PEGylation is considered one of the most successful techniques to improve the characteristics of protein drugs including to increase the circulating half-life of proteins in blood and to decrease their immunogenicity and antigenicity. One known PEG modification method is to attach PEG to the free amino group, typically at lysine residues or at the N-terminal amino acid with no selectivity, resulting in a heterogeneous product mixture. This lack of selectivity can present problems when a therapeutic PEGylated protein is being developed, because predictability of activity and manufacturing reproducibility are needed for regulatory approval. Enzymatic PEGylation of proteins is one route to overcome this limitation. Transglutaminases (TGase) are enzyme candidates for site-specific PEGylation. We use human interferon alpha 2a (IFN α2a) as a test case, and predict that the potential modification residues are Gln101 by computational approach as it contains 12 potential PEGylation sites. IFN α2a was PEGylated by Y shaped PEG40k-NH2 mediated by microbial transglutaminase. Our results show that the microbial transglutaminase mediated PEGylation of IFN α2a was site-specific only at the site of Gln101 in IFN α2a, yielding the single mono-conjugate PEG-Gln101-IFN α2a with a mass of 59 374.66 Da. Circular dichroism studies showed that PEG-Gln101-IFN α2a preserved the same secondary structures as native IFN α2a. As expected, the bioactivity and pharmacokinetic profile in rats of PEG-Gln101-IFN α2a revealed a significant improvement to unmodified IFN α2a, and better than PEGASYS.
Animals ; Antiviral Agents ; Humans ; Interferon alpha-2 ; metabolism ; Interferon-alpha ; biosynthesis ; pharmacokinetics ; Polyethylene Glycols ; pharmacokinetics ; Protein Structure, Secondary ; Rats ; Recombinant Proteins ; biosynthesis ; pharmacokinetics ; pharmacology ; Reproducibility of Results ; Transglutaminases ; metabolism

OBJECTIVE: To explore the genetic cause for a child with congenital ichthyosis. METHODS: The child was subjected to next generation sequencing using a specific gene panel. Suspected mutation was validated by Sanger sequencing. RESULTS: The proband was found to harbor compound heterozygous mutations c.327delG (p.Met109Ilefs*2) and c.791G>A (p.Arg264Gln) of the TGM1 gene, which were respectively inherited from his mother and father. The same mutations were not found among 101 healthy controls. c.327delG was not reported previously. By bioinformatic analysis, both mutations are likely to impair the function of TGase-1 protein. CONCLUSION The compound heterozygous mutations of c.327delG and c.791G> A of the TGM1 gene probably underlie the ichthyosis in the proband. The result has facilitated prenatal diagnosis for this pedigree.
Female ; Humans ; Ichthyosiform Erythroderma, Congenital ; genetics ; Infant, Newborn ; Mutation ; Pedigree ; Phenotype ; Pregnancy ; Transglutaminases ; genetics

BACKGROUND: Juvenile xanthogranuloma is a benign, self-limited disorder that usually occurs in infants and young children. Xanthogranuloma is rare in adults, and therefore studies reporting adult xanthogranuloma are limited. OBJECTIVE: We investigated the clinical, histopathological, and immunohistochemical characteristics of adult xanthogranuloma. METHODS: In this study, we evaluated 20 lesions in 19 patients with adult xanthogranuloma. RESULTS: A male predominance was observed (male : female ratio 1.4 : 1), and the mean age of patients was 35.1±16.3 years (range 15∼66 years), with the peak incidence observed in patients in their 20s. Notably, 65.0% of the lesions developed on the head and neck. The nodular form was more common than the papular form of this condition. Histopathological examination revealed dense monomorphic histiocytic infiltration without lipidization and scattered eosinophils without multinuclear giant cells in 5 lesions (25.0%), foamy histiocytic infiltration with variations of completely developed Touton giant cells in 10 lesions (50.0%), and fibrohistiocytic proliferation in 3 lesions (15.0%). On immunohistochemical examination, histiocytes including giant cells showed positive test results with Factor XIIIa (90.9%), vimentin (100%), and CD68 (100%) and negative test results with CD1a, smooth muscle actin, and S-100 protein stains. Tumor excision was the treatment for choice. CONCLUSION: Adult xanthogranuloma most commonly manifested as the nodular form of the disease on the head and neck of men in their late 20s. Histopathologically, the classic Touton cell-rich stage was most commonly observed, followed by the stage of early predominantly mononuclear infiltration. This was a single-center, small-sized retrospective study; however, we expect the results of this study to contribute to a better understanding of adult xanthogranuloma.
Actins ; Adult ; Child ; Coloring Agents ; Eosinophils ; Factor XIIIa ; Female ; Giant Cells ; Head ; Histiocytes ; Humans ; Incidence ; Infant ; Male ; Muscle, Smooth ; Neck ; Retrospective Studies ; S100 Proteins ; Vimentin ; Xanthogranuloma, Juvenile

OBJECTIVETo explore the genetic cause for a Uyghur Chinese child with collodion skin.

METHODSG-banded chromosomal karyotyping was carried out for the child and his parents. High-throughput sequencing for 25 genes related to ichthyosis and ichthyosiform dermatosis was also performed for the child.

RESULTSNo karyotypic abnormality was found in the child and his parents. High-throughput sequencing has detected in the patient a previously described pathogenic mutation c.919C>T (p.Arg307Trp) and a novel c.856C>T (p.Arg286Trp) mutation in the TGM1 gene. By Sanger sequencing, the child was verified to have carried both mutations. His father was found to be a heterozygous carrier of the c.856C>T (p.Arg286Trp) mutation, while neither mutation was found in the mother.

CONCLUSIONCongenital ichthyosis associated with the TGM1 gene may show an autosomal recessive inheritance. The collodion condition of the child is probably due to the compound heterozygous mutations of the TGM1 gene.

Child ; Chromosome Banding ; Female ; High-Throughput Nucleotide Sequencing ; Humans ; Ichthyosis, Lamellar ; genetics ; Infant ; Karyotyping ; Mutation ; Transglutaminases ; genetics

BACKGROUND: Dermatofibrosarcoma protuberance (DFSP) must be differentiated from dermatofibroma (DF). However, especially in cases of superficial biopsy and cellular dermatofibroma, this is difficult by using histopathology alone since both are composed of neoplastic spindle cells. Although a panel of immunostains is useful, the expressions of conventional markers often overlap. A previous study showed that novel D2-40 immunostain may be useful for differentiating between DF and DFSP. OBJECTIVE: To evaluate the usefulness of D2-40 immunohistochemical staining for differentiating DFSP from DF and compare the results with other commonly used immunostains (CD34 and factor XIIIa). METHODS: Twenty-eight cases of DF and 15 cases of DFSP were selected from clinicopathologically proven cases reviewed by the Department of Dermatology at our medical center and Daegu Catholic University Medical Center. D2-40, CD34, and factor XIIIa immunohistochemical staining was performed. The immunopositivity was measured throughout the entire lesion. RESULTS: Seventeen cases (60.7%) of DF and no cases of DFSP showed immunoreactivity to D2-40 in the spindle cells. Three (10.7%) cases of DF and 13 (86.7%) cases of DFSP showed immunoreactivity to CD34 in the spindle cells. Twenty-five (89.3%) cases of DF and four (26.7%) cases of DFSP showed immunoreactivity to factor XIIIa in the spindle cells. A total of 60.7% of cases of DF were positive on D2-40 staining, 89.3% were negative on CD34 staining, and 89.3% were positive on factor XIIIa staining. All cases (100%) of DFSP were negative by D2-40 staining, 86.7% were positive by CD34 staining, and 73.3% were negative by factor XIIIa staining. CONCLUSION: D2-40 immunostaining may be useful for distinguishing between DF and DFSP since the immunoreactivity of DF was significantly higher than that of DFSP (p=0.001). However, the results of our study were not as useful as those of a previous study. Therefore, further studies are needed to address this issue.
Academic Medical Centers ; Biopsy ; Daegu ; Dermatofibrosarcoma* ; Dermatology ; Factor XIIIa* ; Histiocytoma, Benign Fibrous*

Transglutaminase (TG) is a kind of calcium-dependent enzymes. The TGase family found in rodents and human contains 9 types, including TG1-7, blood coagulation factor XIIIa and erythrocyte membrane protein 4.2, with the former 8 types possessing catalytic activity. TG catalyzes various conversion reactions of glutamine, including transamination, deamination and esterification, and participates in post-transcriptional modification of proteins such as cross-linking peptides glutamine residue and lysyl-residue, stabilizing protein structure and catalyzing formation of protein aggregates. TGase has been found to contribute to a variety of important physiological and pathological processes and play a role in the pathogenesis of multiple diseases. Notably, neurodegenerative diseases such as Huntington's disease, spinocerebellar ataxia, Alzheimer's disease and Parkinson's disease, have a close connection with TGase's role in the human body.
Animals ; Brain ; enzymology ; Humans ; Neurodegenerative Diseases ; enzymology ; genetics ; Transglutaminases ; genetics ; metabolism

BACKGROUND: A few recent studies have demonstrated a possible role of transglutaminase 2 (TG2) in tumorigenesis or progression of renal cell carcinoma (RCC). The aim of this study was to examine TG2 expression and its clinicopathologic significance in a large number of human clear cell RCCs (CCRCCs). METHODS: We analyzed 638 CCRCC patients who underwent partial or radical nephrectomy between 1995 and 2005. The expression of TG2 was determined by immunohistochemistry and categorized into four groups, according to staining intensity: negative (0), mild (1+), moderate (2+), and strong (3+). RESULTS: TG2 staining intensity was negative in 8.5% of CCRCC (n=54), 1+ in 32.6% (n=208), 2+ in 50.5% (n=322), and 3+ in 8.5% (n=54). Strong TG2 expression was correlated with high Fuhrman nuclear grade (p=.011), high T category (p=.049), metastasis (p=.043) and male sex (p<.001) but not with N category.The survival analysis showed a significant association between strong TG2 expression and worse overall and cancer-specific survival (p=.027 and p=.010, respectively). On multivariate analysis, strong TG2 expression was a marginally significant prognostic indicator for Fuhrman nuclear grade and TNM staging (p=.054). CONCLUSIONS: Our study is the first to demonstrate the clinicopathologic significance of TG2 expression in a large number of human CCRCC samples. Strong TG2 expression was associated with high nuclear grade and poor prognosis.
Carcinogenesis ; Carcinoma, Renal Cell* ; Humans ; Immunohistochemistry ; Male ; Multivariate Analysis ; Neoplasm Metastasis ; Neoplasm Staging ; Nephrectomy ; Prognosis ; Transglutaminases

BACKGROUND: Dermatofibroma (DF) comprises a heterogeneous group of mesenchymal tumors, with fibroblastic and histiocytic elements present in varying proportions. The cell of origin of DF has been investigated, but remains unclear. OBJECTIVE: The present study attempted to investigate the expression of leukocyte-specific protein 1 (LSP1), a marker of fibrocytes, in DF. Additionally, we evaluated the effectiveness of LSP1 in the differential diagnosis of DF from dermatofibrosarcoma protuberans (DFSP). METHODS: Immunohistochemical staining was performed on 20 cases of DF using antibodies against LSP1, CD68, and factor XIIIa (FXIIIa). In addition, the expression of LSP1 and FXIIIa was evaluated in 20 cases of DFSP. RESULTS: Eighteen of 20 cases (90%) of DF stained positive for LSP1, with variation in the intensity of expression. CD68 was positive in 10 cases (50%), and FXIIIa was expressed in all cases of DF. There were differences between the regional expression patterns of the three markers in individual tumors. In contrast, only 2 of 20 cases of DFSP expressed LSP1, and none of DFSP cases stained positive for FXIIIa. CONCLUSION: The LSP1-positive cells in DF could potentially be fibrocyte-like cells. FXIIIa and CD68 expression suggests that dermal dendritic cells and histiocytes are constituent cells of DF. It is known that fibrocytes, dermal dendritic cells and histiocytes are all derived from CD14+ monocytes. Therefore, we suggest that DF may originate from CD14+ monocytes. Additionally, the LSP1 immunohistochemical stain could be useful in distinguishing between DF and DFSP.
Antibodies ; Dermatofibrosarcoma ; Diagnosis* ; Diagnosis, Differential ; Factor XIIIa ; Fibroblasts ; Histiocytes ; Histiocytoma, Benign Fibrous* ; Langerhans Cells ; Monocytes

Chronic rhinosinusitis (CRS) is one of the most common chronic diseases in adults and severely affects quality of life in patients. Although various etiologic and pathogenic mechanisms of CRS have been proposed, the causes of CRS remain uncertain. Abnormalities in the coagulation cascade may play an etiologic role in many diseases, such as asthma and other inflammatory conditions. While studies on the relationship between asthma and dysregulated coagulation have been reported, the role of the coagulation system in the pathogenesis of CRS has only been considered following recent reports. Excessive fibrin deposition is seen in nasal polyp (NP) tissue from patients with chronic rhinosinusitis with nasal polyp (CRSwNP) and is associated with activation of thrombin, reduction of tissue plasminogen activator (t-PA) and upregulation of coagulation factor XIII-A (FXIII-A), all events that can contribute to fibrin deposition and crosslinking. These findings were reproduced in a murine model of NP that was recently established. Elucidation of the mechanisms of fibrin deposition may enhance our understanding of tissue remodeling in the pathophysiology of NP and provide new targets for the treatment of CRSwNP.
Adult ; Asthma ; Blood Coagulation Factors ; Chronic Disease ; Factor XIIIa ; Fibrin ; Fibrinolysis ; Humans ; Nasal Polyps ; Quality of Life ; Thrombin ; Tissue Plasminogen Activator ; Up-Regulation

This study was performed to examine the role of transglutaminase 2 (TG2) in ventilator-induced lung injury (VILI). C57BL/6 mice were divided into six experimental groups: 1) control group; 2) lipopolysaccharide (LPS) group; 3) lung protective ventilation (LPV) group; 4) VILI group; 5) VILI with cystamine, a TG2 inhibitor, pretreatment (Cyst+VILI) group; and 6) LPV with cystamine pretreatment (Cyst+LPV) group. Acute lung injury (ALI) score, TG2 activity and gene expression, inflammatory cytokines, and nuclear factor-kappaB (NF-kappaB) activity were measured. TG2 activity and gene expression were significantly increased in the VILI group (P < 0.05). Cystamine pretreatment significantly decreased TG2 activity and gene expression in the Cyst+VILI group (P < 0.05). Inflammatory cytokines were higher in the VILI group than in the LPS and LPV groups (P < 0.05), and significantly lower in the Cyst+VILI group than the VILI group (P < 0.05). NF-kappaB activity was increased in the VILI group compared with the LPS and LPV groups (P < 0.05), and significantly decreased in the Cyst+VILI group compared to the VILI group (P = 0.029). The ALI score of the Cyst+VILI group was lower than the VILI group, but the difference was not statistically significant (P = 0.105). These results suggest potential roles of TG2 in the pathogenesis of VILI.
Acute Lung Injury/pathology ; Animals ; Cystamine/therapeutic use ; Cytokines/analysis ; Enzyme Inhibitors/therapeutic use ; Enzyme-Linked Immunosorbent Assay ; GTP-Binding Proteins/*antagonists & inhibitors/genetics/metabolism ; Gene Expression ; Lipopolysaccharides/toxicity ; Male ; Mice ; Mice, Inbred C57BL ; NF-kappa B/metabolism ; Respiration, Artificial ; Transglutaminases/*antagonists & inhibitors/genetics/metabolism ; Ventilator-Induced Lung Injury/*enzymology/pathology/prevention & control