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Clinical trials of Treg therapy in transplantation are currently entering phases IIa and IIb, with the majority of these employing polyclonal Treg populations that harbor a broad specificity. Enhancing Treg specificity is possible with the use of chimeric antigen receptors (CARs), which can be customized to respond to a specific human leukocyte antigen (HLA). In this study, we build on our previous work in the development of HLA‐A2 CAR‐Tregs by further equipping cells with the constitutive expression of interleukin 10 (IL‐10) and an imaging reporter as additional payloads. Cells were engineered to express combinations of these domains and assessed for phenotype and function. Cells expressing the full construct maintained a stable phenotype after transduction, were specifically activated by HLA‐A2, and suppressed alloresponses potently. The addition of IL‐10 provided an additional advantage to suppressive capacity. This study therefore provides an important proof‐of‐principle for this cell engineering approach for next‐generation Treg therapy in transplantation., Chimeric antigen receptor (CAR) constructs can be modified to incorporate elements for enhanced activity. Here, human Tregs were modified to express an anti‐HLA‐A2 CAR as well as IL‐10 and an imaging reporter. These cells maintained a stable Treg phenotype after lentiviral transduction, were specifically activated by cells expressing HLA‐A2, and had enhanced in vitro suppressive potency compared with CAR Tregs without constitutive IL‐10 expression.

Because apoptotic evasion is a central feature of prostate cancer, there is an urgent need for increased understanding of the key regulatory molecules that control the life/death decision of prostate cells. Functional expression cloning permits the isolation of genes that control the rate-limiting steps of cell death and offers a possible solution to this problem. This technique requires the availability of prostate cells that meet several stringent requirements. Therefore, the main objective was to obtain prostate cell clones that undergo cell death with minimal survival of spontaneously resistant cells and that can be infected at a high efficiency with viral vectors. Initial characterization of 5 prostate cell lines with a range of apoptotic inducers revealed cell line—dependent and treatment-dependent effects. In general, the colony-forming ability of nontumorigenic PNT2C2 cells showed the highest sensitivity to most chemical agents and ultraviolet (UV) irradiation, whereas the metastases-derived cell lines, LNCaP and PC-3, showed resistance to UV and etoposide, respectively. Clones of PNT2C2, 22Rv1, and PC-3 were produced, which displayed heterogeneous responses to UV irradiation. Further characterization of UV-sensitive clones revealed at least 1 clone per cell line with high sensitivity (mean clonogenic survival ≤0.02% control cells) to 3 or more apoptotic inducers. These clones could be infected at a high efficiency (>90%) with a lentiviral vector. In conclusion, we have isolated clones of nontumorigenic prostate cells (PNT2C2), androgen-sensitive prostate cancer cells (22Rv1), and androgen-independent, metastatic prostate cancer cells (PC-3), which are suitable as host cells for functional cloning studies to address cell death control mechanisms in the prostate during cancer progression.

Hepatocyte growth factor (HGF) mediates cancer cell invasion and metastasis. This study characterised the down-regulation of HGF expression by pyrrolidine dithiocarbamate (PDTC), which markedly reduced HGF mRNA expression and protein production in MRC-5 cells. Reporter gene studies revealed that PDTC inhibited HGF gene transcription and that the response element is located in the region −75 to +42bp flanking the transcription initiation site. Electrophoretic mobility shift assay identified three specific protein complexes binding in this region, which were abrogated by exposure of cells to PDTC. PDTC deserves further investigation as a novel therapeutic agent for HGF-driven cancers.

Background The current treatments for hepatocellular carcinoma (HCC) are poor, particularly for metastatic HCC. Intraportal transfusion of adeno-associated virus (AAV) leads to long-term and persistent transgenic expression in livers. Kallistatin, a novel angiogenesis inhibitor, exhibits anti-tumor activity. The aim of the study was to investigate whether intraportal injection of AAV-kallistatin could suppress local and metastatic HCC in mice. Methods An AAV vector encoding kallistatin was constructed, and its transduction efficiency by intraportal transfusion in livers was examined by RT-PCR, immunohistochemical and Western blotting analysis. The anti-tumor activity was tested in three HCC models including hepatic and subcutaneous human Hep3B HCC tumors in BALB/c athymic (nu/nu) mice, and subcutaneous mouse BNL HCC tumors in BALB/c mice. Tumor cell proliferation in situ was examined by anti-Ki-67 staining, and apoptosis by TUNEL. Results Gene transfection by rAAV-kallistatin inhibited proliferation of human umbilical vein endothelial cells and HCC cells in vitro. Intraportal injection of rAAV-kallistatin resulted in persistent and specific expression of kallistatin in livers detected by RT-PCR and immunohistochemical analysis, and kallistatin protein in circulation detected by Western blotting analysis. Intraportal injection of rAAV-kallistatin significantly suppressed angiogenesis and growth of hepatic Hep3B tumors. The kallistatin released by hepatocytes into the circulation suppressed remote Hep3B and BNL tumors established subcutaneously. The rAAV-kallistatin gene therapy significantly inhibited tumor cell proliferation and induced apoptosis. Conclusions Intraportal injection of rAAV-kallistatin suppressed hepatic and subcutaneous HCC tumors, relying on its anti-angiogenic and anti-proliferative activities. Copyright © 2008 John Wiley & Sons, Ltd.

Objective To investigate whether chorionic villus sampling (CVS) is associated with an increase in fetomaternal cell trafficking. Design Prospective study. Setting King's College London School of Medicine, King's College Hospital. Sample Eighteen singleton pregnancies undergoing CVS for fetal karyotyping at 11–14 weeks of gestation and subsequently found to have chromosomal defects. Method Maternal blood samples were obtained immediately before and at 3–14 (median 5) days after CVS. Fetal erythroblasts were isolated using triple density gradient separation and anti-CD71 magnetic cell sorting techniques. The enriched erythroblasts were stained with Kleihauer–Giemsa and with fluorescent antibodies for the epsilon (e) and gamma (γ) globin chains. The percentage of fetal cells positive for each stain was calculated. Fluorescence in situ hybridisation (FISH) for X- and Y-chromosomes was also performed. Comparison was made in the proportion of enriched fetal cells between the pre-CVS and post-CVS samples. Main outcome measures The proportion of fetal erythroblasts in maternal blood. Results The percentage of erythroblasts enriched from maternal blood that stained positive for e and γ globin chains and with Kleihauer–Giemsa was significantly higher in the post-CVS samples compared with the pre-CVS samples. FISH analysis for the Y-chromosome confirmed the increase in fetal cell proportion in the post-CVS samples. The percentage difference in fetal cells decreased significantly with time interval from CVS. Conclusion CVS results in an increase in fetomaternal cell trafficking, which continues to be present for several days after the procedure.

This study examines GATA-4 gene expression in cardiac tissue from fetuses with trisomy 21 presenting with increased nuchal translucency thickness at 10-14 weeks of gestation. mRNA was extracted from cardiac tissue after termination of pregnancy at 10-18 weeks of gestation in ten trisomy 21 fetuses and 29 normal controls. Northern and slot blots were performed and densitometric analysis of slot blots was used to determine the steady-state levels of expression of GATA-4. GATA-4 transcript levels were also compared with ANP and BNP, which have previously been measured in the same panel of samples. GATA-4 expression increased significantly with gestation but there was no significant difference between fetuses with trisomy 21 and controls. There was no significant association between GATA-4 expression and the steady-state level of transcripts for the natriuretic peptides.

Chromosomal deletions, associated with the loss of normal function of tumour suppressor genes, have been identified in a variety of both familial and sporadic human cancers. Although the molecular pathology of ovarian cancer is not understood, several studies have reported deletions in chromosome 17 in ovarian tumours. We have used 13 restriction site polymorphic, microsatellite, and variable number tandem repeat markers to make a detailed analysis of chromosome 17 deletions in 12 benign and 19 malignant ovarian tumours. Two benign and 11 malignant tumours were informative for at least one marker on each arm of the chromosome. Loss of heterozygosity (LOH) was detected in both arms (by all informative markers) in 5 malignant tumours from four women (three with the disease at FIGO stage la). In a further bilateral ovarian tumour a partial LOH affecting 17q22-q25 was present in one ovary only. By contrast to a number of previous studies, none of the 19 malignant and 12 benign tumours showed ERBB2 (17q12ndash;22) amplification. The data presented show that the loss of a whole copy of chromosome 17 is a frequent and relatively early event in the development of some ovarian cancers. This suggests the possible involvement of multiple chromosome 17 loci in the pathogenesis of ovarian cancer. Equally plausible is that the loss of a whole chromosome copy could be the product of chromosomal instabilities induced by loss of the normal allele of tumour suppressors, such as TP53, located on this chromosome. © 1993 Wiley-Liss, Inc.

Objective In about 80% of fetuses with trisomies, Turner syndrome, and in fetuses with skeletal abnormalities, genetic syndromes, structural abnormalities and cardiac defects, there is increased collection of fluid in the neck region. This can be visualized sonographically at 11–14 weeks of gestation as increased nuchal translucency thickness. The pathophysiology of this common phenotypic expression of different chromosomal abnormalities is uncertain, but there is some evidence that the underlying mechanism may be altered composition of extracellular matrix components and/or cardiac failure. Methods We performed a number of studies investigating nuchal skin tissue for various extracellular matrix components and cardiac heart failure using molecular techniques. We used northern blotting, immunohistochemistry, electron-microscopy and in situ hybridization. Results Studies investigating the heart found increased levels of mRNA for ANP and BNP, and reduced levels of Calcium ATPase, whereas transcript levels for GATA-4 were unchanged. Studies investigating components of the extracellular matrix in nuchal skin of trisomic fetuses found overexpression of ecm genes in trisomies (collagen type VI, collagen type IV, laminin) or an altered ratio of genes between normals and abnormals (collagen type VI). Conclusions The present data provide some evidence, that chromosomally abnormal fetuses with increased nuchal translucency at 12–14 week may suffer from an altered extracellular matrix of the heart and skin or transient heart failure.

Cellular differentiation in a number of eukaryotic systems is associated with changes in the number of DNA-strand breaks and involves the activity of adenosine diphosphoribosyl transferase (ADPRT). DNA-strand breaks are essential for activation of nuclear ADPRT, the activity of which is required for efficient religation of DNA-strand breaks. In this study we demonstrate the dynamic nature of DNA-strand breaks formed in the genome of differentiating avian skeletal muscle cells and quiescent human lymphocytes. Inhibition of ADPRT activity blocks DNA-strand ligation in both cell types and leads to the accumulation of a higher number of strand breaks. DNA-strand breakADP-ribosylationDifferentiationMitogen activation