Active immunization with tumor cells transduced by a novel AAV plasmid-based gene delivery system

Ex vivo genetically engineered cytokine-secreting tumor cell vaccines have been shown to prevent metastatic disease in animal models of lung and breast cancer. Because of the inefficiency of existing modes of gene delivery in transducing primary human tumor cells, it has been difficult to clinically apply this strategy. In this study, liposome-mediated delivery of an adeno-associated virus (AAV)-based plasmid containing the sequence for murine gamma-interferon (gamma-IFN) (pMP6A-mIFN-gamma) was used to generate cytokine-secreting murine tumor cell vaccines. High levels of gamma-IFN and elevated class I major histocompatibility complex expression after transfer of pMP6A-mIFN-gamma into the murine lung cancer cell line, D122, was demonstrated. The efficiency of gene transfer was determined by two different methods and was estimated to be 10-15%. Irradiated gamma-IFN D122 cells generated by this novel gene delivery system (D122/pMP6A-mIFN-gamma) and also by standard retroviral methods (DIF2) were administered as weekly vaccinations by intraperitoneal injection to animals bearing 7-day-old intrafootpad D122 tumors. Hindlimb amputation was performed when footpad diameters reached 7 mm, and lungs were harvested 28 days later. Animals vaccinated with gamma-IFN-secreting D122 cells produced by AAV-based plasmids delivery demonstrated a significant delay in footpad tumor growth when compared with controls and DIF2 cells. Fifty-seven percent of animals vaccinated with D122/pMP6A-mIFN-gamma were free of pulmonary metastases 28 days after amputation, significantly improved from the 0, 7, and 15% observed in animals vaccinated with irradiated parental D122 cells, irradiated D122 cells lipofected with an empty-cassette vector (pMP6A), or DIF2 cells, respectively. These results and the ability to transfer genes with this delivery system to a broad range of tumor types support its use in the generation of cytokine-secreting tumor cell vaccinations for use in clinical trials.     

A continuous colorimetric assay for rhinovirus-14 3C protease using peptide p-nitroanilides as substrates

Primary salivary gland lymphomas are almost always of B lineage, with most being represented by low grade B-cell lymphoma of mucosa-associated lymphoid tissue. This study characterizes the rare non-B-cell lymphomas of the salivary gland based on an analysis of six cases. All patients were men, with a mean age of 53.5 years. They presented with submandibular or parotid mass, which on histological examination showed extensive interstitial infiltration by small, medium-sized, or large lymphoid cells. There was prominent invasion and expansion of the ducts and acini in five cases. Angioinvasion was evident in two cases. Three cases were of T lineage and were CD56 negative; one of these cases expressed CD30. Three cases showed an immunophenotype of CD2+ CD3(f)- CD3(p)+ CD56+, consistent with T/natural killer (NK) cell lymphoma. In situ hybridization for Epstein-Barr virus (EBV)-encoded early nuclear RNA (EBER) showed positive reaction exclusively in the three CD56+ cases. Clonal T-cell populations were shown in two CD56-negative cases by polymerase chain reaction on paraffin sections using primers for the T-cell-receptor (TCR) gamma-chain gene, but not in the other four cases (the three CD56+ cases and one CD56- case). Four patients (two CD56+ and two CD56-) died within 3 years, and two were disease free at 4 and 1.5 years, respectively. This study shows that salivary gland T- or T/NK-cell lymphomas cannot be reliably distinguished from B-cell lymphomas on morphological grounds alone, because both can show prominent lymphoepithelial lesions. It appears that T/NK-cell lymphomas, which are often extranodal in localization and strongly associated with Epstein-Barr virus (EBV), show a predilection to involve the salivary glands as well.     

Continued competency--a responsibility of the profession

For the past decade the Continued Competency Committee of the American Association of Dental Examiners has explored issues in continued competency for the dental profession. The efforts have focused on creating policy and standards which must be met by any continued competency assessment mechanisms. Nine potential systems are under review. Some, such as examination for diplomate status in a recognized dental specialty are already in place. The development and pilot testing of four new mechanisms--simulations, continuing education with measurable outcomes, case presentation, and in-office audit--is being encouraged.     

AP-3: an adaptor-like protein complex with ubiquitous expression

Equine herpesvirus type 2 (EHV-2) is a slow-growing, cytopathogenic gammaherpesvirus, which is suggested to be ubiquitous in the equine population. However, its precise role as a pathogen and its tissue tropism remains uncertain. To estimate the prevalence of EHV-2 in Germany and to investigate the possible pathogenicity of the virus, peripheral blood leucocytes (PBL) from 172 horses were examined for EHV-2 DNA by a sensitive and specific nested PCR based on the EcoRI-N genomic fragment and by classical cocultivation. PBL samples from 51% of the horses were positive by PCR and virus was isolated from 31% of the horses by cocultivation. However, almost all animals were seropositive for EHV-2. This may indicate that PBL do not harbour EHV-2 indefinitely after infection. Furthermore, a correlation between clinical signs and EHV-2 as a causative agent could not be determined. Nevertheless, the prevalence of virus was high among horses with upper respiratory tract disease, abortion and severe ataxia. The products of the second round of the PCR reactions showed size polymorphism. Sequencing of the products revealed that these size differences were due to repetition of the motif (AGACAGGGGCCATGCTGGC) between 9-16 times depending on the isolate, suggesting that the nested PCR might be a useful tool for the differentiation of EHV-2 isolates.     

Mycobacterium genavense infections in pet animals

Mycobacterium genavense, a recently reported cause of a wasting illness in patients with AIDS, was isolated from a cervical lymph mode from a dog with severe hind limb weakness and from trachael tissue from a parrot with acute onset respiratory distress. Physicians caring for immunocompromised patients should consider birds and dogs potential sources of M. genavense infection and submit appropriate specimens for culture.     

Retrograde root filling with dentin-bonded modified resin composite

A retrograde root-end cover with a special resin composite (Retroplast) combined with the dentin bonding agent Gluma (Bayer AG) has been used since 1984 by the authors. Its content of silver, added to promote radiopacity, has been found to lower the working time and storage stability of the composite and might cause discolorations. Since 1990, silver has therefore been replaced with ytterbium trifluoride, which eliminates these side effects. The purpose of this study was to compare the clinical results obtained with these two resin composites and to evaluate the healing results after several years in function. Apical fillings (351) with the modified Retroplast showed the following radiographic healing pattern 1 yr after surgery: 80% complete healing, 2% scar tissue, 12% uncertain healing, and 6% failure. No significant difference in this healing pattern was found, compared with that obtained with the silver-containing Retroplast. Cases with ytterbium trifluoride classified as scar tissue and uncertain healing at 1 yr when examined at 2 to 4 yr postoperatively showed 89% complete healing. 0% scar tissue, 1% uncertain healing, and 9% failure. This result is significantly different from that obtained 1 yr after surgery. Based on calculations, it was predicted that with time 90% will become complete healing, whereas 10% will become failure.     

Stress in employed women: impact of marital status and children at home on neurohormone output and home strain

We isolated peroxisome biogenesis mutants from Chinese hamster ovary (CHO) cells, using the 9-(1'-pyrene)nonanol/ultraviolet (P9OH/ UV) method and wild-type CHO-K1 cells that had been stably transfected with cDNA encoding Pex2p (formerly peroxisome assembly factor-1, PAF-1). Three mutant cell clones, ZP110, ZP111, and ZP114, showed cytosolic localization of catalase, thereby indicating a defect in peroxisome biogenesis, whereas ZP112 and ZP113 contained fewer but larger catalase-positive particles. Mutant ZP115 displayed an aberrant, tubular structure immunoreactive to anti-catalase antibody. Mutants lacking morphologically recognizable peroxisomes also showed the typical peroxisome assembly-defective phenotype such as severe loss of catalase latency and resistance to 12-(1'-pyrene)dodecanoic acid (P12)/UV treatment. ZP110 and ZP111, and ZP114 were found to belong to two novel complementation groups, respectively, by complementation group analysis with cDNA transfection and cell fusion. Cell fusion with fibroblasts from patients with peroxisome biogenesis disorders such as Zellweger syndrome revealed that ZP110 and ZP114 could not be classified to any of human complementation groups. Thus, ZP110/ZP111 and ZP114 are the first, two peroxisome-deficient cell mutants of newly identified complementation groups distinct from the ten mammalian groups previously characterized.     

Thyroid hormone-induced expression of specific somatostatin receptor subtypes correlates with involution of the TtT-97 murine thyrotrope tumor

Following the protracted hypothyroid state, treatment with thyroid hormone will induce a decline in TSH and reduce thyrotrope hyperplasia. Somatostatin is a hypothalamic peptide that has been implicated in the negative regulation of TSH secretion in the thyrotrope. Moreover, analogs of native somatostatin have potent TSH-reducing and growth-retarding effects on human thyrotropinomas. The TtT-97 tumor is an in vivo murine thyrotropic model that has retained its physiological response to thyroid hormone. This study investigates the regulation of somatostatin receptor subtypes in this tumor. TtT-97 tumors, actively growing in hypothyroid mice, did not express any significant somatostatin receptor messenger RNA (mRNA) or protein. T4 administration resulted in a reduction in TSH beta mRNA expression and a marked degree of tumor involution. Analysis of residual tumors from thyroid hormone-treated mice showed the specific up-regulation of SSTR1 and SSTR5 mRNA subtypes and the appearance of abundant, high affinity SSTR receptor-binding sites within the tumor. Thus, the TtT-97 tumor provides a thyrotrope-specific model in which to study the regulation of somatostatin receptor subtypes by thyroid hormone and correlate this expression with both antisecretory and antiproliferative effects.     

Structurally similar Drosophila alpha-tubulins are functionally distinct in vivo

We used transgenic analysis in Drosophila to compare the ability of two structurally similar alpha-tubulin isoforms to support microtubule assembly in vivo. Our data revealed that even closely related alpha-tubulin isoforms have different functional capacities. Thus, in multicellular organisms, even small changes in tubulin structure may have important consequences for regulation of the microtubule cytoskeleton. In spermatogenesis, all microtubule functions in the postmitotic male germ cells are carried out by a single tubulin heterodimer composed of the major Drosophila alpha-84B tubulin isoform and the testis-specific beta 2-tubulin isoform. We tested the ability of the developmentally regulated alpha 85E-tubulin isoform to replace alpha 84B in spermatogenesis. Even though it is 98% similar in sequence, alpha 85E is not functionally equivalent to alpha 84B. alpha 85E can support some functional microtubules in the male germ cells, but alpha 85E causes dominant male sterility if it makes up more than one-half of the total alpha-tubulin pool in the spermatids. alpha 85E does not disrupt meiotic spindle or cytoplasmic microtubules but causes defects in morphogenesis of the two classes of singlet microtubules in the sperm tail axoneme, the central pair and the accessory microtubules. Axonemal defects caused by alpha 85E are precisely reciprocal to dominant defects in doublet microtubules we observed in a previous study of ectopic germ-line expression of the developmentally regulated beta 3-tubulin isoform. These data demonstrate that the doublet and singlet axoneme microtubules have different requirements for alpha- and beta-tubulin structure. In their normal sites of expression, alpha 85E and beta 3 are coexpressed during differentiation of several somatic cell types, suggesting that alpha 85E and beta 3 might form a specialized heterodimer. Our tests of different alpha-beta pairs in spermatogenesis did not support this model. We conclude that if alpha 85E and beta 3 have specialized properties required for their normal functions, they act independently to modulate the properties of microtubules into which they are incorporated.