from their normal tissue counterparts. Such putative tumor-specific antigens should be recognizable by the immune system. However, malignant cells are of self origin and only poorly immunogenic, which limits their capability to induce an anticancer immune response. To overcome this problem, tumor cells have been isolated, genetically engineered to secrete cytokine gene products and administered as cancer vaccines. We used adenovirus-enhanced transferrinfection (AVET), which allows high-level transient transgene expression, to introduce cytokine gene expression vectors into murine melanoma cells. The efficiency of AVET makes laborious selection and cloning procedures obsolete. We administered such modified tumor cells as cancer vaccines to syngeneic animals and investigated their impact on the induction of anticancer immunity. We found that IL-2 or GM-CSF gene-transfected murine melanoma cells are highly effective vaccines. Both of these cytokine-secreting vaccines cured 80% of animals which bore a subcutaneous micrometastasis prior to treatment, and induced potent antitumor immunity. The generation of antitumor immunity by these cytokine-secreting vaccines requires three different steps: (1) tumor antigen uptake and processing by antigen-presenting cells (APCs) at the site of vaccination; (2) migration of these APCs into the regional lymph nodes where T-cell priming occurs; (3) recirculation of specific, activated T-cells that recognize distinct tumor load and initiate its elimination. Extending our previously reported studies, we have now comprehensively analysed the requirements for effective antitumor vaccination in animals. This may also become the basis for treatment of human cancer patients.

[Oral vaccines against poliomyelitis and vaccination-related paralytic poliomyelitis in Germany. Do we need a new immunization strategy?]. / Schluckimpfung gegen Poliomyelitis und impfassoziierte paralytische Poliomyelitis in Deutschland. Brauchen wir eine neue Impfstrategie?

Oral vaccination against poliomyelitis, which was carried out worldwide, lead to eradication of poliomyelitis in the United States, in South America and parts of Europe; in other parts of the world, paralytic poliomyelitis is still a severe risk of health. In those countries where poliomyelitis has been eradicated, it is presently discussed whether the vaccination schedules should be changed to an inactivated polio vaccine (IPV), as in polio-free countries only cases of paralytic poliomyelitis after vaccinations have been reported. Behringwerke's data from a 30-year period of analysing adverse drug reaction reveal the following: using the trivalent oral polio vaccine (OPV), based on WHO case definition, the risk for vaccine-associated paralytic poliomyelitis with permanent damage is approximately 1 case for 4.5 million vaccinations (0.22 per million) in vaccinees, and approximately 1 case for 11 million (0.09 per million) in contact persons. This low risk is in line with the data ascertained worldwide.

The use of recombinant adeno-associated viral vectors for the transduction of epithelial tumor cells.

Using hight-titer recombinant adeno-associated viral vectors (rAAV), we have investigated the feasibility of cancer vaccines from tumor explants. In a first set of experiments, rAAV vectors expressing firefly luciferase reporter genes were used to transduce different human tumor cell lines. At day three post transduction, all of the human tumor cell lines tested showed high levels of luciferase expression. To further evaluate rAAV-mediated gene transfer efficiency into primary tumor cells, we transduced freshly isolated tumor cells from malignant melanoma and ovarian carcinoma patients. As a remarkable result, reporter gene expression in primary tumor cells was significantly higher than in the tested established tumor cell lines. These data could also be reproduced with a rAAV/lacZ vector, since the portion of successfully transduced primary tumor was higher than 90%. Taken together, our data demonstrate that rAAV-mediated gene transfer is a very efficient method for the transduction of freshly isolated human tumor cells and may allow the generation of potent autologous cancer vaccines.