Search

Background: Gene therapies based on adeno-associated viruses (AAV) are a therapeutic option to successfully treat monogenetic diseases. However, the influence of pre-existing immunity to AAV can compromise the application of AAV gene therapy, most notably by the presence of neutralizing antibodies (NAb) to AAV., Methods: In the following study, we investigated to what extent the treatment by immunoadsorption (IA) would reduce the levels of human anti-AAV antibodies to AAV2 and AAV5. To that end, we screened blood sera from 40 patients receiving IA treatment because of underlying autoimmune disease or transplant rejection, with detectable AAV-antibodies in 23 patients (22 by NAb detection, and 1 additionally by anti-AAV5 ELISA analysis)., Results: Our results show that IA efficiently depleted anti-AAV2 NAb with a mean reduction of 3.92 ± 1.09 log2 titer steps (93.4%) after three to five single IA treatments, 45% of seropositive subjects had an anti-AAV2 titer below the threshold titer of 1:5 after the IA treatment series. Anti-AAV5 NAb were reduced to below the threshold titer of 1:5 in all but one of five seropositive subjects. Analysis of total anti-AAV5 antibodies by ELISA demonstrated an anti-AAV5 antibody reduction over the IA treatment series of 2.67 ± 1.16 log2 titer steps (84.3%)., Conclusion: In summary, IA may represent a safe strategy to precondition patients with pre-existing anti-AAV antibodies to make this population eligible for an effective AAV-based gene therapy., (© 2023 The Authors. Journal of Clinical Apheresis published by Wiley Periodicals LLC.)

Engineered T cell therapies have shown significant clinical success. However, current manufacturing capabilities present a challenge in bringing these therapies to patients. Furthermore, the cost of development and manufacturing is still extremely high due to complexity of the manufacturing process. Increased automation can improve quality and reproducibility while reducing costs through minimizing hands-on operator time, allowing parallel manufacture of multiple products, and reducing the complexity of technology transfer. In this article, we describe the results of a strategic alliance between GSK and Miltenyi Biotec to develop a closed, automated manufacturing process using the CliniMACS Prodigy for autologous T cell therapy products that can deliver a high number of cells suitable for treating solid tumor indications and compatible with cryopreserved apheresis and drug product. We demonstrate the ability of the T cell Transduction - Large Scale process to deliver a significantly higher cell number than the existing process, achieving 1.5 × 10 10 cells after 12 days of expansion, without affecting other product attributes. We demonstrate successful technology transfer of this robust process into three manufacturing facilities., Competing Interests: N.F., S.N., and A.K. are current or past employees and/or shareholders of GSK. M.B., S.P., C.H., A.B., A.R., T.O., S.L., K.I., B.G., and S.W. are current employees of Miltenyi Biotec., (© 2023 GlaxoSmithKline plc.)

Background: There is high medical need for safe long-term immunosuppression monotherapy in kidney transplantation. Selective targeting of post-transplant alloantigen-(re)activated effector-T cells by anti-TNF antibodies after global T cell depletion may allow safe drug minimization, however, it is unsolved what might be the best maintenance monotherapy., Methods: In this open, prospective observational single-centre trial, 20 primary deceased donor kidney transplant recipients received 2x20 mg Alemtuzumab (d0/d1) followed by 5 mg/kg Infliximab (d2). For 14 days all patients received only tacrolimus, then they were allocated to either receive tacrolimus (TAC, n = 13) or sirolimus (SIR, n = 7) monotherapy, respectively. Protocol biopsies and extensive immune monitoring were performed and patients were followed-up for 60 months., Results: TAC-monotherapy resulted in excellent graft survival (5yr 92%, 95%CI: 56.6-98.9) and function, normal histology, and no proteinuria. Immune monitoring revealed low intragraft inflammation (urinary IP-10) and hints for the development of operational tolerance signature in the TAC- but not SIR-group. Remarkably, the TAC-monotherapy was successful in all five presensitized (ELISPOT+) patients. However, recruitment into SIR-arm was stopped (after n = 7) because of high incidence of proteinuria and acute/chronic rejection in biopsies. No opportunistic infections occurred during follow-up., Conclusions: In conclusion, our novel fast-track TAC-monotherapy protocol is likely to be safe and preliminary results indicated an excellent 5-year outcome, however, a full-scale study will be needed to confirm our findings., Trial Registration: EudraCT Number: 2006-003110-18., Competing Interests: O.V. recived lecture fees from Sanofi. S.T., S.S., U.J., and B.G. were employed by Miltenyi Biotec GmbH, Germany. This does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.

Introduction: Chemotherapy resistance resulting in incomplete pathologic response is associated with high risk of metastasis and early relapse in breast cancer. The aim of this study was to identify and evaluate biomarkers of treatment-resistant tumor cells., Methods: We performed a cell surface marker screen in triple-negative breast cancer patient-derived xenograft models treated with standard care genotoxic chemotherapy. Global expression profiling was used to further characterize the identified treatment-resistant subpopulations., Results: High expression of sialyl-glycolipid stage-specific embryonic antigen 4 (SSEA4) was found in residual tumor cells surviving chemotherapy and in samples from metastatic patients who relapsed after neoadjuvant chemotherapy. Gene and microRNA (miRNA) expression profiling linked SSEA4 positivity with a mesenchymal phenotype and a deregulation of drug resistance pathways. Functional assays demonstrated a direct link between epithelial-mesenchymal transition (EMT) and SSEA4 expression. Interestingly, SSEA4 expression, EMT, and drug resistance seemed to be regulated posttranscriptionally. Finally, high expression of CMP-N-acetylneuraminate-β-galactosamide-α-2,3-sialyltransferase 2 (ST3GAL2), the rate-limiting enzyme of SSEA4 synthesis, was found to be associated with poor clinical outcome in breast and ovarian cancer patients treated with chemotherapy., Conclusions: In this study, we identified SSEA4 as highly expressed in a subpopulation of tumor cells resistant to multiple commonly used chemotherapy drugs, as well as ST3GAL2, the rate-limiting enzyme of SSEA4 synthesis, as a predictive marker of poor outcome for breast and ovarian cancer patients undergoing chemotherapy. Both biomarkers and additionally identified regulatory miRNAs may be used to further understand chemoresistance, to stratify patient groups in order to avoid ineffective and painful therapies, and to develop alternative treatment regimens for breast cancer patients.

Background: Clinically useful biomarkers for patient stratification and monitoring of disease progression and drug response are in big demand in drug development and for addressing potential safety concerns. Many diseases influence the frequency and phenotype of cells found in the peripheral blood and the transcriptome of blood cells. Changes in cell type composition influence whole blood gene expression analysis results and thus the discovery of true transcript level changes remains a challenge. We propose a robust and reproducible procedure, which includes whole transcriptome gene expression profiling of major subsets of immune cell cells directly sorted from whole blood., Methods: Target cells were enriched using magnetic microbeads and an autoMACS® Pro Separator (Miltenyi Biotec). Flow cytometric analysis for purity was performed before and after magnetic cell sorting. Total RNA was hybridized on HGU133 Plus 2.0 expression microarrays (Affymetrix, USA). CEL files signal intensity values were condensed using RMA and a custom CDF file (EntrezGene-based)., Results: Positive selection by use of MACS® Technology coupled to transcriptomics was assessed for eight different peripheral blood cell types, CD14+ monocytes, CD3+, CD4+, or CD8+ T cells, CD15+ granulocytes, CD19+ B cells, CD56+ NK cells, and CD45+ pan leukocytes. RNA quality from enriched cells was above a RIN of eight. GeneChip analysis confirmed cell type specific transcriptome profiles. Storing whole blood collected in an EDTA Vacutainer® tube at 4°C followed by MACS does not activate sorted cells. Gene expression analysis supports cell enrichment measurements by MACS., Conclusions: The proposed workflow generates reproducible cell-type specific transcriptome data which can be translated to clinical settings and used to identify clinically relevant gene expression biomarkers from whole blood samples. This procedure enables the integration of transcriptomics of relevant immune cell subsets sorted directly from whole blood in clinical trial protocols.

MicroRNAs are important negative regulators of protein-coding gene expression and have been studied intensively over the past years. Several measurement platforms have been developed to determine relative miRNA abundance in biological samples using different technologies such as small RNA sequencing, reverse transcription-quantitative PCR (RT-qPCR) and (microarray) hybridization. In this study, we systematically compared 12 commercially available platforms for analysis of microRNA expression. We measured an identical set of 20 standardized positive and negative control samples, including human universal reference RNA, human brain RNA and titrations thereof, human serum samples and synthetic spikes from microRNA family members with varying homology. We developed robust quality metrics to objectively assess platform performance in terms of reproducibility, sensitivity, accuracy, specificity and concordance of differential expression. The results indicate that each method has its strengths and weaknesses, which help to guide informed selection of a quantitative microRNA gene expression platform for particular study goals.

CD133+ cells are hemangioblasts that have capacity to generate into both hematopoietic and endothelial cells (ECs). Hypoxia/normoxia has shown to be the regulator of the balance between stemness and differentiation. In this study we performed Agilent's whole human genome oligo microarray analysis and examined the differentiation potential of the bone-marrow-derived CD133+ cells after hypoxic/normoxic preconditioning of CD133+ cells. Results showed that there was no significant increase in erythroid colony forming unit (CFU-E) and CFU-granulocyte, erythrocyte, monocyte, and megakaryocyte formation with cells treated under hypoxia/normoxia. However, a significant increment of EC forming unit at 24 h (143.2 +/- 8.0%) compared to 0 h (100 +/- 11.4%) was observed in CFU-EC analysis. Reverse transcription-polymerase chain reaction and immunostaining analysis showed that the differentiated cells diminished hematopoietic stem cell surface markers and acquired the gene markers and functional phenotype of ECs. The transcriptome profile revealed a cluster of 232 downregulated and 498 upregulated genes in cells treated for 24 h under hypoxia. The upregulated genes include angiogenic genes, angiogenic growth factor genes, angiogenic cytokine and chemokine genes, as well as angiogenic-positive regulatory genes, including FGFBP1, PDGFB, CCL15, CXCL12, CXCL6, IL-6, PTN, EREG, ERBB2, EDG5, FGF3, FHF2, GDF15, JUN, L1CAM, NRG1, NGFR, and PDGFB. On the other hand, angiogenesis inhibitors and related genes, including IL12A, MLLT7, STAB1, and TIMP2, are downregulated. Taken together, hypoxic/normoxic preconditioning may lead to the differentiation of CD133+ cells toward endothelial lineage, which may improve the current clinical trial studies.

Identifying transplant recipients in whom immunological tolerance is established or is developing would allow an individually tailored approach to their posttransplantation management. In this study, we aimed to develop reliable and reproducible in vitro assays capable of detecting tolerance in renal transplant recipients. Several biomarkers and bioassays were screened on a training set that included 11 operationally tolerant renal transplant recipients, recipient groups following different immunosuppressive regimes, recipients undergoing chronic rejection, and healthy controls. Highly predictive assays were repeated on an independent test set that included 24 tolerant renal transplant recipients. Tolerant patients displayed an expansion of peripheral blood B and NK lymphocytes, fewer activated CD4+ T cells, a lack of donor-specific antibodies, donor-specific hyporesponsiveness of CD4+ T cells, and a high ratio of forkhead box P3 to alpha-1,2-mannosidase gene expression. Microarray analysis further revealed in tolerant recipients a bias toward differential expression of B cell-related genes and their associated molecular pathways. By combining these indices of tolerance as a cross-platform biomarker signature, we were able to identify tolerant recipients in both the training set and the test set. This study provides an immunological profile of the tolerant state that, with further validation, should inform and shape drug-weaning protocols in renal transplant recipients.

In order to identify novel genes involved in appetite and body weight regulation we have developed a microarray based method suitable for detecting small changes in gene expression in discrete groups of hypothalamic neurons. The method is based on a combination of stereological sampling, laser capture microdissection (LCM), PCR based amplification (SuperAmp), and one-color cDNA microarray analysis. To validate the method we assessed and compared fasting induced changes in mRNA levels of Neuropeptide Y (NPY) and proopiomelanocortin (POMC) in the hypothalamic arcuate nucleus (ARC) of diet-induced obese rats using cDNA microarrays, quantitative PCR and in situ hybridization. All methods revealed statistically significant fasting-induced changes in NPY and POMC expression. An additional 3480 differentially expressed probes (fold change >1.22, t-test p=0.05) were identified in the microarray analysis. Our findings demonstrate a consistent gene expression pattern across three different gene expression detection methods and strongly suggest that LCM coupled microarray analysis combined with SuperAmp can be used as a semi-quantitative mRNA profiling tool. Importantly, the sensitivity of the method greatly improves the usefulness of the microarray technology for gene expression profiling in non-homogeneous tissues such as the brain.

The induction of heme oxygenase 1 (HO-1) by a single treatment with cobalt protoporphyrin (CoPPIX) protects against inflammatory liver failure and ischemia reperfusion injury after allotransplantation. In this context, the HO-1-mediated inhibition of donor-derived dendritic cell maturation and migration is discussed as one of the key events of graft protection. To investigate the poorly understood mechanism of CoPPIX-induced HO-1 activity in more detail, we performed gene expression analysis in murine liver, revealing the up-regulation of STAT3 after CoPPIX treatment. By using wild-type and HO-1-deficient dendritic cells we demonstrated that LPS-induced maturation is dependent on STAT3 phosphorylation and independent of HO-1 activity. In summary, our observations revise our understanding of the anti-inflammatory properties of HO-1 and highlight the immunomodulatory capacity of STAT3, which might be of further interest for targeting undesired immune responses, including ischemia reperfusion injury.

Ischemia/reperfusion injury (IRI) has a major impact on short- and long-term renal allograft survival by increasing graft immunogenicity. Donor preconditioning by inducing heme oxygenase 1 (HO-1) has been proven to exert cytoprotective and antiinflammatory effects on the graft, thus resulting in reduced graft immunogenicity. The study analyzed the effects and mechanisms of HO-1-mediated cytoprotection in rat kidney transplants exposed to cold preservation. We studied the differential gene-expression patterns of allografts after either short or long cold ischemia using a customized cDNA microarray. Prolonged cold ischemia led, 12 h after engraftment, to enhanced levels of adhesion molecules, heat-shock proteins, chemokines (CXCL10), and a remarkable upregulation of immunoproteasomes. Next we addressed the question whether induction of HO-1 or its byproduct carbon monoxide (CO) in organ donors targets these candidate markers related to enhanced immunogenicity. Induction of HO-1 or CO in organ donors 24 h before organ harvesting resulted in reduced mRNA levels of immunoproteasomes, MHC class II expression, and co-stimulatory molecules in the recipient's spleen, suggesting diminished migration and activation of donor dendritic cells. This observation suggests that HO-1/CO induction protects marginal allografts by inhibiting the immunogenicity of donor-derived dendritic cells.

Allogeneic and autoimmune islet destruction limits the success of islet transplantation in autoimmune diabetic patients. This study was designed to investigate whether ex vivo gene transfer of viral interleukin-10 (vIL-10) protects BioBreeding (BB) rat islets from autoimmune destruction after transplantation into diabetic BB recipients. Islets were transduced with adenoviral constructs (Ad) expressing the enhanced green fluorescent protein (eGFP), alpha-1 antitrypsin (AAT) or vIL-10. Transduction efficiency was demonstrated by eGFP-positive cells and vIL-10 production. Islet function was determined in vitro by measuring insulin content and insulin secretion and in vivo by grafting AdvIL-10-transduced islets into syngeneic streptozotocin (SZ)-diabetic, congenic Lewis (LEW.1 W) rats. Finally, gene-modified BB rat islets were grafted into autoimmune diabetic BB rats. Ad-transduction efficiency of islets increased with virus titre and did not interfere with insulin content and insulin secretion. Ad-transduction did not induce Fas on islet cells. AdvIL-10-transduced LEW.1 W rat islets survived permanently in SZ-diabetic LEW.1 W rats. In diabetic BB rats AdvIL-10-transduced BB rat islets were rapidly destroyed. Prolongation of islet culture prior to transplantation improved the survival of gene-modified islets in BB rats. Several genes including those coding for chemokines and other peptides associated with inflammation were down-regulated in islets after prolonged culture, possibly contributing to improved islet graft function in vivo. Islets transduced ex vivo with vIL-10 are principally able to cure SZ-diabetic rats. Autoimmune islet destruction in diabetic BB rats is not prevented by ex vivo vIL-10 gene transfer to grafted islets. Graft survival in autoimmune diabetic rats may be enhanced by improvements in culture conditions prior to transplantation.

Gene expression profiling in animal models exposed to cigarette mainstream smoke (CS) shapes up as a promising tool for investigating the molecular mechanisms involved in the onset and development of CS-related disease and may aid in the identification of disease candidate genes. Here we report on differential gene expression in lungs of rats exposed for 2, 7, and 13 weeks to 300 and 600 microg total particulate matter/l CS with sacrifice 2, 6, or 20 h after the last exposure. Regarding antioxidant and xenobiotic-metabolizing (phase I/II) enzymes, a stereotypic, mostly transient, expression pattern of differentially expressed genes was observed after each exposure period. The expression patterns were generally dose dependent for antioxidant and phase II genes and not dose dependent for phase I genes at the CS concentrations tested. However, with increasing length of exposure, there was a distinct, mostly sustained and dose-sensitive, expression of genes implicated in innate and adaptive immune responses, clearly pointing to an emerging inflammatory response. Notably, this inflammatory response included the expression of lung disease-related genes not yet linked to CS exposure, such as galectin-3, arginase 1, and chitinase, as well as genes encoding proteolytic enzymes. Finally, our experiments also revealed a CS exposure-dependent shift in the cyclical expression of genes involved in controlling the circadian rhythm. Altogether, these results provide further insight into the molecular mechanisms of CS-dependent disease onset and development and thus may also be useful for defining CS-specific molecular biomarkers of disease.

The underlying molecular mechanisms of renal cell carcinoma (RCC) are poorly understood and more reliable markers for early diagnosis are needed. Hence, alternative strategies for biomarker discovery with appropriate validation technologies have to be performed. To elucidate genesis and progression of RCC we used high parallel chip based gene expression profiling comparing normal and tumour tissues. We compared corresponding control and tumour tissue samples from 10 patients with clear cell RCC. We isolated RNA from histologically well characterised tissue sections and performed reverse transcription, labelling and linear RNA amplification. Samples were hybridised on microarrays containing 642 human cDNAs. Of the 352 differentially expressed genes found, CD70 and FRA2 were selected for further evaluation by real-time RT-PCR. The analysis all showed a high potential to discriminate between normal and tumour tissue. Moreover, increased CD70 mRNA expression in tumour cells could be correlated to its expression at the protein level. Immunohistochemistry (IHC) showed very strong expression of CD70 in all tumour samples but no expression in adjacent normal kidney tissue. With our combined approach we were able to identify CD70 as a new marker for RCC, which may be useful in the future for improved immunohistochemical diagnosis.

Cigarette smoke (CS) is known to cause cancer and other diseases, but little is known about the global molecular and cellular changes that occur prior to the appearance of clinically detectable symptoms. Using DNA microarrays covering 2031 cDNA probes, we investigated differential gene expression in tissues of the rat respiratory tract, i.e. respiratory nasal epithelium (RNE) and lungs of rats exposed either acutely (3 h) or subchronically (3 h/day, 5 days/week, 3 weeks) to mainstream CS with death either immediately or at 20 h after exposure. Differential gene expression was most evident in RNE of rats exposed once and was characterized by strong up-regulation of genes encoding oxidative stress-responsive and Phase II drug-metabolizing enzymes, such as haem oxygenase-1 and NAD(P)H:quinone oxidoreductase, which are all, at least in part, transcriptionally regulated by NF-E2-related factor 2 (Nrf2). After 3 weeks of exposure, the strength of expression of this class of genes was markedly reduced, pointing to an adaptive response. The generally lower response in the lungs of exposed rats is indicative of a deposition gradient of active smoke constituents from the upper to the lower respiratory tract. In sharp contrast to the CS-induced expression of oxidative stress and Phase II-responsive genes, induction of the genes encoding the Phase I drug-metabolizing enzymes cytochrome P450 (CYP)1A1 and aldehyde dehydrogenase-3 was not reduced after 3 weeks of exposure and was similarly high in lungs and RNE. Gene expression patterns in rats allowed to recover for 20 h showed that the CS-induced transcriptional changes observed immediately after exposure returned almost completely to normal, even after 3 weeks of repeated CS exposure. In general, these results demonstrate that CS induces a specific differential gene expression pattern in vivo, which may be instrumental in identifying the molecular mechanisms leading to the onset of inflammatory and/or morphological changes.

There is an increasing demand for tissue samples that, after having been used for conventional histologic examination, are also suited for molecular analyses. As to formalin-fixed, paraffin embedded (FFPE) tissue, the latter applications are very limited. The HOPE (Hepes-Glutamic acid buffer mediated Organic solvent Protection Effect) technique comprises a new protection-solution with an organic buffer, with acetone as the only dehydrating agent, and pure paraffin of 52-54 degrees C melting temperature, allowing for all pathologic routine investigations. In contrast to FFPE tissue, the HOPE-technique allows for the application of molecular methods, such as high molecular DNA and RNA isolation, which can be used for PCR and reverse transcription PCR (RT-PCR). In this study, we investigated whether RNA from HOPE-fixed tissue samples is suitable for Northern blot and microarray analyses. RNAs of two HOPE-fixed breast cancer specimens of different histologic grade were used to carry out an array experiment. It turned out that RNA from HOPE-fixed tissue is of high quality and can be successfully used for array experiments. In addition, by detecting GAPDH and high mobility group protein gene B1 (HMGB1)-specific transcripts, we were able to demonstrate that RNA from HOPE-fixed tissue can also be used for Northern blot hybridization.

About 10% of hip endoprostheses will loosen after 10 years. Prosthesis loosening is caused by two different pathomechanisms: aseptic loosening (AL) and septic loosening (SL). This study evaluated differences in gene expression in AL and SL. Eight hybridizations were performed on PIQOR cDNA arrays. Objects of the study were periprosthetic interface tissue samples from two patients with SL and three patients with AL. Tissue parts directly adjacent to the site of RNA isolation were analyzed immuno/histopathologically in order to overcome the problem of tissue heterogeneity. Thirty-three genes were found constantly differentially expressed, among which were cd11b, cd18, cd68, osteopontin and ferritin heavy-chain upregulated in AL and collagen types 1alpha-1, 3alpha-1, integrin alpha-1, thrombospondin2 and nidogen upregulated in SL. The most striking finding was the strong upregulation (from 20-fold to 323-fold) of megakaryocyte stimulating factor (msf) in all aseptic cases and one of the two septic cases, which was confirmed by real-time reverse transcription-polymerase chain reaction. In this study, msf is linked to prosthesis loosening for the first time. The upregulation in AL suggests an important pathogenetic role: the msf splice product lubricin is responsible for the lubrication of healthy joints, but its excellent lubrication ability may disturb the tight interaction between bone and prosthesis and thereby contribute to prosthesis loosening.

The two discoidin domain receptors, DDR1 and DDR2, are tyrosine kinases that are activated by collagen and are essential regulators of cell-matrix communication. However, the target genes downstream of activated DDRs and their physiological significance are largely unknown. Here, we describe a novel method to dissect signaling pathways induced by extracellular matrix (ECM) receptors. Using the doxycycline-inducible repression system (tet-off), we generated human fibrosarcoma and mouse fibroblast cell lines over-expressing DDR1 or DDR2. These cell lines were employed for gene expression analysis using microarrays specific for human and mouse genes coding for ECM proteins or ECM-interacting factors. We found that approximately 10% of the genes studied were up- or down-regulated more than twofold in response to signals generated by over-expressing DDRs. A common event downstream of DDR1 and DDR2 in human and mouse cells was the up-regulation of P-selectin glycoprotein ligand. Key target genes repressed upon DDR activation were agrin, syndecan-1 and alpha3 integrin. ECM-specific microarrays were found a valuable tool to dissect gene expression changes induced by collagen-receptor signaling pathways.

Members of the transforming growth factor beta/bone morphogenetic protein (TGF-beta/BMP) family are involved in the control of hair follicle (HF) morphogenesis and cycling. The activities of several members of this family activins and BMP-2, -4, -7, and -11) are controlled by antagonists such as follistatin. Because follistatin-deficient mice show abnormalities in vibrissae development, we explored the role of follistatin and activin in pelage HF development and cycling. We show here that during HF development follistatin mRNA was prominently expressed by hair matrix and outer root sheath keratinocytes as well as by interfollicular epidermal cells, whereas activin betaA mRNA was mainly expressed in dermal papilla cells. Compared with age-matched wild-type controls, both follistatin knockout mice and activin betaA transgenic mice showed a significant retardation of HF morphogenesis. Treatment of wild-type embryonic skin explants with follistatin protein stimulated HF development. This effect was inhibited by addition of recombinant activin A protein. Activin betaA transgenic mice demonstrated retardation of catagen entry, down-regulation of BMP-2, and up-regulation of expression of its antagonist matrix GLA protein. These observations suggest that follistatin and activin interaction plays an important role in both HF development and cycling, possibly in part by regulating expression of BMP-2 and its antagonist.

About 5 to 12 % of hip endoprostheses will loosen after ten years. The periprosthetic membran between bone and prosthesis plays a crucial role in prosthesis loosening. Different pathomechanisms lead to the growth of such a membran, which can be discriminated by different histomorphologies: wear particle induced type, infectious type, combined type, indifferent type. 8 hybridizations were performed on PIQOR cDNA arrays. Objects of the study were periprosthetic interface tissue samples from 3 patients with particle induced and 2 patients with infectious prosthesis loosening. Tissue parts directly adjacent to the site of RNA-isolation were analyzed immuno-/ histopathologically in order to overcome the problem of tissue heterogeneity. 34 genes were found constantly differentially expressed, among which were cd9, cd11b, cd18, cd68, osteopontin, ferritin heavy-chain upregulated in the particle induced membrane and collagen types 1alpha-1, 3alpha-1, integrin alpha-1, thrombospondin 2 and nidogen upregulated in the infectious membrane. The most striking finding was the strong upregulation (from 20 fold to 323 fold) of megakaryocyte stimulating factor (msf) in all wear particle cases and 1 out of 2 infectious cases, which was confirmed by real-time RT-PCR. The upregulation of msf suggests an important pathogenetic role: The msf splice product lubricin is responsible for the lubrification of healthy joints, but its excellent lubrification ability may disturb the tight interaction between bone and prosthesis and thereby contribute to prosthesis loosening.

The antitumoral and immunostimulating properties of rViscumin (recombinant mistletoe lectin) were investigated in two mouse tumor models. After intravenous inoculation with RAW-117-P or L-1 sarcoma cells in Balb/c mice, rViscumin was given s.c. at non-toxic doses ranging from 0.3 to 150 ng rViscumin/kg. One set of experiments was performed to investigate the survival of rViscumin-treated animals. Another set was carried out to analyze the effect of rViscumin treatment on the number of tumor colonies in infiltrated lungs (RAW-117P) or liver (L-1) and the activation of immune cell subsets, respectively. An overall prolonged survival time after treatment with rViscumin and a reduction in the number of tumor colonies after administration of certain rViscumin doses was observed. Immunophenotyping of the peripheral leukocytes of treated mice revealed increased numbers of T-lymphocytes, pan-NK cells and activated monocytes. The results indicate that rViscumin has antineoplastic properties and might therefore be a promising candidate in cancer therapy.

Efficient T-cell activation requires two signals. The first signal, which confers specificity, is provided by interaction of the T-cell receptor with peptides presented by MHC molecules. One of the second costimulatory signals is induced by binding of B7 proteins on the surface of antigen-presenting cells to CD28 on the T-cell surface. Expression of B7 molecules on tumor cells can result in the activation of tumor specific T lymphocytes and induce protective antitumor immunity. However, at present such gene-therapeutic approaches are limited by the inability to selectively target B7 gene expression to cancer cells. As an alternative approach we exploited recombinant antibody fragments to localize a costimulatory B7 molecule to the surface of tumor cells. We constructed chimeric proteins that contain in a single polypeptide chain a portion of human B7-2 (CD86) genetically fused to single-chain (sc) Fv antibody domains specific for the tumor-associated antigens epidermal growth factor receptor and the closely related ErbB2 receptor tyrosine kinase. A small recombinant fragment of human CD86 was characterized that corresponds to amino acid residues 1-111 (CD86(111)) of the mature protein. CD86(111) produced in the yeast Pichia pastoris and CD86(111) expressed in bacteria was functionally active and displayed specific binding to B7 counter receptors. Bacterially expressed CD86(111)-scFv fusion proteins specifically localized to the respective target antigens on the surface of tumor cells and markedly enhanced the proliferation of primary T cells when bound to immobilized tumor antigen.

T cells require at least two signals for activation and clonal expansion. The first signal conferring specificity is initiated by interaction of the T cell receptor with peptide-bearing MHC molecules. The second, costimulatory signal can be provided by cell-surface molecules on antigen-presenting cells such as B7-1 (CD80) and B7-2 (CD86), which interact with CD28 on T cells. To direct the costimulatory B7-2 molecule to the surface of tumor cells we have constructed a chimeric fusion protein, which consists of the extracellular domain of human B7-2 fused to a single-chain antibody domain (scFv) specific for the ErbB2 protein, a type I growth factor receptor overexpressed in a high percentage of human adenocarcinomas. This B7-2(225)-scFv(FRP5) molecule, expressed in the yeast Pichia pastoris and purified from culture supernatants, binds to B7 counter-receptors and to ErbB2. B7-2(225)-scFv(FRP5) localizes specifically to the surface of ErbB2-expressing target cells, thereby providing a costimulatory signal, which results in enhanced proliferation of syngeneic T cells.

Resistance to chemotherapy is a major cause for failure in the treatment of lung cancer. Compared to conventional cytotoxic drugs, immunotoxins act by different mechanisms and thus might be promising for the treatment of chemoresistant cancer. The monoclonal antibody MOC31 recognises the epithelial glycoprotein-2 (EGP-2), a cell-surface antigen associated with small-cell lung cancer (SCLC) and a major fraction of lung adenocarcinomas. An immunotoxin composed of MOC31 and a recombinant from of Pseudomonas exotoxin A lacking the cell-binding domain (ETA252-613) was prepared and its effect on lung cancer cell lines examined. MOC31 ETA252-613 was selectively cytotoxic to EGP-2-positive SCLC and adenocarcinoma cell lines inhibiting proliferation by 50% at concentrations ranging from 0.01 nM to 0.3 nM. Moreover, the immunotoxin reduced the number of clonogenic tumour cells from cultures by factors of 10(4) and 10(5) during a 24-h and a 3-week exposure respectively. In athymic mice, the immunotoxin, which revealed a serum half-life of approximately 4 h, caused substantial regression of small (40 mm3) chemoresistant tumour xenografts and significantly delayed the growth of larger tumours (120 mm3). This finding indicates that MOC31-ETA252-613 may be useful for the treatment of lung cancer in the setting of chemoresistant minimal residual disease.

We are developing strategies to use naive T lymphocytes in cancer therapy. For this purpose, we are deriving T cells with specificity of recognition for defined tumor cells. To direct effector lymphocytes toward tumor cells, we have manipulated the recognition specificity of naive rat and mouse T lymphocytes and a mouse T-cell line. The cells were stably transduced with a chimeric T-cell receptor (TCR) component. The zeta chain of the TCR consists of a single transmembrane protein with a short extracellular domain and an intracellular domain for TCR signaling. We provided an extracellular tumor cell recognition domain to the zeta chain. Human heregulin beta1 (ligand to the erbB-3 and erbB-4 receptors) and three different single-chain antibodies specific for the human and rat Neu/erbB-2 receptors were used. One single-chain antibody (C11) is directed against the rat Neu protein, and one single-chain antibody (FRP5) is directed against the human erbB-2 receptor. The single-chain antibody (R-AK) directed against the Mr 14,000 fusion protein of orthopox viruses served as a control. An efficient procedure was devised to introduce the chimeric genes into primary rat and mouse T lymphocytes. Retrovirus-producing packaging cell lines were cocultured with the T cells activated by phytohemagglutinin and interleukin 2. T-cell lines were transduced by exposure to retrovirus-containing supernatants from helper cell lines. Expression of the fusion genes was determined by fluorescence-activated cell sorting analysis. More than 80% of the naive rat and mouse T cells and 85-100% of the cells from the established T-cell lines expressed the fusion genes within 48 h after infection. The expression of the fusion genes was maintained for at least 10 days after infection. Target cells expressing Neu/erbB-2, erbB-3, or erbB-4 were lysed in vitro with high specificity by T cells expressing the corresponding recognition proteins. No selection of a marker gene is necessary to confer a predetermined recognition specificity. The described experiments are important for a gene therapy approach to cancer treatment with autologous T cells.