Junctional epidermolysis bullosa (JEB) is a severe and often lethal genetic disease caused by mutations in genes encoding the basement membrane component laminin-332. Surviving patients with JEB develop chronic wounds to the skin and mucosa, which impair their quality of life and lead to skin cancer. Here we show that autologous transgenic keratinocyte cultures regenerated an entire, fully functional epidermis on a seven-year-old child suffering from a devastating, life-threatening form of JEB. The proviral integration pattern was maintained in vivo and epidermal renewal did not cause any clonal selection. Clonal tracing showed that the human epidermis is sustained not by equipotent progenitors, but by a limited number of long-lived stem cells, detected as holoclones, that can extensively self-renew in vitro and in vivo and produce progenitors that replenish terminally differentiated keratinocytes. This study provides a blueprint that can be applied to other stem cell-mediated combined ex vivo cell and gene therapies.
Pseudoxanthoma elasticum (PXE) is a heritable disorder characterized by calcification of elastic fibres in skin, arteries and retina that results in dermal lesions with associated laxity and loss of elasticity, arterial insufficiency and retinal haemorrhages leading to macular degeneration. PXE is usually found as a sporadic disorder, but examples of both autosomal recessive and autosomal dominant forms of PXE have been observed. Partial manifestations of the PXE phenotype have also been described in presumed carriers in PXE families. Linkage of both dominant and recessive forms of PXE to a 5-cM domain on chromosome 16p13.1 has been reported (refs 8,9). We have refined this locus to an 820-kb region containing 6 candidate genes. Here we report the exclusion of five of these genes and the identification of the first mutations responsible for the development of PXE in a gene encoding a protein associated with multidrug resistance (ABCC6).
Over 2.5 billion people live in areas at high risk for Dengue and related flavivirus infections (1). Dengue hemorrhagic fever, a severe complication present in ϳ5% of cases, claims more lives annually than all other hemorrhagic fevers combined (2); much of the damage is caused by death of infected cells. The fate of infected cells depends on cell type. Although flavivirus induces apoptosis of neurons and macrophages, infected hepatocytes and epithelial cells do not die. We find that flavivirus up-regulates autophagy in MDCK 3 renal epithelial cells and other cell types and subsequently protects them. We further identify nonstructural protein NS4A of both Dengue-2 and Modoc viruses as the sole viral mediator of autophagy up-regulation and protection against death. Up-regulation of autophagosomes by either live virus infection or NS4A expression depends on PI3K and is important for replication of flavivirus in renal epithelial cells. Flaviviruses often persist in liver and kidney following the acute phase of infection, suggesting that infected cells evade destruction by the host immune response, likely by flavivirusinduced up-regulation of autophagy in these cells. In vitro infection of hepatocytes and fibroblasts leads to up-regulation in autophagy and protection against death (3, 4). More relevant, Dengue-2 viruses replicate within the hepatocyte autophagosomes (5), and inhibition of autophagy attenuates virus replication (3). We extend these findings by identifying the nonstructural viral protein NS4A as the virus-encoded protein that up-regulates autophagy and thus protects the host cell against death, providing a well protected host cell for long term replication of virus.The flavivirus genome contains 10 genes encoding an ϳ11-kDa polyprotein precursor that binds to the ER membrane after translation at the rough ER as follows: three structural proteins (capsid (core) protein, pre-membrane protein ,and envelope glycoprotein) and seven nonstructural genes (NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5) that mediate viral replication, assembly, and evasion of the host immune system. The active proteins are cleaved from the ER-bound viral polyprotein by cellular proteases signalase and furin and viral protease NS3/ 2B. NS3 is both a viral protease (with required cofactor NS2B (6)) and a viral ATP-dependent helicase (7, 8); NS5 is an RNAdependent RNA polymerase and methyltransferase (9) responsible for virus genome replication, whereas NS1 is possibly a part of the viral replication complex (10). The small hydrophobic flavivirus proteins (NS2A, NS4A, and NS4B) remain the most poorly characterized. NS2A is required for the assembly of new flavivirus virions; NS4A associates with the virus replication complex and induces ER membrane rearrangements (discussed below), and NS4B is an antagonist of interferon. Flavivirus NS4A is an ϳ16-kDa membrane-associated protein consisting of four transmembrane helices and an N-terminal cytosolic region. Once the viral genome is translated in the ER,
In patients infected by SARS-CoV-2 who experience an exaggerated inflammation leading to pneumonia, monocytes likely play a major role but have received poor attention. Thus, we analyzed peripheral blood monocytes from patients with COVID-19 pneumonia and found that these cells show signs of altered bioenergetics and mitochondrial dysfunction, had a reduced basal and maximal respiration, reduced spare respiratory capacity, and decreased proton leak. Basal extracellular acidification rate was also diminished, suggesting reduced capability to perform aerobic glycolysis. Although COVID-19 monocytes had a reduced ability to perform oxidative burst, they were still capable of producing TNF and IFN-c in vitro. A significantly high amount of monocytes had depolarized mitochondria and abnormal mitochondrial ultrastructure. A redistribution of monocyte subsets, with a significant expansion of intermediate/pro-inflammatory cells, and high amounts of immature monocytes were found, along with a concomitant compression of classical monocytes, and an increased expression of inhibitory checkpoints like PD-1/PD-L1. High plasma levels of several inflammatory cytokines and chemokines, including GM-CSF, IL-18, CCL2, CXCL10, and osteopontin, finally confirm the importance of monocytes in COVID-19 immunopathogenesis.
SummaryWe report a long-term follow-up (6.5 years) of a phase I/II clinical trial envisaging the use of autologous genetically modified cultured epidermal stem cells for gene therapy of junctional epidermolysis bullosa, a devastating genetic skin disease. The critical goals of the trial were to evaluate the safety and long-term persistence of genetically modified epidermis. A normal epidermal-dermal junction was restored and the regenerated transgenic epidermis was found to be fully functional and virtually indistinguishable from a normal control. The epidermis was sustained by a discrete number of long-lasting, self-renewing transgenic epidermal stem cells that maintained the memory of the donor site, whereas the vast majority of transduced transit-amplifying progenitors were lost within the first few months after grafting. These data pave the way for the safe use of epidermal stem cells in combined cell and gene therapy for genetic skin diseases.
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