High-resolution computed tomography and pure-tone audiometry in patients with otosclerosis in the spongiotic phase.

OBJECTIVE: There is no consensus in the literature regarding the relationship between high-resolution computed tomography findings and hearing thresholds in pure-tone audiometry in otosclerosis. This study evaluated the association between high-resolution computed tomography findings and pure-tone audiometry in otosclerosis in the spongiotic phase. METHODS: A cross-sectional study was conducted of 57 ears with surgically confirmed stapes fixation and tomographic findings. Air conduction and bone conduction thresholds on audiometry, and air-bone gap, were analysed. RESULTS: There were no correlations between sites affected by otospongiosis and air conduction threshold, bone conduction threshold or air-bone gap in the analysed tomographic images, but the diameter of the otospongiotic focus was greater in the presence of extension of the otospongiotic foci to the cochlear endosteum. CONCLUSION: There were no relevant associations between high-resolution computed tomography findings and pure-tone audiometric measurements. However, the diameter of the otospongiotic focus was greater in the presence of extension of the otospongiotic foci to the cochlear endosteum.

Interferon gamma inhibits apoptotic cell death in B cell chronic lymphocytic leukemia.

The malignant, CD5+ B lymphocytes of B cell chronic lymphocytic leukemia (B-CLL) die by apoptosis in vitro. This is in contrast to the prolonged life span of the leukemic cells in vivo and likely reflects the lack of essential growth factors in the tissue culture medium. We found that interferon gamma (IFN-gamma) inhibits programmed cell death and promotes survival of B-CLL cells in culture. This effect may also be important in vivo: increased serum levels of IFN-gamma, ranging from 60 to > 2,200 pg/ml, were found in 7 of 10 B-CLL samples tested, whereas the sera of 10 healthy individuals did not contain detectable levels of this cytokine (< 20 pg/ml). High levels of IFN-gamma message were detected in RNA from T cell-depleted B-CLL peripheral blood samples by Northern blot analysis. Synthesis of IFN-gamma by B-CLL lymphocytes was confirmed by in situ hybridization and flow cytometry. The majority of B-CLL cells (74-82%) expressed detectable levels of IFN-gamma mRNA, and CD19+ B-CLL cells were labeled with anti-IFN-gamma monoclonal antibodies. These results show that IFN-gamma inhibits programmed cell death in B-CLL cells and suggest that the malignant cells are able to synthesize this cytokine. By delaying apoptosis, IFN-gamma may extend the life span of the malignant cells and thereby contribute to their clonal accumulation.

Differential behaviour of lipid based and polycation based gene transfer systems in transfecting primary human fibroblasts: a potential role of polylysine in nuclear transport.

DNA delivery systems for gene therapy applications have to be able to trigger the uptake of plasmid DNA into the nucleus. We have tested two types of non-viral vector systems, lipofection (cationic lipid-based, using Lipofectamine) and polyfection (cationic polymer-based, using glycerol enhanced transferrinfection), for their ability to transfect confluent, contact inhibited primary human fibroblasts. While both systems worked well with growing fibroblasts, polyfection was superior with confluent cells. A slight reduction in cell associated plasmid DNA was observed with resting cells, but it was similar for both types of complexes. Lipofectamine showed a prevalence for transfecting cycling cells as judged by costaining transfected cells with cell cycle markers. No such bias was observed when glycerol enhanced transferrinfection was used. Microinjection of plasmid DNA/polylysine complexes into the cytoplasm of fibroblasts resulted in a higher percentage of expressing cells than injection of plasmid DNA, offering an explanation for the higher transfection levels obtained with transferrinfection in non-growing cells.

Evidence for pluripotent stem cell origin of idiopathic myelofibrosis: clonal analysis of a case characterized by a N-ras gene mutation.

Three cases of idiopathic myelofibrosis were screened for the presence of mutations at codon 12, 13, or 61 of the ras gene family by a rapid method based on polymerase chain reaction and hybridization to mutation-specific oligonucleotides. PB cells of one patient showed a point mutation at codon 12 of the N-ras oncogene. This molecular genetic hallmark was used to investigate the clonal relationship of different cell lineages by cell separation analysis. Presence of the N-ras 12 mutation in granulocytes, monocytes, B cells, and T lymphocytes, as well as erythroblasts, indicates that idiopathic myelofibrosis originates from a pluripotent stem cell, at least in this patient.

Acute myeloid leukemia: analysis of ras gene mutations and clonality defined by polymorphic X-linked loci.

In vitro DNA amplification and synthetic oligonucleotide hybridization was used to analyze 57 acute myelocytic leukemias (AML) for the presence of ras gene mutations. We demonstrated mutated alleles in 19% of primary AMLs (10/51) as well as in five of six secondary leukemias. Mutations occurred predominantly at N-ras codons 12, 13, or 61 (13 cases) and twice at Ki-ras codons 12 and 13. Ras gene mutations were preferentially associated with an M4 morphology according to the FAB (French-American-British) classification, but no particular correlation was observed with respect to clinical parameter (sex, age, course of disease) or immunophenotype and karyotype. Mutated ras alleles were absent in nine mutation-positive cases analyzed during remission. However, a more complex pattern emerged from the five patients analyzed in relapse exhibiting identical ras mutations in three cases, absence of a mutated allele in one patient, and acquisition of a N-ras mutation in yet another case, in which no mutation had been detected initially. Moreover, restriction fragment length polymorphisms (RFLP) of the X-chromosome genes hypoxanthine phosphoribosyl transferase (HPRT) and phosphoglycerate kinase (PGK) were studied in 19 of the AML patients. Nine cases (47%) were heterozygous for BglI or BamHI RFLPs at the PGK or HPRT loci, respectively, and therefore suitable for clonal analysis investigating X-chromosome inactivation. All of the patients exhibited a monoclonal leukemic cell population at presentation. In addition, five of seven cases studied in remission showed reemergence of a polyclonal pattern. However, two children exhibited persistence of monoclonal hematopoiesis despite complete clinical/hematological remission and a corresponding loss of a mutated ras allele in one of the patients. These data indicate the value of molecular genetic approaches for evaluation of the heterogeneous nature of remission and relapse in AML.

Retrovirus-mediated gene transfer as an approach to analyze neuroblastoma relapse after autologous bone marrow transplantation.

Disseminated neuroblastoma is a malignancy of children often treated by intensive chemotherapy/radiotherapy followed by autologous bone marrow transplantation (ABMT). A high proportion of those treated subsequently relapse. It is unknown if relapse is a consequence of residual disease in the patient or of contaminating malignant cells remaining in the infused marrow, which, of necessity, is harvested and stored prior to ablative chemotherapy/radiotherapy. The assumption that residual cells in the infused marrow contribute to relapse has lead to the adoption of marrow purging prior to reinfusion. However, neither the necessity nor the efficacy of the procedure have been established. We now show how retroviral-mediated gene transfer using the LNL6 vector may resolve this issue. Clonogenic neuroblastoma cells in patient marrow can be transduced and the NEOR gene detected by observing individual neuroblastoma cell colony growth in G418, and by polymerase chain reaction (PCR) of individual colonies. Efficiency of transduction is between 0 and 13.5%. If marrow is exposed to LNL6 prior to infusion and marked cells are detected at the time of relapse, this would demonstrate that infused marrow contributed to disease recurrence. The technique could then be used to analyze the efficacy of marrow purging techniques. Since normal progenitor cells from these patients are also marked, the technique can be used to study factors that modify reconstitution and transducibility of infused marrow. Clinical studies using this approach have now begun.

Receptor-mediated gene transfer into human T lymphocytes via binding of DNA/CD3 antibody particles to the CD3 T cell receptor complex.

Retrovirus-mediated gene transfer is currently the method of choice for the transfection of human T lymphocytes for applications in gene therapy. Use of retroviral vectors, however, is hampered by limits on the size of the genetic material to be transferred, the requirement of dividing target cells, and by potential safety questions. Synthetic peptide-enhanced or adenovirus-enhanced receptor-mediated transferrinfection of DNA (SPET and AVET, respectively) is a powerful method for the introduction of genetic material into mammalian cells. Although transferrin has proven to be a useful ligand for gene transfer in many cell types, gene expression in T cells with transferrin/DNA complexes is usually not satisfactory. To improve gene transfer to T cells, antibodies directed against the CD3-T cell receptor complex were tested for their ability to function as ligands for DNA delivery. In T cell lines, up to 50% of the cells expressed a beta-galactosidase reporter gene using anti-CD3 gene transfer complexes. Applying optimized conditions, prestimulated primary peripheral blood lymphocytes were also transfected successfully, although at a lesser efficiency (5%).

Immunotherapy of metastatic malignant melanoma by a vaccine consisting of autologous interleukin 2-transfected cancer cells: outcome of a phase I study.

We performed a phase I trial to evaluate the safety and tolerability of repeated skin injections of IL-2-transfected autologous melanoma cells into patients with advanced disease. Cell suspensions, propagated from excised metastases, were IL-2 gene transfected by adenovirus-enhanced transferrinfection and X-irradiated prior to injection. Vaccine production was successful in 54% of the patients. Fifteen patients (37%) received two to eight skin vaccinations of either 3 x 10(6) (intradermal) or 1 x 10(7) (half intradermal, half subcutaneous) transfected melanoma cells per vaccination (secreting 140-17,060 biological response modifier program units of IL-2/10(6) cells/24 hr). Analyses of safety and efficacy were carried out in 15 and 14 patients, respectively. Overall, the vaccine was well tolerated. All patients displayed modest local reactions (erythema, induration, and pruritus) and some experienced flu-like symptoms. Apart from newly appearing (4 of 14) and increasing (5 of 14) anti-adenovirus and newly detectable anti-nuclear antibody titers (1 of 15), recipients developed de novo or exhibited increased melanoma cell-specific delayed-type hypersensitivity (DTH) reactions (8 of 15) and vitiligo (3 of 15) and showed signs of tumor regression (3 of 15). This supports the idea of a vaccine-induced or -amplified anti-cancer immune response. None of the patients exhibited complete or partial regressions, but five of them experienced periods of disease stabilization. Three of these individuals received more than the four planned vaccinations and their mean survival time was 15.7 +/- 3.5 months as compared to 7.8 +/- 4.6 months for the entire patient cohort. These data indicate that IL-2-producing, autologous cancer cells can be safely administered to stage IV melanoma patients and could conceivably be of benefit to patients with less advanced disease.

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.

Transfection and gene expression in normal and malignant primary B lymphocytes.

Although many different protocols for transfection of lymphoid cell lines exist, successful DNA transfer into primary B lymphocytes has, to date, not been demonstrated. We now describe a simple method for gene transfer into highly purified normal and malignant B lymphocytes by electroporation. Using a powerful expression vector containing two copies of the cytomegalovirus (CMV) immediate early enhancer linked to the human T cell lymphotropic virus I (HTLV I) promoter, we could demonstrate transfected gene expression in both high density small 'resting' B cells and in low density 'activated' B cells. Successful transfection was detected by expression of chloramphenicol acetyl transferase and by immunofluorescence. The neoplastic cells of B cell chronic lymphocytic leukemia could also be transfected with an efficiency of 5-10%, but only after preactivation. This method of transfection will permit analysis of the contribution of individual genes and their products to normal and malignant B cell growth and differentiation.

Small-fragment genomic libraries for the display of putative epitopes from clinically significant pathogens.

Taking advantage of whole genome sequences of bacterial pathogens in many thriving diseases with global impact, we developed a comprehensive screening procedure for the identification of putative vaccine candidate antigens. Importantly, this procedure relies on highly representative small-fragment genomic libraries that are expressed to display frame-selected epitope-size peptides on a bacterial cell surface and to interact directly with carefully selected disease-relevant high-titer sera. Here we describe the generation of small-fragment genomic libraries of Gram-positive and Gram-negative clinically significant pathogens, including Staphylococcus aureus and Staphylococcus epidermidis, Streptococcus pyogenes, Streptococcus agalactiae, and Streptococcus pneumoniae, Enterococcus faecalis, Helicobacter pylori, Chlamydia pneumoniae, the enterotoxigenic Escherichia coli, and Campylobacter jejuni. Large-scale sequencing revealed that the libraries, which provide an average of 20-fold coverage, were random and, as demonstrated with two S. aureus libraries, highly representative. Consistent with the comprehensive nature of this approach is the identification of epitopes that reside in both annotated and putatively novel open reading frames. The use of these libraries therefore allows for the rapid and direct identification of immunogenic epitopes with no apparent bias or difficulty that often associate with conventional expression methods.

Improvement of the enzymatic stability of a cytotoxic T-lymphocyte-epitope model peptide for its oral administration.

Oral administration of peptide antigens, to provide proper mucosal and/or systemic immunity, is largely ineffective. This is mainly due to the very small quantity of antigen that survives degradation in the intestine and that crosses the intestinal absorption membrane. The present study focuses on the improvement of the enzymatic stability of a 13 amino acid long peptide containing a cytotoxic T-lymphocytes (CTL)-epitope. Within this study, it is shown, that simple chemical modification at the N- and C-terminus of the peptide can provide significant stability towards enzymatic attack by intestinal exopeptidases. Around 50% of the modified peptide resisted enzymatic attack on native porcine intestinal mucosa within 3h of incubation at pH 6.8 and 37 degrees C, whereas unmodified control peptide was almost completely degraded within the same time period. Additionally, a mucoadhesive drug carrier matrix with specific inhibitory properties towards luminally secreted endopeptidases has been generated. The incorporation of the simply modified peptide in this delivery system should enhance the amount of biologically active antigen being available at the mucosal site for further presentation to immunomodulating systems. This might open the door for a successful oral immunotherapy.

Differential expression of TRAP Isoenzyme in B-CLL Cells Treated with Different Inducers.

Purified B-cells from normal tonsils and from the peripheral blood of eight patients with B-chronic lymphocytic leukemia (B-CLL) were treated in vitro with the protein kinase C (PKC) activators TPA, Bryostatin 1 (Bryo), mezerein, with the calcium ionophore A23187, and with the cytokines interleukin-1/2, interferon-alpha/gamma, tumor necrosis factor. The induction of the lysosomal enzyme tartrate-resistant acid phosphatase (TRAP) was examined at the RNA (by Northern blotting analysis) and the protein level (by isoelectric focusing). TRAP mRNA and protein were induced by treatment with PKC activators while the combination of PKC stimulator and calcium ionophore was not effective, TRAP mRNA was detected as early as 2 hours after initiation of the culture. This induction of positivity for TRAP which is the enzymatic hallmark of hairy cell leukemia (HCL) by TPA or Bryo is consistent with the previously reported acquisition of HCL-type cellular features in TPA-driven CLL cells; CLL cells exposed to the double stimulus of TPA or Bryo + A23187 have previously been described to differentiate to plasmacytoid cells which is in accord with their remaining TRAP negative. The present data provide evidence that 1) B-CLL cells can be induced by direct stimulation of PKC to convert to HCL-type cells; 2) TPA/Bryo-exposed B-CLL cells display phenotypes different from those of cells treated with combinations containing calcium ionophore; 3) the phosphoinositol signal transduction pathway distal to PKC can be activated effectively in B-CLL cells; 4) Bryo, a new natural PKC activator, induces responses in CLL similar to those caused by TPA; 5) TRAP is an inducible indicator of cellular activation. These observations provide support for the idea that HCL and HCL-like cells might differentiate along a separate branch of B-cell lineage and might represent mature "activation end stage" cells.

Delayed Induction of Proto-Oncogene Expression in B-CLL Cells by Tumor Necrosis Factor.

In-vitro treatment of B-chronic lymphocytic leukaemia (B-CLL) cells with tumour necrosis factor (TNF) induced the transcription of the proto-oncogenes c-fos, c-jun and c-myc. The products of these oncogenes appear to be involved in the signal transmission initiated via the phosphoinositol lipid and protein kinase C (PKC) signal transduction pathway. In contrast to the rapid induction of these oncogene products by the phorbol ester TPA (which directly activates PKC) alone or in combination with the calcium ionophore A23187, stimulation of c-fos, c-jun and c-myc mRNA expression by TNF is significantly delayed. A later onset of action mediated by TNF has similarly been reported for the induction of DNA synthesis and of gene expression following gene transfer in B-CLL cells. The delay in the response of B-CLL cells to TNF might be due to the delayed stimulation of the oncogene signal transmitters fos, jun and myc by TNF. The mechanism for this delay remains to be elucidated.

Bilateral narrow duplication of the internal auditory canal.

AIM: To demonstrate diagnostic imaging of an extremely rare presentation of bilateral narrow duplication of the internal auditory canal. CASE REPORT: An adolescent boy with profound sensorineural hearing loss presented for hearing rehabilitation. Imaging studies (i.e. multidetector computed tomography and magnetic resonance imaging) clearly demonstrated bilateral duplication of the internal auditory canals, with narrowing of the lower canals, unilateral cochlear and vestibular dysplasia, bilateral superior semicircular canal malformation, and bilateral absence of the posterior semicircular canals. CONCLUSION: To our knowledge, this is only the third such case described in the literature. Considering that the vestibulocochlear nerve has been unable to be demonstrated in almost all cases of duplicated internal auditory canal (unilateral and bilateral), our case supports the hypothesis that vestibulocochlear nerve aplasia or hypoplasia leads to internal auditory canal stenosis. We consider this rare presentation of bilateral narrow duplication of the internal auditory canal to represent a contraindication for cochlear implantation.

Depletion of naive T cells of the peripheral lymph nodes abrogates systemic antitumor protection conferred by IL-2 secreting cancer vaccines.

It has been postulated that IL-2 secreting cancer vaccines establish antitumor immunity because the cytokine acting in a paracrine fashion would deliver a helper signal directly to the T cells making contact with the modified tumor cells at the site of vaccination. However, patterns of lymphocyte recirculation cannot be reconciled with the above direct interaction model: only primed memory T cells rather than naive T lymphocytes patrol the periphery, while naive T cells travel to the peripheral lymph nodes, where priming occurs. We have found that in vivo treatment of mice with the antibody MEL-14 directed against L-selectin, which is a molecule expressed at high levels on naive T cells, can completely abrogate protection against a mouse melanoma conferred by an IL-2 secreting vaccine. Since mouse memory CD4 and CD8 T cells are L-selectin-low, only migration of naive T cells is perturbed by the in vivo antibody blockade. Thus, priming of naive T cells in the draining lymph node is a critical step for the successful vaccination by IL-2 secreting cancer vaccines. Such priming is performed most efficiently by professional antigen presenting cells; consequently, these data also imply that allogeneic origin of tumor vaccines may not exclude successful vaccination.

Adenovirus-enhanced receptor-mediated transferrinfection for the generation of tumor vaccines.

Cancer vaccines are genetically modified tumor cells that, by cytokine secretion or by expression of costimulatory molecules, are capable of mobilizing the host's immune system to destroy tumor cells. We have used adenovirus-enhanced transferrinfection (AVET) for the generation of cancer vaccines. This is a highly efficient method to deliver various genes into a large proportion of tumor cells, making further selection unnecessary. We found in the mouse M-3 melanoma model that two consecutive vaccinations with transfected cells secreting IL-2 protect animals from tumor development by a subsequent challenge, and result in long-lasting tumor-specific immunity dependent on both CD4+ and CD8+ T cells. Patterns of lymphocyte recirculation and the need for CD4+ T cells indicated that the role of IL-2 is not merely local 'replacement of help', as has been proposed before. Instead, our findings suggest a three-stage process for the generation of effector T cells after vaccination with IL-2 secreting tumor cells: (1) tumor antigen uptake and processing at the site of injection by APCs, (2) migration of APCs into the regional draining lymph nodes where T-cell priming occurs, and (3) recirculation of activated cytotoxic T cells, that recognize and eliminate distant tumor cells. This model also implies that allogeneic tumor cells or synthetic tumor antigens may be used with success in future cancer vaccines.

Defined synthetic vaccines.

Although vaccines have proven very successful in preventing certain infectious diseases, progress in the field has been slowed by the tediousness of developing classical vaccines consisting of whole pathogens. Thus, there is great need for improvement in several areas: firstly, the range of diseases which can be treated has to be expanded. Secondly, antigens have to be defined to make the use of whole pathogens as antigen obsolete. And thirdly, new adjuvants have to be developed which show low toxicity, high potency and are also able to drive the immune response in the desired direction. Ideally, a vaccine would only consist of well-characterized, synthetic materials. This review summarizes the different approaches for the development of completely defined synthetic vaccines.