Laminin 332-Dependent YAP Dysregulation Depletes Epidermal Stem Cells in Junctional Epidermolysis Bullosa

Laminin 332-deficient junctional epidermolysis bullosa (JEB) is a severe genetic skin disease. JEB is marked by epidermal stem cell depletion, the origin of which is unknown. We show that dysregulation of the YAP and TAZ pathway underpins such stem cell depletion. Laminin 332-mediated YAP activity sustains human epidermal stem cells, detected as holoclones. Ablation of YAP selectively depletes holoclones, while enforced YAP blocks conversion of stem cells into progenitors and indefinitely extends the keratinocyte lifespan. YAP is dramatically decreased in JEB keratinocytes, which contain only phosphorylated, inactive YAP. In normal keratinocytes, laminin 332 and alpha 6 beta 4 ablation abolish YAP activity and recapitulate the JEB phenotype. In JEB keratinocytes, laminin 332-gene therapy rescues YAP activity and epidermal stem cells in vitro and in vivo. In JEB cells, enforced YAP recapitulates laminin 332-gene therapy, thus uncoupling adhesion from proliferation in epidermal stem cells. This work has important clinical implication for ex vivo gene therapy of JEB

Prospective Newborn Screening for SCID in Germany: A First Analysis by the Pediatric Immunology Working Group (API)

Background: T-cell receptor excision circle (TREC)-based newborn screening (NBS) for severe combined immunodeficiencies (SCID) was introduced in Germany in August 2019. / Methods: Children with abnormal TREC-NBS were referred to a newly established network of Combined Immunodeficiency (CID) Clinics and Centers. The Working Group for Pediatric Immunology (API) and German Society for Newborn Screening (DGNS) performed 6-monthly surveys to assess the TREC-NBS process after 2.5 years. / Results: Among 1.9 million screened newborns, 88 patients with congenital T-cell lymphocytopenia were identified (25 SCID, 17 leaky SCID/Omenn syndrome (OS)/idiopathic T-cell lymphocytopenia, and 46 syndromic disorders). A genetic diagnosis was established in 88%. Twenty-six patients underwent hematopoietic stem cell transplantation (HSCT), 23/26 within 4 months of life. Of these, 25/26 (96%) were alive at last follow-up. Two patients presented with in utero onset OS and died after birth. Five patients with syndromic disorders underwent thymus transplantation. Eight syndromic patients deceased, all from non-immunological complications. TREC-NBS missed one patient, who later presented clinically, and one tracking failure occurred after an inconclusive screening result. / Conclusion: The German TREC-NBS represents the largest European SCID screening at this point. The incidence of SCID/leaky SCID/OS in Germany is approximately 1:54,000, very similar to previous observations from North American and European regions and countries where TREC-NBS was implemented. The newly founded API-CID network facilitates tracking and treatment of identified patients. Short-term HSCT outcome was excellent, but NBS and transplant registries will remain essential to evaluate the long-term outcome and to compare results across the rising numbers of TREC-NBS programs across Europe

Decreased Toll-like receptor 8 expression and lower TNF-alpha synthesis in infants with acute RSV infection

<p>Abstract</p> <p>Background</p> <p>Toll-like receptors (TLRs) are part of the innate immune system, able to recognize pathogen-associated molecular patterns and activate immune system upon pathogen challenge. Respiratory syncytial virus (RSV) is a RNA virus particularly detrimental in infancy. It could cause severe lower respiratory tract disease and recurrent infections related to inadequate development of anti-viral immunity. The reason could be inadequate multiple TLRs engagement, including TLR8 in recognition of single-stranded viral RNA and diminished synthesis of inflammatory mediators due to a lower expression.</p> <p>Methods</p> <p>Intracellular TLR8 expression in peripheral blood monocytes from RSV-infected infants was profiled and compared to healthy adults and age matched controls. Whether the observed difference in TLR8 expression is a transitory effect, infants in convalescent phase (4-6 weeks later) were retested. Specific TLR8-mediated TNF-α production in monocytes during an acute and convalescent phase was analyzed.</p> <p>Results</p> <p>RSV-infected and healthy infants had lower percentage of TLR8-expressing monocytes than healthy adults whereas decreased of TLR8 protein levels were detected only for RSV-infected infant group. Lower protein levels of TLR8 in monocytes from RSV-infected infants, compared to healthy infants, negatively correlated with respiratory frequency and resulted in lower TNF-α synthesis upon a specific TLR8 stimulation. In the convalescent phase, levels of TLR8 increased, accompanied by increased TNF-α synthesis compared to acute infection.</p> <p>Conclusions</p> <p>Lower TLR8 expression observed in monocytes, during an acute RSV infection, might have a dampening impact on early anti-viral cytokine production necessary to control RSV replication, and subsequently initiate an adaptive Th1 type immune response leading to severe disease in infected infants.</p

Primary Human mDC1, mDC2, and pDC Dendritic Cells Are Differentially Infected and Activated by Respiratory Syncytial Virus

Respiratory syncytial virus (RSV) causes recurrent infections throughout life. Vaccine development may depend upon understanding the molecular basis for induction of ineffective immunity. Because dendritic cells (DCs) are critically involved in early responses to infection, their interaction with RSV may determine the immunological outcome of RSV infection. Therefore, we investigated the ability of RSV to infect and activate primary mDCs and pDCs using recombinant RSV expressing green fluorescent protein (GFP). At a multiplicity of infection of 5, initial studies demonstrated ∼6.8% of mDC1 and ∼0.9% pDCs were infected. We extended these studies to include CD1c−CD141+ mDC2, finding mDC2 infected at similar frequencies as mDC1. Both infected and uninfected cells upregulated phenotypic markers of maturation. Divalent cations were required for infection and maturation, but maturation did not require viral replication. There is evidence that attachment and entry/replication processes exert distinct effects on DC activation. Cell-specific patterns of RSV-induced maturation and cytokine production were detected in mDC1, mDC2, and pDC. We also demonstrate for the first time that RSV induces significant TIMP-2 production in all DC subsets. Defining the influence of RSV on the function of selected DC subsets may improve the likelihood of achieving protective vaccine-induced immunity

Effects of Human Respiratory Syncytial Virus, Metapneumovirus, Parainfluenza Virus 3 and Influenza Virus on CD4+ T Cell Activation by Dendritic Cells

BACKGROUND: Human respiratory syncytial virus (HRSV), and to a lesser extent human metapneumovirus (HMPV) and human parainfluenza virus type 3 (HPIV3), re-infect symptomatically throughout life without antigenic change, suggestive of incomplete immunity. One causative factor is thought to be viral interference with dendritic cell (DC)-mediated stimulation of CD4+ T cells. METHODOLOGY, PRINCIPAL FINDINGS: We infected human monocyte-derived DC with purified HRSV, HMPV, HPIV3, or influenza A virus (IAV) and compared their ability to induce activation and proliferation of autologous CD4+ T cells in vitro. IAV was included because symptomatic re-infection without antigenic change is less frequent, suggesting that immune protection is more complete and durable. We examined virus-specific memory responses and superantigen-induced responses by multiparameter flow cytometry. Live virus was more stimulatory than inactivated virus in inducing DC-mediated proliferation of virus-specific memory CD4+ T cells, suggesting a lack of strong suppression by live virus. There were trends of increasing proliferation in the order: HMPV<HRSV<HPIV3<IAV, and greater production of interferon-γ and tumor necrosis factor-α by proliferating cells in response to IAV, but differences were not significant. Exposure of DC to HRSV, HPIV3, or IAV reduced CD4+ T cell proliferation in response to secondary stimulus with superantigen, but the effect was transitory and greatest for IAV. T cell cytokine production was similar, with no evidence of Th2 or Th17 skewing. CONCLUSIONS, SIGNIFICANCE: Understanding the basis for the ability of HRSV in particular to symptomatically re-infect without significant antigenic change is of considerable interest. The present results show that these common respiratory viruses are similar in their ability to induce DC to activate CD4+ T cells. Thus, the results do not support the common model in which viral suppression of CD4+ T cell activation and proliferation by HRSV, HMPV, and HPIV3 is a major factor in the difference in re-infectability compared to IAV

Long survival in leigh syndrome: new cases and review of literature

Item does not contain fulltextLeigh syndrome (MIM 25600), also known as infantile subacute necrotizing encephalomyelopathy, is a neurodegenerative disorder with characteristic bilateral symmetric lesions in basal ganglia and subcortical brain regions. It is commonly associated with systemic cytochrome c oxidase (COX) deficiency and mutations in the SURF1 gene (MIM 185620), encoding a putative assembly or maintenance factor of COX. The clinical course is dominated by neurodevelopmental regression, brain stem, and basal ganglia involvement (e.g., dystonia, apnea) with death often occurring before the age of 10 years. Herein, we present three sisters carrying a previously reported homozygous SURF1 mutation (c.868_869insT) that is predicted to result in a truncated protein with loss of function. Our patients show heterogeneous clinical findings with different distribution patterns of metabolic lesions in brain magnetic resonance imaging (MRI) as well as a Chiari malformation with hydrocephalus in one patient. However, all three siblings show an unusual long survival (12 years and > 16 years). COX activity was not detectable in one patient and strongly reduced in the other two. We discuss these findings with respect to a review of the literature. A total of 15 additional patients with survival > 14 years have been reported so far. Overall, no clear genotype-phenotype correlations are detectable among these patients