Design of a syringe extension device (Chloe SED®) for low-resource settings in sub-Saharan Africa: a circular economy approach.

Underfunded healthcare infrastructures in low-resource settings in sub-Saharan Africa have resulted in a lack of medical devices crucial to provide healthcare for all. A representative example of this scenario is medical devices to administer paracervical blocks during gynaecological procedures. Devices needed for this procedure are usually unavailable or expensive. Without these devices, providing paracervical blocks for women in need is impossible resulting in compromising the quality of care for women requiring gynaecological procedures such as loop electrosurgical excision, treatment of miscarriage, or incomplete abortion. In that perspective, interventions that can be integrated into the healthcare system in low-resource settings to provide women needing paracervical blocks remain urgent. Based on a context-specific approach while leveraging circular economy design principles, this research catalogues the development of a new medical device called Chloe SED® that can be used to support the provision of paracervical blocks. Chloe SED®, priced at US$ 1.5 per device when produced in polypropylene, US$ 10 in polyetheretherketone, and US$ 15 in aluminium, is attached to any 10-cc syringe in low-resource settings to provide paracervical blocks. The device is designed for durability, repairability, maintainability, upgradeability, and recyclability to address environmental sustainability issues in the healthcare domain. Achieving the design of Chloe SED® from a context-specific and circular economy approach revealed correlations between the material choice to manufacture the device, the device's initial cost, product durability and reuse cycle, reprocessing method and cost, and environmental impact. These correlations can be seen as interconnected conflicting or divergent trade-offs that need to be continually assessed to deliver a medical device that provides healthcare for all with limited environmental impact. The study findings are intended to be seen as efforts to make available medical devices to support women's access to reproductive health services.

Crigler-Najjar syndrome in The Netherlands: identification of four novel UGT1A1 alleles, genotype-phenotype correlation, and functional analysis of 10 missense mutants.

Crigler-Najjar syndrome (CN), caused by deficiency of UGT isoform 1A1 (UGT1A1), is characterized by severe unconjugated hyperbilirubinemia. In this study we have analyzed 19 CN patients diagnosed in The Netherlands (18) and in Belgium (1), and have identified 14 different UGT1A1 mutations, four of which are novel. Two mutations were present in several unrelated patients, suggesting the presence of two founder effects in The Netherlands. In addition, we show linkage of the UGT1A1 *28 promoter polymorphism (rs5719145insTA) to three structural mutations. Functional studies of partial active UGT1A1 mutants are limited. Therefore, we performed in vitro studies to determine the functional activity of seven missense mutants identified in this study and of three reported previously. In addition to bilirubin, we also determined their activity toward eight other UGT1A1 substrates. We demonstrate that five mutants have residual activity that, depending on the substrate, varies from not detectable to 94% of wild-type UGT1A1 activity. The identification of four novel pathogenic mutations and the analysis of residual activity of 10 UGT1A1 missense mutants are useful for clinical diagnosis, and provides new insights in enzyme activity, whereas the identification of two founder mutations will speed up genetic counseling for newly identified CN patients in The Netherlands.

A fiber modified adenovirus vector that targets to the EphrinA2 receptor reveals enhanced gene transfer to ex vivo pancreatic cancer.

Pancreatic cancer is an aggressive malignancy with a dismal prognosis. To improve treatment options new treatments, such as adenoviral (Ad) gene therapy are necessary. However, low expression of the coxsackie and adenovirus receptor (CAR) in pancreatic cancer cells (PC) limits the therapeutic efficacy of these vectors. The aim of this study was to improve transduction of PC by recombinant adenoviruses by inserting peptides into the HI loop that binds to receptors highly expressed on pancreatic cancer and were shown to target these carcinomas in vivo. We report the successful incorporation into the HI loop of peptide Tyr-Ser-Ala (YSA), a peptide ligand targeting the EphrinA2 (EphA2) receptor, and K237, a peptide targeting to the vascular endothelial growth factor receptor-II (VEGFRII). Subsequently, we showed that both peptides enhanced the transduction of a number of human PC lines that abundantly express the targeted receptor. Additional competition studies confirmed that the YSA peptide redirects Ad-YSA from CAR and specifically targets the EphA2 receptor. Due to this transduction efficiency of Ad-YSA is increased not only in human pancreatic cancer cell lines but more importantly also in pancreatic cancer resection specimens. Since the YSA peptide has been shown to specifically target pancreatic cancer in patients, it may be expected that Ad-YSA will also display increased tropism for this tumour.

Ephrin A2 receptor targeting does not increase adenoviral pancreatic cancer transduction in vivo.

AIM: To generate an adenoviral vector specifically targeting the EphA2 receptor (EphA2R) highly expressed on pancreatic cancer cells in vivo. METHODS: YSA, a small peptide ligand that binds the EphA2R with high affinity, was inserted into the HI loop of the adenovirus serotype 5 fiber knob. To further increase the specificity of this vector, binding sites for native adenoviral receptors, the coxsackie and adenovirus receptor (CAR) and integrin, were ablated from the viral capsid. The ablated retargeted adenoviral vector was produced on 293T cells. Specific targeting of this novel adenoviral vector to pancreatic cancer was investigated on established human pancreatic cancer cell lines. Upon demonstrating specific in vitro targeting, in vivo targeting to subcutaneous growing human pancreatic cancer was tested by intravenous and intraperitoneal administration of the ablated adenoviral vector. RESULTS: Ablation of native cellular binding sites reduced adenoviral transduction at least 100-fold. Insertion of the YSA peptide in the HI loop restored adenoviral transduction of EphA2R-expressing cells but not of cells lacking this receptor. YSA-mediated transduction was inhibited by addition of synthetic YSA peptide. The transduction specificity of the ablated retargeted vector towards human pancreatic cancer cells was enhanced almost 10-fold in vitro. In a subsequent in vivo study in a nude (nu/nu) mouse model however, no increased adenoviral targeting to subcutaneously growing human pancreas cancer nodules was seen upon injection into the tail vein, nor upon injection into the peritoneum. CONCLUSION: Targeting the EphA2 receptor increases specificity of adenoviral transduction of human pancreatic cancer cells in vitro but fails to enhance pancreatic cancer transduction in vivo.

Ex-vivo evaluation of gene therapy vectors in human pancreatic (cancer) tissue slices.

AIM: To culture human pancreatic tissue obtained from small resection specimens as a pre-clinical model for examining virus-host interactions. METHODS: Human pancreatic tissue samples (malignant and normal) were obtained from surgical specimens and processed immediately to tissue slices. Tissue slices were cultured ex vivo for 1-6 d in an incubator using 95% O(2). Slices were subsequently analyzed for viability and morphology. In addition the slices were incubated with different viral vectors expressing the reporter genes GFP or DsRed. Expression of these reporter genes was measured at 72 h after infection. RESULTS: With the Krumdieck tissue slicer, uniform slices could be generated from pancreatic tissue but only upon embedding the tissue in 3% low melting agarose. Immunohistological examination showed the presence of all pancreatic cell types. Pancreatic normal and cancer tissue slices could be cultured for up to 6 d, while retaining viability and a moderate to good morphology. Reporter gene expression indicated that the slices could be infected and transduced efficiently by adenoviral vectors and by adeno associated viral vectors, whereas transduction with lentiviral vectors was limited. For the adenoviral vector, the transduction seemed limited to the peripheral layers of the explants. CONCLUSION: The presented system allows reproducible processing of minimal amounts of pancreatic tissue into slices uniform in size, suitable for pre-clinical evaluation of gene therapy vectors.

Adeno-associated virus vector serotypes mediate sustained correction of bilirubin UDP glucuronosyltransferase deficiency in rats.

Crigler-Najjar (CN) patients have no bilirubin UDP glucuronosyltransferase (UGT1A1) activity and suffer brain damage because of bilirubin toxicity. Vectors based on adeno-associated virus (AAV) serotype 2 transduce liver cells with relatively low efficiency. Recently, AAV serotypes 1, 6, and 8 have been shown to be more efficient for liver cell transduction. We compared AAV serotypes 1, 2, 6, and 8 for correction of UGT1A1 deficiency in the Gunn rat model of CN disease. Adult Gunn rats were injected with CMV-UGT1A1 AAV vectors. Serum bilirubin was decreased over the first year by 64% for AAV1, 16% for AAV2, 25% for AAV6, and 35% for AAV8. Antibodies to UGT1A1 were detected after injection of all AAV serotypes. An AAV1 UGT1A1 vector with the liver-specific albumin promoter corrected serum bilirubin levels but did not induce UGT1A1 antibodies. Two years after injection of AAV vectors all animals had large lipid deposits in the liver. These lipid deposits were not seen in age-matched control animals. AAV1 vectors are promising candidates for CN gene therapy because they can mediate a reduction in serum bilirubin levels in Gunn rats that would be therapeutic in humans.

Selection of tumour specific promoters for adenoviral gene therapy of cholangiocarcinoma.

BACKGROUND/AIMS: The majority of cholangiocarcinoma patients present with advanced incurable disease. Therefore development of new therapeutic modalities including adenoviral gene therapy is of paramount importance. We set out to identify tumour specific promoters which have low activity in human liver cells and retain their specificity in an adenoviral vector. METHODS: mRNA levels of cyclo-oxygenase-2, cytokeratin-19, mucin-1, midkine and telomerase reverse transcriptase (TERT) were determined in human liver, cholangiocarcinoma (resection specimens and cell lines), primary human hepatocytes, cholangiocytes and endothelial cells by Reverse Transcriptase-quantitative PCR. The activity of candidate promoters in adenoviral vectors was then determined in cholangiocarcinoma cell lines, primary human hepatocytes and cholangiocytes. RESULTS: mRNA levels of all tested tumour markers were significantly higher in cholangiocarcinoma than in normal liver. Based on low expression in hepatocytes, either in combination with low expression in primary cholangiocytes or endothelial cells, the cytokeratin-19, mucin-1 and TERT promoters were selected for further analyses. In an adenoviral vector, the activity of the TERT or cytokeratin-19 promoters were low in normal human hepatocytes and cholangiocytes, and high in cholangiocarcinoma cell lines. CONCLUSIONS: The TERT and Cytokeratin-19 promoters are highly expressed in cholangiocarcinoma and seem suitable to restrict adenoviral gene therapy to these intra-hepatic tumours.

Anion exchanger 2 is essential for spermiogenesis in mice.

Na+-independent anion exchangers (AE) mediate electroneutral exchange of Cl- for HCO3- ions across cell membranes, being involved in intracellular pH and cell volume regulation and in transepithelial hydroionic fluxes. Bicarbonate activation of adenylyl cyclase is known to be necessary for sperm motility and sperm capacitation, and a few studies have suggested a possible role of AE carriers in reproduction. Among the four AE genes identified in mammals thus far, only Ae2 (Slc4a2) has been determined to be expressed in the male reproductive system, especially in developing spermatozoa and in epididymal epithelium. Most AE genes drive alternative transcription, which in mouse Ae2 results in several Ae2 isoforms. Here, we generated mice carrying a targeted disruption of Ae2 that prevents the expression of the three AE2 isoforms (Ae2a, Ae2b1, and Ae2b2) normally found in mouse testes. Male Ae2-/- mice (but not female Ae2-/- mice) are infertile. Histopathological analysis of Ae2-/- testes shows an interruption of spermiogenesis, with only a few late spermatids and a complete absence of spermatozoa in the seminiferous tubules. The number of apoptotic bodies is increased in the seminiferous tubules and in the epididymis, which also shows squamous metaplasia of the epididymal epithelium. Our findings reveal an essential role of Ae2 in mouse spermiogenesis and stress the recently postulated involvement of bicarbonate in germ-cell differentiation through the bicarbonate-sensitive soluble-adenylyl-cyclase pathway.