The IFITM5 Ser40Leu variant can manifest as prenatal Caffey disease.

We report on a female neonate with a clinico-radiological presentation in keeping with a lethal form of prenatal Caffey disease (PCH). She had antenatal and postnatal features of severely bowed long bones, small chest, diaphyseal hyperostosis and polyhydramnios and died shortly after birth. Initial testing excluded COL1A1-related PCH, as an OI gene panel, consisting of COL1A1, COL1A2, CRTAP, and P3H1 genes, was negative. Targeted sequencing using a gene panel was performed and a de novo heterozygous, likely pathogenic variant in IFITM5: c.119C > T(p.Ser40Leu) was identified, which was previously described to cause a severe form of progressively deforming osteogenesis imperfect (OI). To our knowledge, variants in IFITM5 have not been reported in infantile Caffey disease (ICH) or PCH. Given that the pathogenesis of PCH is largely unknown, we postulate that a subset of PCH may be associated with variants in IFITM5.

Outcomes of hematopoietic stem cell gene therapy for Wiskott-Aldrich syndrome.

Wiskott-Aldrich syndrome (WAS) is a rare X-linked disorder characterized by combined immunodeficiency, eczema, microthrombocytopenia, autoimmunity, and lymphoid malignancies. Gene therapy (GT) to modify autologous CD34+ cells is an emerging alternative treatment with advantages over standard allogeneic hematopoietic stem cell transplantation for patients who lack well-matched donors, avoiding graft-versus-host-disease. We report the outcomes of a phase 1/2 clinical trial in which 5 patients with severe WAS underwent GT using a self-inactivating lentiviral vector expressing the human WAS complementary DNA under the control of a 1.6-kB fragment of the autologous promoter after busulfan and fludarabine conditioning. All patients were alive and well with sustained multilineage vector gene marking (median follow-up: 7.6 years). Clinical improvement of eczema, infections, and bleeding diathesis was universal. Immune function was consistently improved despite subphysiologic levels of transgenic WAS protein expression. Improvements in platelet count and cytoskeletal function in myeloid cells were most prominent in patients with high vector copy number in the transduced product. Two patients with a history of autoimmunity had flares of autoimmunity after GT, despite similar percentages of WAS protein-expressing cells and gene marking to those without autoimmunity. Patients with flares of autoimmunity demonstrated poor numerical recovery of T cells and regulatory T cells (Tregs), interleukin-10-producing regulatory B cells (Bregs), and transitional B cells. Thus, recovery of the Breg compartment, along with Tregs appears to be protective against development of autoimmunity after GT. These results indicate that clinical and laboratory manifestations of WAS are improved with GT with an acceptable safety profile. This trial is registered at clinicaltrials.gov as #NCT01410825.

A novel NSDHL variant in CHILD syndrome with gastrointestinal manifestations and localized skin involvement.

BACKGROUND: CHILD syndrome is an X-linked dominant disorder associated with pathogenic mutations in the NSDHL gene. The condition is predominantly found in females as it is lethal in males. Most cases present at birth with extensive unilateral ichthyosiform erythroderma involving the trunk and limbs. Milder and less extensive presentations have been reported, leading to misdiagnosis especially during early childhood. METHODS AND RESULTS: We report an adult female of Malay ancestry who presented with minimal skin and limb involvement. She was only diagnosed in adulthood when she presented with gastrointestinal symptoms and worsening of skin manifestations. The clinical diagnosis was suspected after a combination of clinical, pathological and immunohistochemistry correlation, and molecularly confirmed with the discovery of a frameshift variant in NSDHL. The novel variant was inherited from her mother who had some linear hypopigmented patches over the medial aspects of both her arms and right forearm. CONCLUSION: We uncovered a novel frameshift variant associated with presentations that cast a new light on the clinical features of CHILD syndrome.

Micro- and nanotopography with extracellular matrix coating modulate human corneal endothelial cell behavior.

The human corneal endothelium plays an important role in maintaining corneal transparency. Human corneal endothelial cells have limited regenerative capability in vivo. Consequently, endothelial dysfunction can occur following corneal endothelial trauma or inherited diseases. To restore endothelial function, corneal transplantation is needed. However, there is a worldwide shortage of donor corneas, motivating the development of a tissue-engineered graft alternative using cultivated endothelial cells. To induce in vitro cell proliferation, much effort has been made to improve culture conditions and to mimic the native extracellular microenvironment. We incorporated topographical and biochemical cues in our in vitro culture of human corneal endothelial cell line B4G12 (HCEC-B4G12) and hypothesized that manipulation of the extracellular environment can modulate cell proliferation, morphometry and phenotype. The topographies tested were nanopillars, microwells and micropillars on polydimethylsiloxane, while the biochemical factors were extracellular matrix protein coatings of fibronectin-collagen I (FC), FNC® coating mix (FNC) and laminin-chondroitin sulfate (LC). Cellular morphometry, Na(+)/K(+)-ATPase and zona occludens 1 (ZO-1) gene and protein expression were analyzed 3days after cells had formed a confluent monolayer. The cell circularity on all patterns and coatings was above 0.78. On all coatings, cell area was the lowest on micropillars. The coefficient of variation (CV) of the cell area was the lowest on nanopillars with an LC coating. With an FC coating, micropillars induced a better cellular outcome as the cells had the greatest circularity, smallest cell area and highest Na(+)/K(+)-ATPase and ZO-1 gene and protein expression. With the LC coating, HCECs grown on nanopillars resulted in the lowest CV of the cell area and the highest ZO-1 gene expression. Thus, HCEC-B4G12 morphometry and phenotype can be improved using different topographical and biochemical cues.

Non-adsorbing macromolecules promote endothelial adhesion of erythrocytes with reduced sialic acids.

BACKGROUND: Abnormal adhesion of red blood cells (RBCs) to vascular endothelium is often associated with reduced levels of sialic acids on RBC membranes and with elevated levels of pro-adhesive plasma proteins. However, the synergistic effects of these two factors on the adhesion are not clear. In this work, we tested the hypothesis that macromolecular depletion interaction originating from non-adsorbing macromolecules can promote the adhesion of RBCs with reduced sialic acid content to the endothelium. METHODS: RBCs are treated with neuraminidase to specifically remove sialic acids from their surface followed by the evaluation of their deformability, zeta potential and membrane proteins. The adhesion of these enzyme-treated RBCs to cultured human umbilical vein endothelial cells (ECs) is studied in the presence of 70 or 500kDa dextran with a flow chamber assay. RESULTS: Our results demonstrate that removal of sialic acids from RBC surface can induce erythrocyte adhesion to endothelial cells and that such adhesion is significantly enhanced in the presence of high-molecular weight dextran. The adhesion-promoting effect of dextran exhibits a strong dependence on dextran concentration and molecular mass, and it is concluded to originate from macromolecular depletion interaction. CONCLUSION: These results suggest that elevated levels of non-adsorbing macromolecules in plasma might play a significant role in promoting endothelial adhesion of erythrocytes with reduced sialic acids. GENERAL SIGNIFICANCE: Our findings should therefore be of great value in understanding abnormal RBC-EC interactions in pathophysiological conditions (e.g., sickle cell disease and diabetes) and after blood transfusions.

Poloxamer 188 reduces normal and phosphatidylserine-exposing erythrocyte adhesion to endothelial cells in dextran solutions.

Abnormal red blood cell (RBC) adhesion to endothelial cells (ECs) has been correlated with vascular complications in diseases such as sickle cell anemia and diabetes. Poloxamer 188 (P188) has been clinically tested to treat vaso-occlusion. However, the underlying mechanism(s) have not been clarified, making a methodical application difficult. In this study, we investigate how and to what extent P188 reduces RBC adhesion to ECs in plasma-like solutions. RBC adhesion to ECs is studied in solutions containing dextran, which is known to induce adhesion via macromolecular depletion interaction. It is demonstrated that P188 itself does not induce adhesion of normal RBCs to ECs but significantly reduces the adhesion in solutions containing high molecular mass-dextran. In addition, it is shown that P188 can reduce the adhesion of RBCs with enhanced exposure of phosphatidylserine (PS). Measurements of the electrophoretic mobility indicate that P188 increases the local viscosity inside the electric double layer of RBCs. Based on these results this study suggests that P188 reduces macromolecular depletion interaction, via penetrating into the depletion layer. Taking into consideration that dextran mimics the effects of pro-adhesive non-adsorbing plasma proteins and macromolecules, our study therefore suggests a mechanism for the adhesion reducing effect of P188 and should thus be of potential value for a detailed understanding of how cell-cell interactions in pathological conditions can be reduced.

Functional reconstruction of corneal endothelium using nanotopography for tissue-engineering applications.

Dysfunction in the corneal endothelium, which controls the hydration and transparency of the cornea, is one of the common reasons for transplantation. A tissue-engineered corneal endothelium is of interest for corneal regeneration and for in vitro testing of ocular drugs. In the native environment, corneal endothelial cells interact with the nanotopography of the underlying Descemet's membrane. This study showed that nanotopography enhanced bovine corneal endothelial cell (BCEC) responses, creating a monolayer which resembled the healthy corneal endothelium. Topographies of different geometries were first tested to identify those that would elicit the most significant responses. A BCEC monolayer was then generated on both micro- and nanoscale pillars and wells. The BCEC monolayer cultured on topographies exhibited polygonal geometries with well-developed tight junction proteins. Scanning electron microscopy revealed that cells on pillars showed a higher density of microvilli, which was similar to native corneal endothelium. BCECs on nanopillars displayed a lower coefficient of variation of area (0.31) that was within the range of healthy corneal endothelium. More importantly, a BCEC monolayer cultured on nanopillars also had an enhanced Na(+)/K(+)-ATPase immunofluorescence expression, mRNA upregulation and a higher Na(+)/K(+)-ATPase activity. These results suggest that nanopillar substrate topography may provide relevant topographical cues, which could significantly enhance the formation and function of corneal endothelium.

Non-adsorbing macromolecules in plasma induce erythrocyte adhesion to the endothelium.

Red blood cell (RBC) adhesion to the endothelium is usually insignificant. However, an enhanced adhesion can be observed in various pathological conditions such as diabetes mellitus or sickle cell disease, which is often accompanied by elevated levels of pro-adhesive plasma proteins such as fibrinogen. In the past, these proteins have only been considered to act as ligands, cross-linking the corresponding receptors on adjacent cells, but the detailed underlying mechanism often remained obscure. This work demonstrates that the presence of non-adsorbing polymers in plasma can also enhance the adhesion efficiency of RBCs to endothelial cells (ECs) through depletion interaction. Furthermore, adhesion of RBCs to ECs may be likewise promoted by the protein fibrinogen through depletion interaction. We propose an alternative mechanism for the pro-adhesive effects of plasma proteins and indicate that depletion interaction might play a significant role for the stabilization and destabilization of blood flow in health and disease.

Macromolecular depletion modulates the binding of red blood cells to activated endothelial cells.

Adhesion of red blood cells (RBCs) to endothelial cells (ECs) is usually insignificant but an enhanced adhesion has been observed in various diseases associated with vascular complications. This abnormal adhesion under pathological conditions such as sickle cell disease has been correlated with increased levels of various plasma proteins but the detailed underlying mechanism(s) remains unclear. Usually it is assumed that the proadhesive effects of plasma proteins originate from ligand interactions cross-linking receptors on adjacent cells, but explicit results detailing binding sites or receptors for some proteins (e.g., fibrinogen) on either RBC or EC surfaces that would support this model are missing. In this study, the authors tested whether there is an alternative mechanism. Their results demonstrate that dextran 2 MDa promotes the adhesion of normal RBCs to thrombin-activated ECs and that this effect becomes more pronounced with increasing thrombin concentration or with prolonged thrombin incubation time. It is concluded that depletion interaction originating from nonadsorbing macromolecules (i.e., dextran) can modulate the adhesion of red blood cells to thrombin-activated EC. This study thereby suggests macromolecular depletion as an alternative mechanism for the adhesion-promoting effects of nonadsorbing plasma proteins. These findings should not only aid in getting a better understanding of diseases associated with vascular complications but should also have many potential applications in biomedical or biotechnological areas that require the control of cell-cell or cell surface interactions.

Specific binding of red blood cells to endothelial cells is regulated by nonadsorbing macromolecules.

Abnormal adhesion of red blood cells to the endothelium has been linked to the pathophysiology of several diseases associated with vascular disorders. Various biochemical changes, including phosphatidylserine exposure on the outer membrane of red blood cells as well as plasma protein levels, have been identified as being likely to play a key role, but the detailed interplay between plasma factors and cellular factors remains unknown. It has been proposed that the adhesion-promoting effect of plasma proteins originates from ligand interaction, but evidence substantiating this assumption is often missing. In this work, we identified an alternative pathway by demonstrating that nonadsorbing macromolecules can also have a marked impact on the adhesion efficiency of red blood cells with enhanced phosphatidylserine exposure to endothelial cells. It is concluded that this adhesion-promoting effect originates from macromolecular depletion interaction and thereby presents an alternative mechanism by which plasma proteins could regulate cell-cell interactions. These findings should thus be of potential value for a detailed understanding of the pathophysiology of diseases associated with vascular complications and might be applicable to a wide range of cell-cell interactions in plasma or plasma-like media.