Genetic homogeneity of the Camurati-Engelmann disease.
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Camurati-Engelmann Syndrome↗
Biomedical subjects
C Huber
Publications and source records attributed to C Huber.
Coordinate downregulation of multiple MHC class I antigen processing genes in chemical-induced murine tumor cell lines of distinct origin.
In murine tumor cell lines, downregulation of MHC class I surface expression has been frequently detected, but the underlying molecular mechanisms of such deficiencies have not been defined. In this study, murine tumor cell lines of different histology derived from spontaneous or from chemical-induced tumors were analyzed for the expression of multiple components of the major histocompatibility complex (MHC) class I antigen-processing machinery (APM), including the peptide transporter TAP, the interferon (IFN)-gamma inducible proteasome subunits and several chaperones. The tumor cell lines analyzed demonstrated a heterogeneous expression pattern of various APM components. In comparison to control cells an impaired coordinated expression of at least three APM components was detected. In particular, extensive APM deficiencies were found in cell lines derived from chemical-induced tumors. A strong coordinated downregulation of expression and/or function of TAP, the low molecular weight proteins (LMP) subunits, the proteasome activator PA28 and/or tapasin was found in 5 of 10 tumor cells, which was associated with impaired MHC class I surface expression. In contrast, the expression of beta2-microglobulin (beta2-m), PA28beta, the constitutive proteasome subunits X, Y, Z and of the chaperones calnexin, calreticulin, ER60 and phospho disulfide isomerase (PDI) was unaltered or only weakly decreased. The deficient expression of APM components could be corrected by IFN-gamma treatment, which also reconstituted MHC class I surface expression. However, impaired expression of APM molecules appears not to be the only cause of abnormal MHC class I expression, since it could neither be corrected by the addition of exogeneous MHC class I binding peptides nor by incubation at low temperature. These results suggest that one major mechanism of murine tumor cells, in particular chemical-induced tumors, to evade the immune system is the combined dysregulation of various APM components and other factors, which still have to be identified.
Animals↗
Rapid identification of minor histocompatibility antigen HA-1 subtypes H and R using fluorescence-labeled oligonucleotides.
Donor-recipient disparitiy of the minor histocompatibility antigen HA-1 is relevant for the development of graft-versus-host disease after HLA-matched sibling allogeneic bone marrow transplantation in HLA-A*0201-positive individuals. Two different alleles of HA-1 with a single amino acid polymorphism have been identified. Here we describe a time- and cost-efficient method for HA-1 typing of genomic DNA, using site-specific hybridization probes with the LightCycler. This method was compared with standard techniques as sequencing or allele-specific polymerase chain reaction (PCR) and proved to be specific, reliable and reproducible. We conclude that HA-1-subtyping using fluorescent-labeled oligonucleotides represents a attractive method for the screening of samples before allogeneic transplantation in HLA-A*0201-positive individuals.
Fluorescent Dyes↗
Functional deficiencies of components of the MHC class I antigen pathway in human tumors of epithelial origin.
An association between oncogenic transformation and repression of different components of the MHC class I antigen processing machinery (APM) have been described in murine model systems. In order to discover whether a similar correlation exists, human tumor cell lines of distinct histology with altered ras protein were analyzed for the expression of APM components utilizing RT-PCR and Western blot analyses. A heterogeneous expression pattern of MHC class I antigens, TAP peptide transporter, proteasome subunits, proteasome activator PA28 and the chaperones calnexin, calreticulin as well as tapasin was displayed by these tumor cell lines. Single or combined deficiencies in the expression and/or function of TAP, LMP2, LMP10 and tapasin were demonstrated in 11 of 12 cell lines studied, whereas the expression of calnexin, calreticulin, beta2-microglobulin, LMP7 and PA28alpha was unaltered or only weakly decreased. The impaired expression of TAP, LMP subunits and tapasin was not associated with altered ras, but resulted in reduced MHC class I surface expression. In particular, a significant allele- and locus-specific downregulation of the HLA-A and HLA-B haplotypes was found. IFN-gamma treatment corrected the TAP, LMP and tapasin deficiencies and enhanced the constitutive PA28alpha, LMP7, calnexin and calreticulin expression which was accompanied with increased levels of MHC class I antigens. Thus, dysregulation rather than structural alterations of different APM components might be one mechanism of colon carcinoma, small cell lung carcinoma and pancreatic carcinoma cell lines to evade immune recognition.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Hepatic granuloma due to Propionibacterium acnes in a patient with acute myelogenous leukemia.
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Adult↗
CD34+ cell enrichment for autologous peripheral blood stem cell transplantation by use of the CliniMACs device.
Several devices for selection of CD34+ peripheral blood stem cells (PBSC) have been used during the last years for reducing tumor cell contamination of the graft. The new CliniMACS system (magnetic-activated cell separation system by Miltenyi Biotech GmbH, Bergisch-Gladbach, Germany) was recently approved for clinical use in Europe. To evaluate its purging efficiency and engraftment data in the autologous transplant, PBSC from 28 adult patients with various malignant diseases (non-Hodgkin's lymphoma, n = 17; chronic lymphocytic leukemia, n = 5; multiple myeloma, n = 4; acute lymphocytic leukemia, n = 1; medulloblastoma, n = 1) were mobilized by chemotherapy and granulocyte colony-stimulating factor (G-CSF) (10 microg/kg per day). Thirty leukapheresis products from 28 patients with a median of 4.4 x 10(8) nucleated cells/kg body weight (bw)(range 0.6-10.8 x 10(8)/kg bw) and a median of 7.1 x 10(6) CD34+ cells/kg bw (range 2.8 to 18.8 x 10(6)/kg bw) were selected using the Cobe spectra cell separator (Cobe BCT Inc., Lakewood, CO). After the CliniMACS procedure, the median yield of CD34+ selected cells was 4.5 x 10(6)/kg (range 2.2-11.1 X 10(6)/kg bw) with a median recovery of 69.5% (range 46.9-87.3%) and a median purity of 97.7% (range 89.4-99.8%). The procedure did not alter viability of selected cells, which was tested by propidium iodide staining. So far, purified PBSC were used for autologous transplantation in 15 out of 28 patients after total body irradiation and/or high-dose chemotherapy. Median time to reach an absolute neutrophil count > 500/microl was 12 days (range 10-18 days), platelet recovery >50,000/microl occurred at day + 16 (range 11-22). With a median follow-up time of 12 months (range 3-19), 5 patients died of relapse. We confirmed the feasibility and safety of the CliniMACS CD34+ cell enrichment procedure in adult patients with autologous PBSC transplantation.
Adult↗
Multicenter phase II study of oral idarubicin in treated and untreated patients with B-chronic lymphocytic leukemia.
Idarubicin is the first anthracycline that can be administered orally facilitating antineoplastic chemotherapy at an improved quality of life. In different studies idarubicin has proved clinical effectiveness in patients with advanced low grade non Hodgkin's lymphoma. We performed a phase II study in 19 patients with untreated and pretreated B-CLL of Binet stage A-C. Idarubucin was administered orally at a dose of 15 mg/m2 over 3 days every 4 weeks. Of 19 evaluable patients (m:f, 16:3, median age 64 years, range 41-80 years) 7 were previously untreated while 12 patients had received prior therapy with fludarabine, chlorambucil or similar non-anthracycline containing regimens. 12 pts had Binet stage C, 5 Binet stage B and 2 Binet stage A. Five patients achieved a partial remission (26%), 5 patients had stable disease (26%) and 9 patients showed progressive disease (47%), resulting in an overall response of 26% (5/19). There was no correlation of response rate with Binet stages or previous treatment regimens. Treatment associated side effects consisted predominantly of mild nausea and vomiting (26%) as well as minor infections (21%) and diarrhoea (16%). These data demonstrate that oral idarubicin as a single agent is well tolerated but of limited effectiveness in B-CLL. Further studies are needed to assess different doses and schedules of oral idarubucin and to test it in combination with other chemotherapeutic agents.
Administration, Oral↗
Short Report: Engraftment of T-cell-depleted allogeneic haematopoietic stem cells using a reduced intensity conditioning regimen.
Graft-versus-host disease (GVHD) remains a significant complication in patients undergoing allogeneic stem cell transplantation (SCT) using a reduced intensity conditioning regimen. Although T-cell depletion (TCD) reduces the risk of GVHD after a myeloablative conditioning regimen, it is associated with an increased risk of graft failure. We have therefore examined whether TCD compromises engraftment using a fludarabine-based conditioning regimen. Fifteen patients have been transplanted using such a regimen of whom 13 underwent ex vivo TCD. All but one patient demonstrated durable engraftment and no patient receiving a TCD product developed severe GVHD. Thus, TCD may play a role in GvHD prophylaxis using such regimens.
Adult↗
Liposomal amphotericin B for fever and neutropenia.
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Amphotericin B↗
Modified vaccinia virus Ankara for delivery of human tyrosinase as melanoma-associated antigen: induction of tyrosinase- and melanoma-specific human leukocyte antigen A*0201-restricted cytotoxic T cells in vitro and in vivo.
Vaccination with tumor-associated antigens is a promising approach for cancer immunotherapy. Because the majority of these antigens are normal self antigens, they may require suitable delivery systems to promote their immunogenicity. A recombinant vector based on the modified vaccinia virus Ankara (MVA) was used for expression of human tyrosinase, a melanoma-specific differentiation antigen, and evaluated for its efficacy as an antitumor vaccine. Stable recombinant viruses (MVA-hTyr) were constructed that have deleted the selection marker lacZ and efficiently expressed human tyrosinase in primary human cells and cell lines. Tyrosinase-specific human CTLs were activated in vitro by MVA-hTyr-infected, HLA-A*0201-positive human dendritic cells. Importantly, an efficient tyrosinase- and melanoma-specific CTL response was induced in vitro using MVA-hTyr-infected autologous dendritic cells as activators for peripheral blood mononuclear cells derived from HLA-A*0201-positive melanoma patients despite prior vaccination against smallpox. Immunization of HLA-A*0201/Kb transgenic mice with MVA-hTyr induced A*0201-restricted CTLs specific for the human tyrosinase-derived peptide epitope 369-377. These in vivo primed CTLs were of sufficiently high avidity to recognize and lyse human melanoma cells, which present the endogenously processed tyrosinase peptide in the context of A*0201. Tyrosinase-specific CTL responses were significantly augmented by repeated vaccination with MVA-hTyr. These findings demonstrate that HLA-restricted CTLs specific for human tumor-associated antigens can be efficiently generated by immunization with recombinant MVA vaccines. The results are an essential basis for MVA-based vaccination trials in cancer patients.
Animals↗
Induction of immunogenicity of a human renal-cell carcinoma cell line by TAP1-gene transfer.
Reduced expression of the major-histocompatibility-complex(MHC)-class-I antigens has been demonstrated in renal-cell carcinoma (RCC), and appeared to be associated with deficiencies in the expression and function of different components of the MHC-class-I-antigen-processing pathway and poor recognition by cytotoxic T-lymphocytes (CTL). In order to investigate the role of peptide transporters for the immunogenic phenotype of RCC, tumor cells were stably transfected with the human TAP1A gene. While the TAP1 transfectants showed heterogeneous TAP1-transgene expression pattern of mRNA and protein, high TAP1 expression and a TAP-controlled increase in MHC-class-I surface expression could be achieved in selected transfectants. IFN-gamma up-regulates the expression of MHC-class-I antigens and TAP1 both in control and in TAP1-transfected RCC cells to a similar level. No additive effect of TAP1 over-expression was observed in TAP1 transfectants. Although no enhanced CTL-mediated lysis was obtained, cytokine release was substantially increased in response to TAP1-transfected RCC cells, but not to control cells. Furthermore, TAP1 transfectants were able to stimulate the proliferation of allogeneic T cells. These studies suggest that abnormalities of MHC-class-I surface expression due to dysfunctional peptide transporters contribute to the immune escape phenotype of RCC cells and that the immune tolerance of RCC could be altered by TAP1-gene transfer.
ATP Binding Cassette Transporter, Subfamily B, Mem↗
Regulatory elements of the leukaemia inhibitory factor (LIF) promoter in murine bone marrow stromal cells.
Leukaemia inhibitory factor (LIF) plays an important role as a haematopoietically active cytokine. As described earlier in a murine model, interleukin 1 (IL-1) induced LIF mRNA and protein expression. We utilized the murine cell line +/+-1.LDA11 to further define regulatory mechanisms of LIF expression in bone marrow stromal cells. The production of LIF mRNA is stimulated by IL-1beta, TNF-alpha, and the cAMP analogue 8-bromoadenosine 3':5'-monophosphate (8BrcAMP). LIF mRNA expression is controlled at the transcriptional level. Different fragments from -542 to -45 bp 5' upstream of the transcriptional start site of the murine LIF gene were fused to the luciferase gene. All LIF-promoter luciferase constructs exhibited constitutive luciferase activity under serum free conditions. The level of luciferase activity decreased with LIF-promoter constructs of less than 249 bp (pLIF249) in size. When tested with the 314 bp LIF-promoter construct, incubation of stromal cells with IL-1beta (500 U/ml) resulted in a 1.57-fold stimulation, with TNF-alpha (500 U/ml) in 2.06-fold stimulation, and with 8BrcAMP (0.5 mM) in a 3. 42-fold stimulation of luciferase activity. By testing different deletion mutants we could narrow the IL-1 and TNF-alpha responsive promoter areas to the region -249 to -145 bp and the 8BrcAMP responsive area from -145 to -82 bp. Mobility shift experiments revealed that nuclear proteins from stromal cells form a DNA-protein complex by binding to the region from -249 to -145 bp of the LIF promoter.
8-Bromo Cyclic Adenosine Monophosphate↗
Development and use of a PCR assay for detection of the reproductive virus in wild populations of helicoverpa zea (Lepidoptera: noctuidae)
Helicoverpa zea reproductive virus (HzRV) is a nonoccluded bacilliform virus that affects both female and male moths of the corn earworm H. zea. In order to study the biology and host range of HzRV, a bioassay was previously developed to detect the presence of this virus in infected insects. A drawback of this bioassay is that it is time consuming and requires more than a month to complete. Here we describe the development of a polymerase chain reaction (PCR) assay for the rapid detection of HzRV in infected corn earworms. The genome of HzRV was digested with PstI and cloned into a plasmid vector. Sequences from two different clones, P4 and P13, were selected for designing two sets of primers. These primers were used for PCR and their sensitivity and specificity in detecting HzRV DNA were examined. Both sets of primers produced the expected amplification product in samples containing HzRV DNA but not in uninfected corn earworm samples, Spodoptera frugiperda (Sf-9) cells, or samples from the Autographa californica nuclear polyhedrosis virus. In addition, the primer pair of the clone P13 was sensitive enough to detect approximately 175 copies of viral DNA. We then used this assay to examine feral populations of H. zea from seven geographical locations in the United States. HzRV was detected primarily in the Mississippi populations and to a lesser extent in Iowa and Georgia, but none in Maryland, Missouri, and Texas populations. This PCR assay provides a highly specific, sensitive, and rapid way of detecting the presence of HzRV and will be useful in further studying the host range, tissue specificity, and incidence of this virus in wild populations of the corn earworm.
Journal Article↗
The DNA-binding subunit p140 of replication factor C is upregulated in cycling cells and associates with G1 phase cell cycle regulatory proteins.
The DNA-binding subunit of replication factor C (RFCp140) plays an important role in both DNA replication and DNA repair. The mechanisms regulating activation of RFCp140 thereby controlling replication and cellular proliferation are largely unknown. We analyzed protein expression of RFCp140 during cell cycle progression and investigated the association of RFCp140 with cell cycle regulatory proteins in cell lines of various tissue origin and in primary hematopoietic cells. Western and Northern blot analyses of RFCp140 from synchronized cells showed downregulation of RFCp140 when cells enter a G0-like quiescent state and upregulation of RFCp140 in cycling cells. Translocation from the cytoplasmic compartment to the nucleus did not account for the significant increase in RFCp140 protein levels observed in cycling cells. To investigate a potential association of RFCp140 with cell cycle regulatory proteins coimmunoprecipitation assays were performed. These studies demonstrated specific binding of RFCp140 to cdk4-kinase in hematopoietic and fibroblast cell lines. Additional coimmunoprecipitation studies revealed specific association of RFCp140 with cyclin D1, p21, proliferating cell nuclear antigen, and retinoblastoma protein. These findings link DNA replication and repair factor RFCp140 to G1 phase cell cycle regulatory elements critically involved in cell cycle control.
3T3 Cells↗
Inverse regulation of vascular endothelial growth factor and VHL tumor suppressor gene in sporadic renal cell carcinomas is correlated with vascular growth: an in vivo study on 29 tumors.
Tumors associated with the VHL (von Hippel-Lindau) disease, such as hemangioblastomas and renal carcinomas and their sporadic counterparts, are cystic and well vascularized. Mutations of the VHL tumor-suppressor gene and elevated levels of vascular endothelial growth factor (VEGF) have been described in these tumors. The upregulation of VEGF has been shown in vitro as a consequence of alteration of the VHL gene. No comprehensive in vivo analysis has yet been carried out of the factors affecting tumor growth, vascularization, VEGF, and VHL expression. We performed immunohistochemistry and mRNA studies on primary sporadic renal carcinomas and matching normal renal tissue. We semiquantitatively analyzed 29 renal carcinomas (22 clear cell, 5 chromophilic, 2 chromophobic tumors) for VHL mRNA, and VEGF expression for morphology and tumor size. Immunohistochemistry was carried out for VEGF protein expression, vascularization, and macrophage infiltration. Vascularization of the chromophilic renal carcinomas was lower than that of the clear cell type of renal carcinoma. Low VEGF protein expression was seen in four of the five chromophilic renal carcinomas. We found two groups of clear cell renal cell carcinoma: one with reduced VHL mRNA and increased VEGF mRNA, and the other without significantly altered VHL or VEGF mRNAs. Tumor vascularization was correlated with VEGF protein and seemed to be independent of macrophage infiltration. Our in vivo findings support the inverse relationship between the regulation of VHL and that of VEGF. Our data also indicate that there may be an VHL-independent pathway for the induction of tumor vascularization.
Adult↗
CT-guided intratumoral gene therapy in non-small-cell lung cancer.
The objective of this study was to prove the principle of CT-guided gene therapy by intratumoral injection of a tumor suppressor gene as an alternative treatment approach of incurable non-small-cell lung cancer. In a prospective clinical phase I trial six patients with non-small-cell lung cancer and a mutation of the tumor suppressor gene p53 were treated by CT-guided intratumoral gene therapy. Ten milliliters of a vector solution (replication-defective adenovirus with complete wild-type p53 cDNA) were injected under CT guidance. In four cases the vector solution was completely applied to the tumor center, whereas in two cases 2 ml aliquots were injected into different tumor areas. For the procedure the scan room had been approved as a biosafety cabinet. Gene transfer was assessed by reverse transcription and polymerase chain reaction in biopsy specimens obtained under CT guidance 24-48 h after therapy. Potential therapeutic efficacy was evaluated on day 28 after treatment using spiral CT. The CT-guided gene therapy was easily performed in all six patients without intervention-related complications. Besides flu-like symptoms, no significant adverse effects of gene therapy were noted. Three of the four patients with central injection exhibited gene transfer in the posttreatment biopsy. Gene transfer could not be proven in the two patients with multiple 2 ml injections. After 28 days, four of the six patients showed stable disease at the treated tumor site, whereas other tumor manifestations progressed. Computed tomography-guided injections are an adequate and easy-to-perform procedure for intratumoral gene therapy.
Adenoviridae↗
Quanititation of gene expression by means of HPLC analysis of RT-PCR products.
A method for the quantitative analysis of specific mRNA species by reverse transcription-polymerase chain reaction (RT-PCR) and subsequent detection of products by means of ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC) on alkylated micropellicular polystyrene-divinylbenzene particles has been developed. For RT-PCR, we used the EZrTth RNA PCR kit (Perkin Elmer). This method allows reverse transcription and amplification of specific target mRNA in a single reaction tube. RT-PCR products were analyzed qualitatively and quantitatively by means of IP-RP-HPLC and UV detection at 254 nm. The enzymatic amplification combined with chromatographic separation and UV detection permitted high precision (and intra assay CV < 10%), with good practicability (two pipetting steps only). A total RNA preparation of a tissue that highly expressed the sequence of interest and that was stored in multiple aliquots, was diluted to give a standard curve. This external standard curve was used to define when samples have to be diluted, i.e., when the signal is in the plateau phase of amplification. The validity of the method is demonstrated with the example of human retinoic acid receptor mRNA.
Chromatography, High Pressure Liquid↗