TGFBI Research Articles

Human Phenotypically Distinct TGFBI Corneal Dystrophies Are Linked to the Stability of the Fourth FAS1 Domain of TGFBIp

Mutations in the human TGFBI gene encoding TGFBIp have been linked to protein deposits in the cornea leading to visual impairment. The protein consists of an N-terminal Cys-rich EMI domain and four consecutive fasciclin 1 (FAS1) domains. We have compared the stabilities of wild-type (WT) human TGFBIp and six mutants known to produce phenotypically distinct deposits in the cornea. Amino acid substitutions in the first FAS1 (FAS1-1) domain (R124H, R124L, and R124C) did not alter the stability. However, substitutions within the fourth FAS1 (FAS1-4) domain (A546T, R555Q, and R555W) affected the overall stability of intact TGFBIp revealing the following stability ranking R555W>WT>R555Q>A546T. Significantly, the stability ranking of the isolated FAS1-4 domains mirrored the behavior of the intact protein. In addition, it was linked to the aggregation propensity as the least stable mutant (A546T) forms amyloid fibrils while the more stable variants generate non-amyloid amorphous deposits in vivo. Significantly, the data suggested that both an increase and a decrease in the stability of FAS1-4 may unleash a disease mechanism. In contrast, amino acid substitutions in FAS1-1 did not affect the stability of the intact TGFBIp suggesting that molecular the mechanism of disease differs depending on the FAS1 domain carrying the mutation.

Genome-wide scan of granular corneal dystrophy, type II: confirmation of chromosome 5q31 and identification of new co-segregated loci on chromosome 3q26.3

Granular corneal dystrophy, type II (CGD2; Avellino corneal dystrophy) is the most common corneal dystrophy among Koreans, but its pathophysiology is still poorly understood. Many reports showed that even though the causative mutation is the same TGFBI R124H mutation, there are severe and mild phenotypes of the corneal dystrophy. We also observed the phenotype differences in our samples. For this reason, we focused our effort on the identification of unknown genetic factor related to phenotype variation. A total 551 individuals from 59 families were genotyped with SNP chip and used in genome-wide linkage analysis. From single-point linkage analyses, we confirmed the known 5q31 region for TGFBI gene, and selected novel nine candidate loci for CGD2. In simulation analysis, the only 3q26.3 region including neuroligin 1 gene (NLGN1) was supported by empirical statistic significance. To investigate the effect of genetic heterogeneity in linkage analysis, we classified CGD2 families into two subgroups. Although we could not find a significant evidence for correlation between the 3q26.3 region and CGD2 phenotypes, this first genome-wide analysis with CGD2 families in Korea has a very important value for offering insights in genetics of CGD2. In addition, the co-segregating loci with CGD2 including 3q26.3 would be a good target for further study to understand the pathophysiology of CGD2.

Late occurrence of granular dystrophy in bilateral keratoconus: Penetrating keratoplasty and long-term follow-up

We report a rare case of keratoconus with granular dystrophy with a follow-up of two decades, documenting the sequential presentation of two diseases confirmed by histology and genetic studies. A 13-year-old boy was diagnosed in 1988 with keratoconus in both eyes (BE) based on slit-lamp biomicroscopy findings of corneal ectasia in BE accompanied by Fleischer's ring, Vogt's striae, a small, old, healed hydrops. The left eye (LE) had central corneal thinning and scar in the central area involving the mid and posterior stroma secondary to healed hydrops. Penetrating keratoplasty (PKP) was advised. The boy was lost to follow-up till 1991 and presented with white, dot-like opacities in the central cornea in the RE only, suggestive of granular corneal dystrophy. Similar findings of white, dot-like opacities were noted in the LE in 1995 and the patient subsequently underwent PKP in BE. Histopathology of corneal buttons confirmed the presence of patchy, crystal-like orange deposits, which stained bright red with Masson's trichrome. Mutational analysis of the TGFBI gene in patient's DNA revealed a heterozygous mutation corresponding to a change in Arg555Trp in the keratoepithelin protein. Granular dystrophy recurred after 8 years in the RE.

A case of lattice corneal dystrophy due to L527R mutation in the TGFBI gene with asymmetric corneal opacity in eye laterality

A case of lattice corneal dystrophy due to L527R mutation in the TGFBI gene with asymmetric corneal opacity in eye laterality

Founder effect of two families with TGFBI related Thiel-Behnke corneal dystrophy

To investigate the transforming growth factor beta induced (TGFBI; BIGH3) gene mutation and founder effect of two large Chinese families clinically diagnosed as Thiel-Behnke corneal dystrophy. Fifteen members including 13 affected and 2 healthy in family A, 14 members including 6 affected and 8 healthy in family B, as well as 20 other unrelated healthy individuals were tested for TGFBI gene mutation. Haplotype analysis and clinical examination were also carried out in the two families. In exon 12 of the TGFBI gene, 1664G to A change was detected in all the patients, which leads to an amino acid replacement of arginine with glutamine (p.Arg555Gln). Members of the two families share some similar haplotypes. Genetic analysis is helpful in the diagnosis of corneal dystrophy. The two families may come from a same ancestor.

Novel mutation in the TGFBI gene in a patient with a late‐onset lattice corneal dystrophy

Abstract Purpose To report the clinical and molecular findings in a patient with late‐onset lattice corneal dystrophy and a novel Leu565Pro mutation in the TGFBI gene. Methods The diagnosis of lattice corneal dystrophy (LCD) was performed by visualization of characteristic lesions with slit‐lamp examination and by in vivo confocal microscopy. Mutation screening of coding regions of the TGFBI gene was done by PCR amplification and direct sequencing. Results We found a novel c.1694T>C heterozygous missense mutation in exon 13 of the TGFBI gene, which predicts the substitution of a leucine for a proline at codon 565 (Leu565Pro). Sequencing of all remaining exons of the TGFBI gene did not show any alterations. Conclusion The mutant codon 565 is located at the C‐terminus in the region corresponding to the fourth fascilin domain of the TGFBI protein. The novel variant adds to a number of previously reported mutations causing lattice corneal dystrophy and located in this region.

Clinical Findings and Treatments of Granular Corneal Dystrophy Type 2 (Avellino Corneal Dystrophy): A Review of the Literature

To review the literature about clinical findings and treatments of granular corneal dystrophy type 2 (GCD2). Various literatures on clinical findings, exacerbations after refractive corneal surgery, and treatment modalities of GCD2 were reviewed. GCD2 is an autosomal dominant disease. Mutation of transforming growth factor beta-induced gene, TGFBI, or keratoepithelin gene in human chromosome 5 (5q31) is the key pathogenic process in patient with GCD2. Corneal trauma activates TGFBI and then it overproduces transforming growth factor beta-induced gene protein (TGFBIp), which is main component of the corneal opacity. Refractive corneal surgery is a popular procedure to correct refractive error worldwide. However, several cases about exacerbation of GCD2 after corneal refractive surgery such as photorefractive keratectomy, laser in situ keratomileusis, and laser epithelial keratomileusis have been reported. The opacities deteriorate patient's best-corrected visual acuity. Recurrence-free interval varies many factors such as the type of procedure the patient had received and the genotype of the patient. To treat the opacities in GCD2, phototherapeutic keratectomy, lamellar keratoplasty, deep lamellar keratoplasty, and penetrating keratoplasty (PKP) were used. However, the recurrence is still an unsolved problem. Perfect treatment of exacerbation after corneal surface ablation does not exist until now. To prevent exacerbation, refractive surgeons must do a careful preoperative examination of candidates in refractive surgeries.

Genotype of Lattice Corneal Dystrophy (R124C Mutation in TGFBI) in a Patient Presenting With Features of Avellino Corneal Dystrophy

To present a patient with a genotype usually associated with lattice corneal dystrophy but with clinical and histopathologic features of advanced Avellino corneal dystrophy. Penetrating keratoplasty was performed with subsequent histopathologic analysis. For genetic testing, a 5-mL blood sample was taken after informed consent. Genetic sequencing was performed by the John and Marcia Carver Laboratory of the University of Iowa. The mutation was identified by direct sequencing through the positions of the coding sequences of the TGFBI gene that have been previously reported to have genetic variations (exons 4 and 11-14). Corneal examination revealed bilateral lattice and multiple confluent subepithelial and anterior stromal granular opacities. Histopathologic examination showed amyloid deposits by Congo red stain and hyaline deposits by Masson trichrome stain, consistent with a diagnosis of Avellino dystrophy. Automated DNA sequencing revealed a heterozygous Arg124Cys (R124C) mutation in the coding sequence of the TGFBI gene on chromosome 5q31. Recurrent granular deposits developed in the corneal graft 14 months after surgery. Our case presented with clinical and histopathologic findings consistent with a diagnosis of Avellino dystrophy and exhibited a genotype with R124C mutation. Avellino dystrophy has not previously been reported to be associated with the R124C mutation, which is usually associated with lattice corneal dystrophy. This also raises the issue as to whether classification of the corneal stromal dystrophies should be based primarily on phenotype/histopathology or on genotyping.

A variant TGFBI corneal dystrophy from G623D mutation with an unusual amyloidogenic phenotype

Abstract Purpose To present a unique corneal dystrophy never before described in a German family carrying the Gly623Asp mutation of the TGFBI gene with late clinical onset. Methods Clinical documentation and isolation of genomic DNA from peripheral blood leucocytes were obtained from each family member examined. Exons 3, 4, 5, and 11 to 14 of the TGFBI gene were sequenced. 5 corneal buttons of 3 affected siblings were excised at the time of penetrating keratoplasty. Light and electron microscopic examination were performed including immunohistochemistry with antibodies against Keratoepithelin (KE) 2 and 15. Results Specimens showed changes in Bowman's layer and the adjacent stroma. Congo red‐positive amyloid deposits were found within the epithelium in one cornea, in Bowman's layer and in the anterior stroma of all specimens, also showing KE2‐ but not KE15‐immunostaining. EM revealed deposits located in the anterior stroma and Bowman's layer and the basal area of some epithelial cells. These areas were strongly Alcian blue‐positive but negative in the Masson‐Trichrom‐stain. Only affected patients had a heterozygous missense mutation in exon 14 of the TGFBI gene (G‐>A transition at nucleotide 1915) with the change Gly623Asp in the keratoepithelin protein. Conclusion In contrast to the patient carrying the Gly623Asp mutation of the TGFBI gene described by Afshari et al., our cases presented with Salzmann nodular degeneration like clinical features with KE2 positive amyloid. The reason for this now "meeting the expectation histological phenotype" is unclear. The histological findings emphasize that this is a unique corneal dystrophy which shares no clinical characteristics with Reis‐Bücklers dystrophy and should be treated as a distinct entity.

How to tell dystrophies of Bowman's layer (Reis‐Bücklers and Thiel‐Behnke) from one another

Abstract Purpose To discuss difficulties encountered in differentiating two classic dystrophies of the Bowman layer caused by mutations of the TGFBI gene: Thiel–Behnke corneal dystrophy (TBCD) and Reis–Bücklers corneal dystrophy (RBCD). Methods Critical review of published data by the IC3D group and personal experience of the author as an ophthalmic pathologist and clinician. Results It has traditionally been difficult to recognise and distinguish TBCD and RBCD, so much so that some early papers claimed to describe the ultrastructure of RBCD actually analysed tissue from patients with TBCD; the known entity of RBCD was described as an unusual variant of granular corneal dystrophy; and Waardenburg‐Jonkers dystrophy appeared for eyars in textbooks although their patients had TBCD. The main problem is that both dystrophies are fairly rare and clinicians seldom have personal prior experience or adequate literature knowledge of both when they encounter a patient; and the literature with its frequent errors in diagnoses has not been that helpful. Conclusion TBCD and RBCD may be differentiated by careful clinical and ophthalmic pathologic examination if the clinician and pathologist are experienced; genetic or electron microscopic examination is needed for unequivocal confirmation.

Analysis of TGFBI gene mutation in a Chinese family with atypical Reis-Buckler corneal dystrophy

To identify the TGFBI gene mutation and the relationship between genotype and phenotype of a Chinese family with atypical Reis-Buckler corneal dystrophy (RBCD). Four patients, two non-carrier relatives of the family were enrolled in the present study. In addition to ophthalmologic examinations, PCR amplification and DNA sequencing of exons 4, 11, 12, and 14 of the TGFBI gene were carried out. Exon 14 was also sequenced in 100 healthy controls. A G to A transition at codon 623 in all affected members was identified. This mutation resulted in a substitution of glycine (GGC) to aspartic acid (GAC) at the protein level.None of the healthy family members, or any of the 100 control subjects carried this mutation. The G623D mutation of the TGFBI gene caused an atypical Reis-Buckler corneal dystrophy in this family. This mutation is reported in Chinese for the first time.

Clinical and genetic features of TGFBI-linked corneal dystrophies in Mexican population: Description of novel mutations and novel genotype–phenotype correlations

Corneal dystrophies (CDS) are inherited disorders characterized by an altered corneal transparency and refractive index which may be caused by a progressive accumulation of deposits within the different corneal layers. Most CDs are inherited in an autosomal dominant fashion and mutations in the TGFBI gene at chromosome 5q31 cause the majority of CDs affecting the stromal layer. A genotype–phenotype correlation has been identified in most analyzed populations as specific amino acid changes in TGFBI protein cause specific stromal phenotypes. However, analysis of additional populations will help to broaden the mutational spectrum ultimately allowing a better clinical–molecular classification of patients with this group of diseases. In this work, eighteen unrelated Mexican probands suffering from stromal CDs were clinically assessed and their TGFBI gene status investigated. Complete ophthalmologic evaluation, including biomicroscopic inspection and dilated fundus examination, was performed. In addition, detailed genealogical analyses as well as automated DNA sequencing of the entire TGFBI gene were done in all probands. Mutation-carrying exons were examined in 50 first and second degree relatives. Phenotypic analysis disclosed the occurrence of 6 cases of lattice CD, 6 of granular CD, 2 of granular type 2 (Avellino CD), 2 of polymorphic corneal amyloidosis, 1 of Reis–Bucklers CD, and 1 of an unclassifiable phenotype. TGFBI mutations were identified in all 18 probands. A total of six different mutations were observed: p.V113I, p.M502V, p.A546D, p.L550P, p.R555W, and p.H626R. Of these, mutations p.L550P (originated by the change c.1649T>C at exon 12), p.M502V (c.1504A>G, at exon 11), and p.V113I (c.337G>A, at exon 4), are novel TGFBI mutations. All subjects with lattice CD in our sample carried the p.H626R mutation. No instances of defects at codon 124, one of the two most frequently mutated sites in TGFBI-linked CDs, were detected. A distinct TGFBI mutational pattern was identified in Mexican patients with stromal CDs. Novel TGFBI mutations and new genotype–phenotype correlations were also recognized. This study stresses the importance of performing TGFBI genetic analysis in distinct CD populations.

Purification, crystallization and preliminary X-ray diffraction of wild-type and mutant recombinant human transforming growth factor beta-induced protein (TGFBIp).

Transforming growth factor beta-induced protein (TGFBIp) has been linked to several corneal dystrophies as certain point mutations in the protein may give rise to a progressive accumulation of insoluble protein material in the human cornea. Little is known about the biological functions of this extracellular protein, which is expressed in various tissues throughout the human body. However, it has been found to interact with a number of extracellular matrix macromolecules such as collagens and proteoglycans. Structural information about TGFBIp might prove to be a valuable tool in the elucidation of its function and its role in corneal dystrophies caused by mutations in the TGFBI gene. A simple method for the purification of wild-type and mutant forms of recombinant human TGFBIp from human cells under native conditions is presented here. Moreover, the crystallization and preliminary X-ray analysis of TGFBIp are reported.

Corneal dystrophies.

The term corneal dystrophy embraces a heterogenous group of bilateral genetically determined non-inflammatory corneal diseases that are restricted to the cornea. The designation is imprecise but remains in vogue because of its clinical value. Clinically, the corneal dystrophies can be divided into three groups based on the sole or predominant anatomical location of the abnormalities. Some affect primarily the corneal epithelium and its basement membrane or Bowman layer and the superficial corneal stroma (anterior corneal dystrophies), the corneal stroma (stromal corneal dystrophies), or Descemet membrane and the corneal endothelium (posterior corneal dystrophies). Most corneal dystrophies have no systemic manifestations and present with variable shaped corneal opacities in a clear or cloudy cornea and they affect visual acuity to different degrees. Corneal dystrophies may have a simple autosomal dominant, autosomal recessive or X-linked recessive Mendelian mode of inheritance. Different corneal dystrophies are caused by mutations in the CHST6, KRT3, KRT12, PIP5K3, SLC4A11, TACSTD2, TGFBI, and UBIAD1 genes. Knowledge about the responsible genetic mutations responsible for these disorders has led to a better understanding of their basic defect and to molecular tests for their precise diagnosis. Genes for other corneal dystrophies have been mapped to specific chromosomal loci, but have not yet been identified. As clinical manifestations widely vary with the different entities, corneal dystrophies should be suspected when corneal transparency is lost or corneal opacities occur spontaneously, particularly in both corneas, and especially in the presence of a positive family history or in the offspring of consanguineous parents. Main differential diagnoses include various causes of monoclonal gammopathy, lecithin-cholesterol-acyltransferase deficiency, Fabry disease, cystinosis, tyrosine transaminase deficiency, systemic lysosomal storage diseases (mucopolysaccharidoses, lipidoses, mucolipidoses), and several skin diseases (X-linked ichthyosis, keratosis follicularis spinolosa decalvans). The management of the corneal dystrophies varies with the specific disease. Some are treated medically or with methods that excise or ablate the abnormal corneal tissue, such as deep lamellar endothelial keratoplasty (DLEK) and phototherapeutic keratectomy (PTK). Other less debilitating or asymptomatic dystrophies do not warrant treatment. The prognosis varies from minimal effect on the vision to corneal blindness, with marked phenotypic variability.

Novel L558P Mutation of the TGFBI Gene Found in Ukrainian Families with Atypical Corneal Dystrophy

Purpose: To report a novel L558P mutation of the human transforming growth factor β-induced (TGFBI) gene found in Ukrainian families with atypical corneal dystrophy (CD). Methods: Genomic DNA was extracted from peripheral leukocytes of 12 members of 4 unrelated families with atypical CD. We performed genotype analysis of these families with microsatellite markers surrounding the TGFBI locus. Exons of the TGFBI gene were amplified by polymerase chain reaction (PCR), and directly sequenced in 5 patients of 4 unrelated families. We utilized a simple PCR/restriction fragment length polymorphism-based technique for L558P mutation identification. Fifty normal individuals were also analyzed as controls. These assays were complemented by histological analysis of available corneal buttons excised during penetrating keratoplasty. Results: A heterozygous single-base-pair transition (CTC to CCC, leucine to proline) at codon 558 in exon 12 of the TGFBI gene (L558P) was detected in 10 individuals. Eight are affected, and 2 are teenagers with no clinical manifestation of the disease as yet. The mutation was not found in 2 healthy individuals from 2 high-risk CD families, nor in 50 normal controls. Histopathological examination identified amyloid deposits, mostly in the posterior central cornea. Haplotype analysis provided evidence of a common founder of the L558P mutation. The mutation works on the protein level by disrupting an α-helix, which is crucial for the normal functioning of keratoepithelin. Conclusion: A novel L558P mutation in the TGFBI gene causes an atypical type of stromal CD.

A Novel Variant of Combined Granular-Lattice Corneal Dystrophy Associated With the Met619Lys Mutation in the TGFBI Gene

A Novel Variant of Combined Granular-Lattice Corneal Dystrophy Associated With the Met619Lys Mutation in the TGFBI Gene

TGFBI Deficiency Predisposes Mice to Spontaneous Tumor Development

Loss of TGFBI, a secreted protein induced by transforming growth factor-beta, has been implicated in cell proliferation, tumor progression, and angiogenesis by in vitro studies. However, in vivo antitumor functions of TGFBI as well as the underlying molecular mechanism are not well understood. To these aims, we have generated a mouse model with disruption of TGFBI genomic locus. Mice lacking TGFBI show a retarded growth and are prone to spontaneous tumors and 7,12-dimethylbenz(a)anthracene-induced skin tumors. In relation to wild-type (WT) mouse embryonic fibroblasts (MEF), TGFBI(-/-) MEFs display increased frequencies of chromosomal aberration and micronuclei formation and exhibit an enhanced proliferation and early S-phase entry. Cyclin D1 is up-regulated in TGFBI(-/-) MEFs, which correlates with aberrant activation of transcription factor cyclic AMP-responsive element binding protein (CREB) identified by chromatin immunoprecipitation and luciferase reporter assays. TGFBI reconstitution in TGFBI(-/-) cells by either retroviral infection with WT TGFBI gene or supplement with recombinant mouse TGFBI protein in the culture medium leads to the suppression of CREB activation and cyclin D1 expression, and further inhibition of cell proliferation. Cyclin D1 up-regulation was also identified in most of the tumors arising from TGFBI(-/-) mice. Our studies provide the first evidence that TGFBI functions as a tumor suppressor in vivo.

A Corneal Dystrophy Associated with Transforming Growth Factor β–Induced Gly623Asp Mutation: An Amyloidogenic Phenotype

To present the light and electron microscopic findings of a unique corneal dystrophy never before described in a German family carrying the Gly623Asp Mutation of the TGFBI gene with late clinical onset. Experimental study. Four affected and 6 nonaffected family members. Slit-lamp examination, photographic documentation, and isolation of genomic DNA from peripheral blood leucocytes obtained from each family member examined. Exons 3, 4, 5, and 11 to 14 of the TGFBI gene were amplified and sequenced in these family members. Five corneal buttons of 3 affected siblings were excised at the time of penetrating keratoplasty. Light and electron microscopic examination were performed including immunohistochemistry with antibodies against keratoepithelin (KE) 2 and 15. Clinical and histologic characteristics of corneal opacification in affected patients and presence of coding region changes in the TGFBI gene. The specimens showed destructive changes in Bowman's layer and the adjacent stroma. Patchy Congo red-positive amyloid deposits were found within the epithelium in 1 cornea, in Bowman's layer and in the anterior stroma of all specimens also showing KE2, but not KE15, immunostaining. Electron microscopy revealed deposits mainly located in the anterior stroma and Bowman's layer and in small amounts in the basal area of some epithelial cells. The destroyed areas were strongly Alcian blue-positive, the Masson Trichrome stain proved mainly negative for the deposits. All affected but none of the unaffected family members had a heterozygous missense mutation in exon 14 of the TGFBI gene (G-->A transition at nucleotide 1915) replacing glycin by aspartic acid amino acid (Gly623Asp) at position 623 of the KE protein. In contrast with the patient carrying the Gly623Asp mutation of the TGFBI gene described by Afshari et al, our cases presented with Salzmann's nodular degeneration-like clinical features and their specimens contained KE2-positive amyloid. The reason for this now "meeting the expectation histologic phenotype" is unclear. The histologic findings emphasize that this is a unique corneal dystrophy, which shares no clinical characteristics with Reis-Bücklers' dystrophy and should be treated as a distinct entity. The authors have no proprietary or commercial interest in any materials discussed in this article.

Mesenchymal Stromal Cells Promote a Sustained Increase of the CD69 Marker on Activated CD3+ Lymphocytes: Potential Immunomodulatory Role

Mesenchymal stromal cells (MSCs) exert an immune regulatory function and suppress T-cell proliferation in vitro and in vivo. One of the ways by which MSCs may modulate immune responses is by the induction of CD4+CD25+FOXP3+ regulatory T cells (Treg), which suppress alloreactivity in vitro and prevent or attenuate GVHD in animal models. TGF-b (TGFB1 gene), one of the factors secreted by MSC, is known to induce the expression of FOXP3 and to drive the generation of Tregs from CD4+CD25− T cells. T cell activation is characterized by the expression of many surface molecules, and CD69 is one of the earliest markers, transiently expressed following activation. CD69 is selectively expressed at sites of chronic inflammation and recent in vivo and in vitro results indicate that this receptor may modulate the inflammatory response, by inducing TGF-b production. Despite the importance of secreted factors, cell to cell contacts promote increased lymphocyte immune modulation. Interestingly, TGF-b is known to induce the expression of b-IG-H3 (TGFBI gene), a secreted extracellular matrix adaptor protein, whose expression is higher on hematopoietic stem cells adherent to MSC. Given the potential role of CD69 as a regulatory molecule, we explored the effects of MSC on the expression of CD69, TGFB1, FOXP3 and TGFBI on co-cultured T-cells. Peripheral blood mononuclear cells (PBMC) from 6 individuals were co-activated by anti-CD3/CD28 beads and cultured either in the presence or in the absence of MSC (5:1) adhered to the bottom of culture wells. IL2 (20U/ml) was added in the 3rd day for full activation. Cultured PBMC were collected 1, 3 and 5 days after activation. Percentage of CD69+ cells and proliferation of activated lymphocytes (cell division tracking by CFSE) were evaluated by flow cytometry on gated CD3+ lymphocytes. Transcripts levels of TGFB1, TGFBI, FOXP3 and IL10 were determined by real time PCR and normalized using ACTB as an endogenous control. Relative expression levels were calculated in comparison with activated PBMC cultivated alone at the 5th day. Proliferation of lymphocytes co-cultured with MSC was significantly inhibited. As expected, activation of lymphocytes cultured alone was evidenced by the expression of CD69 in 5% of cells in the first day (mean percentage), followed by a decrease in the subsequent days (4% and 3%, respectively). Interestingly, lymphocytes co-cultured with MSC displayed a completely different pattern, with a similar initial activation (7%) which was followed by significant increase in the 3rd day (16%), maintained in the 5th day (14%). Furthermore, TGFBI and IL10 were both expressed at significantly higher levels on PBMC co-cultured with MSC, compared to PBMC alone, in all days evaluated. In addition, their transcript levels decreased faster on PBMC cultured alone. In the other hand, TGFB1 and FOXP3 levels in PBMC cultured alone or in the presence of MSC were similarly higher in the 1st day, but decreased till the 5th day, when their levels were slightly, but significantly higher on PBMC co-cultured with MSC, compared to PBMC cultured alone. We demonstrate for the first time that co-culture with MSC causes a sustained increase of the CD69 marker on CD3+ lymphocytes, which is accompanied by increased levels of TGFB1, TGFBI, FOXP3 and IL10 on total PBMC. Our results are in line with the proposed immunoregulatory role of CD69. In addition, higher TGFBI levels on PBMC may increase lymphocyte adherence to MSC, thus favoring immune modulation. This work was supported by FINEP, CNPq and FAPESP.

TGFBI (BIGH3) gene mutations in German families: two novel mutations associated with unique clinical and histopathological findings

Background:To report the clinical, histopathological and immunohistochemical findings of two novel mutations within the TGFBI gene.Methods:The genotype of 41 affected members of 16 families and nine sporadic cases was investigated...