In silico and in vivo models for Qatari‐specific classical homocystinuria as basis for development of novel therapies

Homocystinuria is a rare inborn error of methionine metabolism caused by cystathionine β‐synthase (CBS) deficiency. The prevalence of homocystinuria in Qatar is 1:1,800 births, mainly due to a founder Qatari missense mutation, c.1006C>T; p.R336C (p.Arg336Cys). We characterized the structure–function relationship of the p.R336C‐mutant protein and investigated the effect of different chemical chaperones to restore p.R336C‐CBS activity using three models: in silico, ΔCBS yeast, and CRISPR/Cas9 p.R336C knock‐in HEK293T and HepG2 cell lines. Protein modeling suggested that the p.R336C induces severe conformational and structural changes, perhaps influencing CBS activity. Wild‐type CBS, but not the p.R336C mutant, was able to restore the yeast growth in ΔCBS‐deficient yeast in a complementation assay. The p.R336C knock‐in HEK293T and HepG2 cells decreased the level of CBS expression and reduced its structural stability; however, treatment of the p.R336C knock‐in HEK293T cells with betaine, a chemical chaperone, restored the stability and tetrameric conformation of CBS, but not its activity. Collectively, these results indicate that the p.R336C mutation has a deleterious effect on CBS structure, stability, and activity, and using the chemical chaperones approach for treatment could be ineffective in restoring p.R336C CBS activity.     

Contrasting behaviors of mutant cystathionine beta-synthase enzymes associated with pyridoxine response

Cystathionine beta-synthase (CBS) deficiency is a recessive genetic disorder characterized by extremely elevated levels in plasma homocysteine. Patients homozygous for the I278T or R266K mutations respond clinically to pharmacologic doses of pyridoxine, the precursor of a cofactor for the enzyme, 5'-pyridoxal phosphate (PLP). Here we test the hypothesis that these mutations are pyridoxine responsive because they lower the affinity of the enzyme for PLP. We show that recombinant R266K has 30 to 100% of the specific activity of the wild-type enzyme, while I278T only has only 1 to 5% activity. Kinetic studies show that the decreased activity in both enzymes is due to reduced turnover rate and not substrate binding. Neither I278T nor R266K appear to greatly affect multimer status of the enzyme. The R266K enzyme has reduced affinity for PLP compared to the wild-type enzyme, providing a mechanism for the pyridoxine response observed in patients. Surprisingly, the I278T enzyme does not have altered affinity for PLP. To confirm that this was not an in vitro artifact, we examined pyridoxine response in mice that stably express human I278T as their sole source of CBS activity. These mice have extremely elevated plasma homocysteine levels and do not respond significantly to large doses of pyridoxine. Our findings suggest that there may be multiple mechanisms involved in response to pyridoxine.     

p62 Deficiency Enhances α‐Synuclein Pathology in Mice

In Lewy body disease (LBD) such as dementia with LBs and Parkinson's disease, several lines of evidence show that disrupted proteolysis occurs. p62/SQSTM1 (p62) is highly involved with intracellular proteolysis and is a component of ubiquitin‐positive inclusions in various neurodegenerative disorders. However, it is not clear whether p62 deficiency affects inclusion formation and abnormal protein accumulation. To answer this question, we used a mouse model of LBD that lacks p62, and found that LB‐like inclusions were observed in transgenic mice that overexpressed α‐synuclein (Tg mice) with or without the p62 protein. p62 deficiency enhanced α‐synuclein pathology with regard to the number of inclusions and staining intensity compared with Tg mice that expressed p62. To further investigate the molecular mechanisms associated with the loss of p62 in Tg mice, we assessed the mRNA and protein levels of several molecules, and found that the neighbor of the brca1 gene (NBr1), which is functionally and structurally similar to p62, is increased in Tg mice without p62 compared with control Tg mice. These findings suggest that p62 and NBR1 affect the pathogenesis of neurodegenerative diseases through the cooperative modulation of α‐synuclein aggregation.     

Control of Schistosoma mansoni egg‐induced inflammation by IL‐4‐responsive CD4+CD25−CD103+Foxp3− cells is IL‐10‐dependent

Host protection to helminth infection requires IL‐4 receptor α chain (IL‐4Rα) signalling and the establishment of finely regulated Th2 responses. In the current study, the role of IL‐4Rα‐responsive T cells in Schistosoma mansoni egg‐induced inflammation was investigated. Egg‐induced inflammation in IL‐4Rα‐responsive BALB/c mice was accompanied with Th2‐biased responses, whereas T‐cell‐specific IL‐4Rα‐deficient BALB/c mice (iLck^creIl4ra^?/lox) developed Th1‐biased responses with heightened inflammation. The proportion of Foxp3⁺ Treg in the draining LN of control mice did not correlate with the control of inflammation and was reduced in comparison to T‐cell‐specific IL‐4Rα‐deficient mice. This was due to IL‐4‐mediated inhibition of CD4⁺Foxp3⁺ Treg conversion, demonstrated in adoptively transferred Rag2^?/? mice. Interestingly, reduced footpad swelling in Il4ra^?/lox mice was associated with the induction of IL‐4 and IL‐10‐secreting CD4⁺CD25^?CD103⁺Foxp3^? cells, confirmed in S. mansoni infection studies. Transfer of IL‐4Rα‐responsive CD4⁺CD25^?CD103⁺ cells, but not CD4⁺CD25^high or CD4⁺CD25^?CD103^? cells, controlled inflammation in iLck^creIl4ra^?/lox mice. The control of inflammation depended on IL‐10, as transferred CD4⁺CD25^?CD103⁺ cells from IL‐10‐deficient mice were not able to effectively downregulate inflammation. Together, these results demonstrate that IL‐4 signalling in T cells inhibits Foxp3⁺ Treg in vivo and promotes CD4⁺CD25^?CD103⁺Foxp3^? cells that control S. mansoni egg‐induced inflammation via IL‐10.     

Disposition of homocysteine in subjects heterozygous for homocystinuria due to cystathionine beta-synthase deficiency: relationship between genotype and phenotype

We have investigated 31 subjects from five unrelated families with one or more members with cystathionine beta-synthase (CBS) deficiency. On the basis of their CBS genotype, the subjects were grouped as normal (n = 11) or heterozygotes (n = 20). Based on pyridoxine effect in the probands, the heterozygotes were further classified as pyridoxine-responsive (n = 9) or non-responsive (n = 11). Heterozygous subjects had normal fasting total plasma homocysteine (tHcy), but median urinary tHcy excretion rate was significantly elevated compared to healthy controls (0.39 micromol/h vs 0.24 micromol/h, P < 0.05). An abnormal tHcy response after methionine loading identified 73% of the pyridoxine non-responsive heterozygotes, but only 33% of the pyridoxine responsive participants. The increase in cystathionine or the change in tHcy relative to cystathionine did not improve diagnostic accuracy of the methionine loading test. After Hcy loading, the maximal increase in tHcy was significantly elevated, whereas t(1/2) was normal in heterozygotes. In conclusion, a single biochemical test cannot discriminate CBS heterozygotes from controls. Abnormal tHcy response after methionine loading was the most sensitive test. Our data suggest that the urinary tHcy excretion rate is a simple, non-invasive approach for studying mild disturbances in Hcy metabolism.     

γδ T cells are indispensable for interleukin‐23‐mediated protection against Concanavalin A‐induced hepatitis in hepatitis B virus transgenic mice

Hepatitis B virus surface antigen (HBsAg) carriers are highly susceptible to liver injury triggered by environmental biochemical stimulation. Previously, we have reported an inverse correlation between γδ T cells and liver damage in patients with hepatitis B virus (HBV). However, whether γδ T cells play a role in regulating the hypersensitivity of HBsAg carriers to biochemical stimulation‐induced hepatitis is unknown. In this study, using HBV transgenic (HBs‐Tg) and HBs‐Tg T‐cell receptor‐δ‐deficient (TCR‐δ^−/−) mice, we found that mice genetically deficient in γδ T cells exhibited more severe liver damage upon Concanavalin A (Con A) treatment, as indicated by substantially higher serum alanine aminotransferase levels, further elevated interferon‐γ (IFN‐γ) levels and more extensive necrosis. γδ T‐cell deficiency resulted in elevated IFN‐γ in CD4⁺ T cells but not in natural killer or natural killer T cells. The depletion of CD4⁺ T cells and neutralization of IFN‐γ reduced liver damage in HBs‐Tg and HBs‐Tg‐TCR‐δ^−/− mice to a similar extent. Further investigation revealed that HBs‐Tg mice showed an enhanced interleukin‐17 (IL‐17) signature. The administration of exogenous IL‐23 enhanced IL‐17A production from Vγ4 γδ T cells and ameliorated liver damage in HBs‐Tg mice, but not in HBs‐Tg‐TCR‐δ^−/− mice. In summary, our results demonstrated that γδ T cells played a protective role in restraining Con A‐induced hepatitis by inhibiting IFN‐γ production from CD4⁺ T cells and are indispensable for IL‐23‐mediated protection against Con A‐induced hepatitis in HBs‐Tg mice. These results provided a potential therapeutic approach for treating the hypersensitivity of HBV carriers to biochemical stimulation‐induced liver damage.     

Targeted inhibition of IL‐10‐secreting CD25− Treg via p38 MAPK suppression in cancer immunotherapy

Cancer‐induced immunotolerance mediated by inducible Treg (iTreg) is a major obstacle to cancer immunotherapy. In a basic study of immunotolerance, injection of an endogenous superantigen, i.e. the minor lymphocyte stimulatory (Mls)‐1^a, into specific TCR Vβ8.1‐Tg mice enabled generation of anergic CD25⁻ iTreg, the immunosuppressive function of which was maintained by IL‐10 production via p38‐MAPK activation. Interestingly, although p38‐chemical inhibitor (p38‐inhibitor) is capable of breaking CD25⁻ iTreg‐induced immunotolerance, the p38‐inhibitor had hardly any immunotolerance breaking effect when CD25⁺ Treg were present, suggesting that depletion of CD25⁺ Treg is necessary for p38‐inhibitor to be effective. Peptide OVA_323–339 iv.‐injection into its specific TCR‐Tg (OT‐II) mice also induced adaptive tolerance by iTreg. Peptide immunotherapy with p38‐inhibitor after CD25⁺ Treg‐depletion was performed in an OVA‐expressing lymphoma E.G7‐bearing tolerant model established by adoptive transfer of OT‐II CD25⁻ iTreg, which resulted in suppression of tumor growth. Similarly, the antitumor immunity induced by peptide immunotherapy in colon carcinoma CT26‐bearing mice, in which the number of IL‐10‐secreting iTreg is increased, was augmented by treatment with p38‐inhibitor after CD25⁺ Treg‐depletion and resulted in inhibition of tumor progression. These results suggest that simultaneous inhibition of two distinct Treg‐functions may be important to the success of cancer immunotherapy.     

Targeting SIRP‐α protects from type 2‐driven allergic airway inflammation

The interplay between innate and adaptive immune responses is essential for the establishment of allergic diseases. CD47 and its receptor, signal regulatory protein α (SIRP‐α), govern innate cell trafficking. We previously reported that administration of CD47^+/+ but not CD47^−/− SIRP‐α⁺ BM‐derived DC (BMDC) induced airway inflammation and Th2 responses in otherwise resistant CD47‐deficient mice. We show here that early administration of a CD47‐Fc fusion molecule suppressed the accumulation of SIRP‐α⁺ DC in mediastinal LN, the development of systemic and local Th2 responses as well as airway inflammation in sensitized and challenged BALB/c mice. Mechanistic studies highlighted that SIRP‐α ligation by CD47‐Fc on BMDC did not impair Ag uptake, Ag presentation and Ag‐specific DO11.10 Tg Th2 priming and effector function in vitro, whereas in vivo administration of CD47‐Fc or CD47‐Fc‐pretreated BMDC inhibited Tg T‐cell proliferation, pinpointing that altered DC trafficking accounts for defective Th priming. We conclude that the CD47/SIRP‐α axis may be harnessed in vivo to suppress airway SIRP‐α⁺ DC homing to mediastinal LN, Th2 responses and allergic airway inflammation.     

Antigenic strength controls the generation of antigen‐specific IL‐10‐secreting T regulatory cells

Administration of peptides i.n. induces peripheral tolerance in Tg4 myelin basic protein‐specific TCR‐Tg mice. This is characterized by the generation of anergic, IL‐10‐secreting CD4⁺ T cells with regulatory function (IL‐10 Treg). Myelin basic protein Ac1–9 peptide analogs, displaying a hierarchy of affinities for H‐2 A^u (Ac1–9[4K]<<[4A]<[4Y]), were used to investigate the mechanisms of tolerance induction, focusing on IL‐10 Treg generation. Repeated i.n. administration of the highest affinity peptide, Ac1–9[4Y], provided complete protection against EAE, while i.n. Ac1–9[4A] and Ac1–9[4K] treatment resulted in only partial protection. Ac1–9[4Y] was also the most potent stimulus for IL‐10 Treg generation. Although i.n. treatment with Ac1–9[4A] gave rise to IL‐10‐secreting CD4⁺ T cells, the population as a whole was also capable of secreting IFN‐γ after an in vitro recall response to Ac1–9[4A] or [4Y]. In addition to IL‐10 production, other facets of tolerance, namely, anergy and suppression (both in vitro and in vivo), were affinity dependent, with i.n. Ac1–9[4Y]‐, [4A]‐ or [4K]‐treated CD4⁺ T cells being the most, intermediate and least anergic/suppressive, respectively. These findings demonstrate that the generation of IL‐10 Treg in vivo is driven by high signal strength.     

Antigen dose‐dependent suppression of murine IgE responses is mediated by CD4−CD8− double‐negative T cells

Background The IgE response against protein antigens is profoundly influenced by the dose used for sensitization. Objective The aim of the study was to identify immune cells that are involved in antigen dose‐dependent regulation of IgE formation. Methods Wild‐type mice as well as T helper (Th)1‐deficient IL‐12p40^?/? and IFN‐γ^?/? mice were immunized by repeated intraperitoneal injection of either low doses (K01 mice) or high doses (K100 mice) of keyhole limpet haemocyanin adsorbed to aluminium hydroxide. Splenocytes of immunized mice were restimulated in vitro and antigen‐dependent T cell proliferation and cytokine production were measured. The frequency of regulatory T cell subsets among splenocytes from K01 and K100 mice was compared using fluorocytometry and RT‐PCR analysis. Splenocytes or T cell subpopulations were transferred into naïve mice and the effect of lymphocyte transfer on IgE production after priming of recipients with low antigen doses was determined. Results Specific IgE production was considerably impaired in K100 mice. Antigenic restimulation revealed hypoproliferation of K100 splenocytes and reduced production of Th2 cytokines IL‐4, IL‐5 and IL‐13, but no induction of IFN‐γ production. Moreover, lymphocytes from K01 and K100 mice did not show significant differences in the expression of molecules associated with the phenotype or activity of conventional regulatory T cells. Transfer of splenocytes or purified T cells from K100 mice substantially suppressed the induction of IgE production in the recipients in an antigen‐ and isotype‐specific manner. Neither CD4⁺ nor CD8⁺ T cells from K100 mice were able to inhibit IgE formation; instead, we identified CD4^?CD8^? double‐negative T cells (dnT cells) as the principal T cell population, which potently suppressed IgE production. Conclusion Our data demonstrate that CD4^?CD8^? dnT cells play a major role in the regulation of IgE responses induced by high antigen doses.     

Diffusion MRI detects early brain microstructure abnormalities in 2‐month‐old 3×Tg‐AD mice

The 3×Tg‐AD mouse is one of the most studied animal models of Alzheimer's disease (AD), and develops both amyloid beta deposits and neurofibrillary tangles in a temporal and spatial pattern that is similar to human AD pathology. Additionally, abnormal myelination patterns with changes in oligodendrocyte and myelin marker expression are reported to be an early pathological feature in this model. Only few diffusion MRI (dMRI) studies have investigated white matter abnormalities in 3×Tg‐AD mice, with inconsistent results. Thus, the goal of this study was to investigate the sensitivity of dMRI to capture brain microstructural alterations in 2‐month‐old 3×Tg‐AD mice. In the fimbria, the fractional anisotropy (FA), kurtosis fractional anisotropy (KFA), and radial kurtosis (K_┴) were found to be significantly lower in 3×Tg‐AD mice than in controls, while the mean diffusivity (MD) and radial diffusivity (D_┴) were found to be elevated. In the fornix, K_┴ was lower for 3×Tg‐AD mice; in the dorsal hippocampus MD and D_┴ were elevated, as were FA, MD, and D_┴ in the ventral hippocampus. These results indicate, for the first time, dMRI changes associated with myelin abnormalities in young 3×Tg‐AD mice, before they develop AD pathology. Morphological quantification of myelin basic protein immunoreactivity in the fimbria was significantly lower in the 3×Tg‐AD mice compared with the age‐matched controls. Our results demonstrate that dMRI is able to detect widespread, significant early brain morphological abnormalities in 2‐month‐old 3×Tg‐AD mice.     

The development of atopic dermatitis is independent of Immunoglobulin E up-regulation in the K14-IL-4 SKH1 transgenic mouse model

Background We have successfully generated an IgE‐associated (extrinsic/allergic) mouse model of atopic dermatitis in K14‐IL‐4‐Tg/CByB6 mice. The newly described subset of non‐IgE‐associated (intrinsic/non‐allergic) atopic dermatitis in human patients raises the question on the role of IgE in the pathogenesis. Objective The aim of this study was to develop a non‐IgE‐associated atopic dermatitis model in K14‐IL‐4‐Tg/SKH1 mice. Methods K14‐IL‐4‐Tg/CByB6 mice were crossed with SKH1 mice to produce K14‐IL‐4‐Tg/SKH1 mice. Phenotypes of clinical and histological, cytokine expression in the skin lesions, and total serum IgE in K14‐IL‐4‐Tg/CByB6 and K14‐IL‐4‐Tg/SKH1 mice were compared. The CD40 and CD40L on T and B cells were also studied to differentiate their roles in IgE production. Results K14‐IL‐4‐Tg/SKH1mice had a normal total serum IgE level and manifested a chronic inflammatory skin phenotype identical to that of K14‐IL‐4‐Tg/CByB6 IgE‐mediated mice in clinical morphology, histology, infiltration of mononuclear cells/eosinophils/mast cells, mast cell degranulation, and up‐regulation of chronic lesional cytokine mRNA expression of IL‐1β, IL‐3, IL‐4, IL‐6, IL‐10, IL‐12, IL‐13, IFN‐γ, TNF‐α, and TNF‐β. We also found that the inability of CD4⁺ T cells of the K14‐IL‐4‐Tg/SKH1mice to up‐regulate CD40L expression upon stimulation might account for their inability to up‐regulate the IgE level. B cell abnormality was ruled out as CD19⁺ B cells of K14‐IL‐4‐Tg/SKH1 mice synthesized the same amount of IgE in vitro compared with K14‐IL‐4‐Tg/CByB6 mice in the presence of IL‐4 and soluble CD40L. Our studies further suggested that the defect of early growth response‐1 in T cells might be responsible for the impaired CD40L up‐regulation in K14‐IL‐4‐Tg/SKH1 mice. Conclusion K14‐IL‐4‐Tg/SKH1 mice developed skin inflammation that resembled human intrinsic atopic dermatitis. Therefore, this model may be suitable to study the pathogenesis of intrinsic atopic dermatitis.     

Cytokines as genetic modifiers in K5–/– mice and in human epidermolysis bullosa simplex

Epidermolysis bullosa simplex (EBS) is a skin disorder caused by fully‐penetrant mutations in the keratin genes KRT5 and KRT14, leading to extensive cytolysis and cell fragility of basal keratinocytes. EBS is subject to environmental conditions and displays high intra‐ and interfamilial variability, suggesting modifying loci. Here, we demonstrate that upregulation of certain cytokines accompanies mutations in keratin 5 (K5) but not in keratin 14 (K14). We find for the first time that cytokines macrophage chemotactic protein (MCP)‐1/[chemokine (C‐C motif) ligand 2] (CCL2), macrophage inflammatory protein (MIP)‐3β/CCL19 and MIP‐3α/CCL20, all regulated by nuclear factor kappa B (NFκB) and involved in the recruitment, maturation, and migration of Langerhans cells (LCs) in the epidermis, are upregulated in the skin of K5^–/–, but not of K14^–/– mice. In neonatal K5^–/– epidermis, the number of LCs was increased two‐fold. At the same time, tumor necrosis factor alpha (TNFα) remained unaltered, demonstrating the specificity of that process. Most remarkably, enhanced LC recruitment within the epidermis was found in five EBS patients carrying mutations in the KRT5 gene but not in EBS patients with KRT14 gene mutations. In agreement with the NFκB‐dependent regulation of these cytokines, we found a decrease in p120‐catenin in the basal epidermis of K5^–/– mice. These data provide the first explanation for distinct, keratin‐type‐specific genotype–phenotype correlations in EBS and represent a rationale to investigate gene loci affecting skin pathology in EBS. Hum Mutat 0, 1–10, 2009. © 2009 Wiley‐Liss, Inc.     

A novel pathogenic peptide of thyroglobulin (2208–2227) induces autoreactive T‐cell and B‐cell responses in both high and low responder mouse strains

Experimental autoimmune thyroiditis (EAT) is commonly induced by thyroglobulin (Tg) or Tg peptides in mice genetically susceptible to thyroiditis. In the present study, we investigated the immunogenic and pathogenic potential of a novel 20mer human Tg peptide, p2208 (amino acids 2208–2227), in mouse strains classified as low (LR) or high (HR) responders in EAT. The peptide was selected for its content in overlapping binding motifs for MHC class II products, associated with either resistance (A^b), or susceptibility (A^s, E^k) to EAT. We therefore immunized LR BALB/c (H‐2^d) and C57BL/6 (H‐2^b) strains, as well as HR CBA/J (H‐2^k) and SJL/J (H‐2^s) mice with 100 nmol of p2208 in adjuvant and collected their sera, lymph nodes and thyroid glands for further analysis. The p2208 peptide was found to contain B‐cell and cryptic T‐cell epitope(s) in two of the four strains examined, one LR and one HR. Specifically, it elicited direct EAT in C57BL/6 mice (two of seven mice, infiltration index 1–3), as well as in SJL/J mice (two of six mice, infiltration index 1–2). Such an EAT model could provide insights into the immunoregulatory cascades taking place in resistant hosts.     

Relation between Plasma Homocysteine Concentration, the 844ins68 Variant of the Cystathionine β-Synthase Gene, and Pyridoxal-5′-Phosphate Concentration

A moderately elevated plasma total homocysteine (tHcy), whether measured during fasting or post-methionine load (PML), is recognized as a risk factor for coronary artery diseases (CAD). Cystathionine β-synthase (CBS), a key enzyme in the transsulfuration pathway, is important for the metabolism of homocysteine. In recent years, a relatively prevalent mutation, the 844ins68 (68-bp insertion), was found to be carried by about 12% of the general population. In the current investigation, we studied 741 individuals with respect to the effect of the 68-bp insertion of the CBS gene on fasting and PML tHcy, and also determined the level of pyridoxal-5′-phosphate (vitamin B₆), a cofactor of the CBS enzyme. Our results showed that the mean fasting and PML increase in tHcy levels were lower in individuals carrying the 844ins68 variant compared to those without the insertion; although only the difference in PML increase in tHcy reached statistical significance (P = 0.02). When these individuals were divided into two groups based on vitamin B₆ concentration, the PML increase in tHcy was significantly lower in individuals heterozygous for the insertion compared to those without the insertion only in the group of individuals whose vitamin B₆ concentrations were below the sample median (38.0 nmol/L). We speculate that the 68-bp insertion is associated with somewhat higher levels of CBS enzyme activity, and that the effect of this becomes more pronounced in the presence of relatively low concentrations of pyridoxal-5′-phosphate, a cofactor of the CBS enzyme.     

Flightless I over‐expression impairs skin barrier development,function and recovery following skin blistering

Development of an intact epidermis is critical for maintaining the integrity of the skin. Patients with epidermolysis bullosa (EB) experience multiple erosions, which breach the epidermal barrier and lead to increased microbial colocalization of wounds, infections and sepsis. The cytoskeletal protein Flightless I (Flii) is a known regulator of both development and wound healing. Using Flii^+/?, WT and Flii^Tg/Tg mice, we investigated the effect of altering Flii levels in embryos and adult mice on the development of the epidermal barrier and, consequently, how this affects the integrity of the skin in EB. Flii over‐expression resulted in delayed formation of the epidermal barrier in embryos and decreased expression of tight junction (TJ) proteins Claudin‐1 and ZO‐2. Increased intercellular space and transepidermal water loss was observed in Flii^Tg/Tg adult mouse skin, while Flii^Tg/Tg keratinocytes showed altered TJ protein localization and reduced transepithelial resistance. Flii is increased in the blistered skin of patients with EB, and over‐expression of Flii in experimental EBA showed impaired Claudin‐1 and ‐4 TJ protein expression and delayed recovery of functional barrier post‐blistering. Immunoprecipitation confirmed Flii associated with TJ proteins and in vivo actin assays showed that the effect of Flii on actin polymerization underpinned the impaired barrier function observed in Flii^Tg/Tg mice. These results therefore demonstrate an important role for Flii in the development and regulation of the epidermal barrier, which may contribute to the impaired healing and skin fragility of EB patients. Copyright © 2013 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd     

A yeast assay for functional detection of mutations in the human cystathionine {beta}-synthase gene

Mutations in the human cystathionine ß-synthase (CBS)gene are known to cause homocystinuria and may also be a significantrisk factor for premature atherosclerosis. We have previouslyshown that the human CBS protein can substitute for the endogenousyeast CBS protein in Saccharomyces cerevisiae. We now show thatexpression of three different CBS mutants known to be associatedwith reduced enzyme activity in humans fail to complement growthin the yeast assay. in addition, we have used the yeast CBSassay to identity eight mutant CBS alleles in cell lines frompatients with CBS deficiency. These mutant alleles include twopreviously identified and five novel CBS mutations. Our resultsalso demonstrate that the yeast CBS assay can detect a largepercentage of individuals heterozygous for mutations in CBS.This system should be useful in determining the relationshipbetween CBS mutations and human disease.