Mechanisms and rescue strategies of calcineurin inhibitor mediated tolerance abrogation induced by anti-CD4 mAb treatment.

To ensure safety tolerance induction protocols are accompanied by conventional immunosuppressive drugs (IS). But IS such as calcineurin inhibitors (CNI), for example, cyclosporin A (CsA), can interfere with tolerance induction. We investigated the effect of an additional transient CsA treatment on anti-CD4mAb-induced tolerance induction upon rat kidney transplantation. Additional CsA treatment induced deteriorated graft function, resulting in chronic rejection characterized by glomerulosclerosis, interstitial fibrosis, tubular atrophy and vascular changes. Microarray analysis revealed enhanced intragraft expression of the B cell attracting chemokine CXCL13 early during CsA treatment. Increase in CXCL13 expression is accompanied by enhanced B cell infiltration with local and systemic IgG production and C3d deposition as early as 5 days upon CsA withdrawal. Adding different CNIs to cultures of primary mesangial cells isolated from glomeruli resulted in a concentration-dependent increase in CXCL13 transcription. CsA in synergy with TNF-α can enhance the B cell attracting and activating potential of mesangial cells. Transient B cell depletion or transfer of splenocytes from tolerant recipients 3 weeks after transplantation could rescue tolerance induction and did inhibit intragraft B cell accumulation, alloantibody production and ameliorate chronic rejection.

High weight differences between donor and recipient affect early kidney graft function--a role for enhanced IL-6 signaling.

The frequency of delayed function of kidney transplants varies greatly and is associated with quality of graft, donor age and the duration of cold ischemia time. Furthermore, body weight differences between donor and recipient can affect primary graft function, but the underlying mechanism is poorly understood. We transplanted kidney grafts from commensurate body weight (L-WD) or reduced body weight (H-WD) donor rats into syngeneic or allogeneic recipients. Twenty-four hours posttransplantation, serum creatinine levels in H-WD recipients were significantly higher compared to L-WD recipients indicating impaired primary graft function. This was accompanied by upregulation of IL-6 transcription and increased tubular destruction in grafts from H-WD recipients. Using DNA microarray analysis, we detected decreased expression of genes associated with kidney function and an upregulation of other genes such as Cyp3a13, FosL and Trib3. A single application of IL-6 into L-WD recipients is sufficient to impair primary graft function and cause tubular damage, whereas immediate neutralization of IL-6 receptor signaling in H-WD recipients rescued primary graft function with well-preserved kidney graft architecture and a normalized gene expression profile. These findings have strong clinical implication as anti-IL6R treatment of patients receiving grafts from lower-weight donors could be used to improve primary graft function.

Effect of zinc deficiency on the mRNA expression pattern in liver and jejunum of adult rats: monitoring gene expression using cDNA microarrays combined with real-time RT-PCR.

In the study presented here, the effect of zinc deficiency on mRNA expression levels in liver and jejunum of adult rats was analyzed. Feed intake was restricted to 8 g/day. The semi-synthetic diet was fortified with pure phytate and contained either 2 microg Zn/g (Zn deficiency, n = 6) or 58 microg Zn/g (control, n = 7). After 29 days of Zn depletion feeding, entire jejunum and liver were retrieved and total RNA was extracted. Tissue specific expression pattern were screened and quantified by microarray analysis and verified individually via real-time RT-PCR. A relative quantification was performed with the newly developed Relative Expression Software Tool Copyright on numerous candidate genes which showed a differential expression. This study provides the first comparative view of gene expression regulation and fully quantitative expression analysis of 35 candidate genes in a non-growing Zn deficient adult rat model. The expression results indicate the existence of individual expression pattern in liver and jejunum and their tissue specific regulation under Zn deficiency. In addition, in jejunum a number of B-cell related genes could be demonstrated to be suppressed at Zn deficiency. In liver, metallothionein subtype 1 and 2 (MT-1 and MT-2) genes could be shown to be dramatically repressed and therefore represent putative markers for Zn deficiency. Expression results imply that some genes are expressed constitutively, whereas others are highly regulated in tissues responsible for Zn homeostasis.

Identification of RELMgamma, a novel resistin-like molecule with a distinct expression pattern.

We have identified RELMgamma, a novel member of the resistin-like molecule/found in inflammatory zone (RELM/FIZZ) family in mice and rats. Microarray and real-time RT-PCR experiments revealed a repression of RELMgamma mRNA in nasal respiratory epithelium of cigarette smoke-exposed versus untreated rats. The analysis of the physiological tissue-specific expression revealed highest expression in hematopoietic tissues, suggesting a cytokine-like role for RELMgamma. RELMgamma is most closely related to RELMalpha/FIZZ1. Despite the high similarity, the expression properties of the two genes are clearly distinct. While RELMgamma (approved symbol retnlg) is expressed in rat white adipose tissue, minute to no expression of RELMalpha was detected in that system. Thus, previous reports analyzing RELMalpha expression in rat adipose tissue might have been influenced by cross-hybridization with RELMgamma. Finally we could demonstrate that white adipose tissue of mice shows strong RELMalpha expression but only low levels of RELMgamma, indicating a species-specific gene regulation.

[Differential gene expression in the wear particle induced and infectious periprosthetic membrane of loosened knee-endoprostheses]. / Differentielle Genexpression in der abriebinduzierten und infektiösen periprothetischen Membran gelockerter Knieendoprothesen.

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.

Anticancer activity of rViscumin (recombinant mistletoe lectin) in tumor colonization models with immunocompetent mice.

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.

Construction and characterization of bispecific costimulatory molecules containing a minimized CD86 (B7-2) domain and single-chain antibody fragments for tumor targeting.

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.

Stable expression of the ecotropic retrovirus receptor in amphotropic packaging cells facilitates the transfer of recombinant vectors and enhances the yield of retroviral particles.

Retroviral vectors are used widely as gene transfer vehicles. Vector particles are generated by packaging cell lines, which supply the structural proteins gag, pol and env needed to package the retroviral vector RNA. The most efficient way to introduce the vector genome into the packaging cell line is cross-infection with a retroviral vector. Since the infection of a packaging cell line by the produced virus is blocked due to the down regulation of the retrovirus receptor by the envelope glycoprotein, the vector genome should be introduced by a virus with a host tropism different from the one of the packaging cell line. The murine ecotropic retrovirus receptor was expressed in the human amphotropic packaging cell line FLYA13 to generate a cell line which can be infected by murine ecotropic retroviruses. Vector transfer can now be facilitated by cross-infection with the appropriate ecotropic retroviral vectors and provides a simple and efficient method for the generation of amphotropic packaging lines.

Costimulation of T cell proliferation by a chimeric B7-2 antibody fusion protein specifically targeted to cells expressing the erbB2 proto-oncogene.

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 antigenic peptides in the context of MHC molecules. The second, costimulatory signal can be provided by cell surface molecules on APCs such as B7-1 (CD80) and B7-2 (CD86), which interact with their counter-receptors on T cells. The absence of costimulatory signals presents one possible mechanism for tumor cells to escape immune surveillance. In experimental models transfection of B7 genes into tumor cells can result in T cell-dependent tumor rejection. We have developed a novel approach to direct the costimulatory B7-2 molecule to the surface of target cells. Our approach is based on a chimeric fusion protein that consists of the extracellular domain of human B7-2 fused to a single-chain Ab 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 is functionally active and 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 that results in enhanced proliferation of syngeneic T cells. Our results suggest that effective tumor vaccines for cancer immunotherapy could be created by targeting such chimeric ligands to the surface of tumor cells.

Construction and expression in the yeast Pichia pastoris of functionally active soluble forms of the human costimulatory molecules B7-1 and B7-2 and the B7 counter-receptor CTLA-4.

We have generated soluble recombinant forms of the costimulatory molecules B7-1 and B7-2, and their counter-receptor CTLA-4 using a yeast Pichia pastoris expression system. Fragments comprising the extracellular domains of human B7-1, B7-2, and CTLA-4 molecules were expressed at high levels and could be purified from culture supernatants following a simple one-step purification protocol. The recombinant proteins retained their functionality and specific binding to their natural counterparts could be demonstrated by FACS analysis. In T cell proliferation assays costimulatory activity of immobilized B7-1 and B7-2 proteins in the presence of an anti-CD3 antibody was observed with the B7-1 protein being more potent than B7-2.

A novel immunotoxin recognising the epithelial glycoprotein-2 has potent antitumoural activity on chemotherapy-resistant lung cancer.

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.

Costimulation of T-cell proliferation by a chimeric B7-antibody fusion protein.

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.

Cytolysis of tumor cells expressing the Neu/erbB-2, erbB-3, and erbB-4 receptors by genetically targeted naive T lymphocytes.

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.