F. Mazurier

Helicobacter pylori Infection Recruits Bone Marrow−Derived Cells That Participate in Gastric Preneoplasia in Mice

BACKGROUND & AIMS Studies in animal models have shown that bone marrow-derived cells (BMDC) could be involved in the formation of carcinomas of the upper gastrointestinal tract, including gastric carcinoma. Most gastric carcinomas in humans have been associated with chronic infection with Helicobacter pylori; we investigated the bacterias potential to induce premalignant lesions in mice and studied the kinetics of BMDC settlement in the gastric epithelium. METHODS C57BL/6J female chimeric mice with BMDCs from male donors that express green fluorescent protein were infected with human-derived and mouse-adapted strains of H pylori and followed. We assessed development of pathologic features and recruitment of BMDC to the gastric mucosa using immunohistochemistry and fluorescent in situ hybridization analyses of gastric tissue sections. RESULTS Infection of mice with different strains of H pylori led to the development of chronic inflammation, hyperplasia, and mucinous metaplasia, and, later in life, of pseudointestinal metaplasia and dysplasia. After 1 year, gastric glands that contained green fluorescent protein-positive male cells were detected in 50%-90% of female chimeric mice infected with H pylori strains; the presence of these glands correlated with the development of pseudointestinal metaplasia. Twenty-two percent of H pylori-induced dysplastic lesions were composed of glands that contained epithelial BMDCs. CONCLUSIONS H pylori infection leads to development of chronic inflammation, hyperplasia, metaplasia, and dysplasia, as well as the recruitment and accumulation of BMDC in the gastric epithelial mucosa. Nearly 25% of dysplastic lesions include cells that originate from the BM.

Gene transfer of the uroporphyrinogen III synthase cDNA into haematopoietic progenitor cells in view of a future gene therapy in congenital erythropoietic porphyria

Congenital erythropoietic porphyria (CEP) is an inherited metabolic disorder characterized by an overproduction and accumulation of porphyrins in bone marrow. This autosomal recessive disease results from a deficiency of uroporphyrinogen III synthase (UROIIIS), the fourth enzyme of the haem biosynthetic pathway. It is phenotypically heterogeneous: patients with mild disease have cutaneous involvement, while more severely affected patients are transfusion dependent. The cloning of UROIIIS cDNA and genomic DNA has allowed the molecular characterization of the genetic defect in a number of families. To date, 22 different mutations have been characterized. Allogeneic bone marrow transplantation is the only curative treatment available for the severe, transfusion-dependent, cases. When bone marrow transplantation cannot be performed owing to the absence of a suitable donor, the autografting of genetically modified cells is an appealing alternative. The best approach to somatic gene therapy in this disease involves the use of recombinant retroviral vectors to transduce cells ex vivo, followed by autologous transplantation of the genetically modified cells. We investigated retroviral transfer in deficient human fibroblasts, immortalized lymphoblasts as well as bone marrow cells, and obtained a complete restoration of the enzymatic activity and full metabolic correction.Using K562 cells, an erythroleukaemic cell line, the expression of the transgene remained stable during 3 months and during erythroid differentiation of the cells. Finally, a 1.6- to 1.9-fold increase in enzyme activity compared to the endogenous level was found in normal CD34+ cells, a population of heterogeneous cells known to contain the progenitor/stem cells for long-term expression. The future availability of a mouse model of the disease will permit ex vivo gene therapy experiments on the entire animal.

Successful therapeutic effect in a mouse model of erythropoietic protoporphyria by partial genetic correction and fluorescence-based selection of hematopoietic cells

Erythropoietic protoporphyria is characterized clinically by skin photosensitivity and biochemically by a ferrochelatase deficiency resulting in an excessive accumulation of photoreactive protoporphyrin in erythrocytes, plasma and other organs. The availability of the Fechm1Pas/Fechm1Pas murine model allowed us to test a gene therapy protocol to correct the porphyric phenotype. Gene therapy was performed by ex vivo transfer of human ferrochelatase cDNA with a retroviral vector to deficient hematopoietic cells, followed by re-injection of the transduced cells with or without selection in the porphyric mouse. Genetically corrected cells were separated by FACS from deficient ones by the absence of fluorescence when illuminated under ultraviolet light. Five months after transplantation, the number of fluorescent erythrocytes decreased from 61% (EPP mice) to 19% for EPP mice engrafted with low fluorescent selected BM cells. Absence of skin photosensitivity was observed in mice with less than 20% of fluorescent RBC. A partial phenotypic correction was found for animals with 20 to 40% of fluorescent RBC. In conclusion, a partial correction of bone marrow cells is sufficient to reverse the porphyric phenotype and restore normal hematopoiesis. This selection system represents a rapid and efficient procedure and an excellent alternative to the use of potentially harmful gene markers in retroviral vectors.

Rapid analysis and efficient selection of human transduced primitive hematopoietic cells using the humanized S65T green fluorescent protein

We have developed an efficient and rapid method to analyze transduction in human hematopoietic cells and to select them. We constructed two retroviral vectors using the recombinant humanized S65T green fluorescent protein (rHGFP) gene. Transduced cells appeared with specific green fluorescence on microscopy or fluorescence-activated cell sorting (FACS) analysis. The rHGFP gene was placed under the control of two different retroviral promotors (LTR) in the LGSN vector and in the SF-GFP vector. Amphotropic retroviruses were tested on NIH/3T3 fibroblasts or human hematopoietic (K562, TF-1) cell lines. Then CD34+ cells isolated from cord blood were infected three times after a 48-h prestimulation with IL-3, IL-6, SCF or with IL-3, IL-6, SCF, GM-CSF, Flt3-L and TPO. After 6 days of expansion, a similar number of total CD34+-derived cells, CD34+ cells and CFC was obtained in non-transduced and transduced cells, demonstrating the absence of toxicity of the GFP. A transduction up to 46% in total CD34+-derived cells and 21% of CD34+ cells was shown by FACS analysis. These results were confirmed by fluorescence of colonies in methyl-cellulose (up to 36% of CFU-GM and up to 25% of BFU-E). The FACS sorting of GFP+ cells led to 83–100% of GFP-positive colonies after 2 weeks of methyl-cellulose culture. Moreover, a mean gene transfer efficiency of 8% was also demonstrated in long-term culture initiating cells (LTC-IC). This rapid and efficient method represents a substantial improvement to monitor gene transfer and retroviral expression of various vectors in characterized human hematopoietic cells.

Premature skin aging features rescued by inhibition of NADPH oxidase activity in XPC-deficient mice.

Xeroderma pigmentosum type C (XP-C) is characterized mostly by a predisposition to skin cancers and accelerated photoaging, but little is known about premature skin aging in this disease. By comparing young and old mice, we found that the level of progerin and p16(INK4a) expression, β-galactosidase activity, and reactive oxygen species, which increase with age, were higher in young Xpc(-/-) mice than in young Xpc(+/+) ones. The expression level of mitochondrial complexes and mitochondrial functions in the skin of young Xpc(-/-) was as low as in control aged Xpc(+/+)animals. Furthermore, the metabolic profile in young Xpc(-/-) mice resembled that found in aged Xpc(+/+) mice. Furthermore, premature skin aging features in young Xpc(-/-) mice were mostly rescued by inhibition of nicotinamide adenine dinucleotide phosphate oxidase 1 (NOX1) activity by using a NOX1 peptide inhibitor, suggesting that the continuous oxidative stress due to overactivation of NOX1 has a causative role in the underlying pathophysiology.

Xeroderma pigmentosum: clues to understanding cancer initiation

Abstract Xeroderma pigmentosum (XP) type C is a rare autosomal recessive disorder that occurs because of inactivation of the xeroderma pigmentosum group C (XPC) protein, which is an important DNA damage recognition protein involved in DNA nucleotide excision repair (NER). This defect, which prevents removal of a wide array of direct and indirect DNA lesions, is associated with a decrease in catalase activity. As a novel photoprotective approach, lentivirus-mediated catalase overexpression in XPC human keratinocytes results in a marked decrease in sunburn cell formation, caspase-3 activation, and p53 accumulation following UVB irradiation. While not correcting the gene defect, indirect gene therapy using antioxidant enzymes may be helpful in limiting photosensitivity in XP type C, as well as in other monogenic/polygenic photosensitive disorders characterized by reactive oxygen species (ROS) accumulation. Hypoxia-inducible factor-1 (HIF-1), a major transcription factor sensitive to oxygen levels, responds to various stress factors. As a common stressor of skin, UVB induces a biphasic HIF-1a variation through ROS generation in keratinocytes. HIF-1a has an important regulator effect on the expression of XPC protein and other NER genes, indicating indirect regulation of NER by ROS. The intrinsic genomic instability arising in XP type C provides a good opportunity to investigate the complex molecular mechanisms underlying the Warburg effect (the shift of mito-chondrial metabolism towards glycolysis). Overall, the monogenic disorder XP type C is a powerful tool for studying photoprotection and cancer.