Engulfment signals and the phagocytic machinery for apoptotic cell clearance

The clearance of apoptotic cells is an essential process for tissue homeostasis. To this end, cells undergoing apoptosis must display engulfment signals, such as ‘find-me' and ‘eat-me' signals. Engulfment signals are recognized by multiple types of phagocytic machinery in phagocytes, leading to prompt clearance of apoptotic cells. In addition, apoptotic cells and phagocytes release tolerogenic signals to reduce immune responses against apoptotic cell-derived self-antigens. Here we discuss recent advances in our knowledge of engulfment signals, the phagocytic machinery and the signal transduction pathways for apoptotic cell engulfment.

Therapeutic Effect of a Recombinant betaig-h3 Fragment-RGD Peptide for Chronic Inflammatory Arthritis

OBJECTIVE: betaig-h3 is a 68kDa extracellular matrix protein which is overexpressed in synovial tissues of rheumatoid arthritis (RA). Previous results proved that betaig-h3 fragments are relevant to adhesion and migration of synovial fibroblast and angiogenesis through interaction with alphavbeta 3 integrin. We designed a recombinant betaig-h3 protein consisting of a fas-1 domain and RGD motif and evaluated the therapeutic efficacy in RA. METHODS: Inhibitory effect of adhesion and migration of NIH3T3 cell line was evaluated in 96 well microtiter and transwell plates coated with betaig-h3. Clinical arthritis index was evaluated after treating CIA mice with MFK12. Immunohistochemical staining in synovial tissues were performed. Expression of transcripts and proteins of inflammatory mediators were analyzed by semi-quantitative RT-PCR and immunoblotting. RESULTS: Recombinant protein consisted of 4th fas-1 domain truncated for H1 and H2 sequences and RGD peptide (MFK12), had M.W. of 10.4kDa. betaig-h3 mediated adhesion and migration of NIH3T3 cell line were significantly inhibited in a dose-dependent manner. Arthritis severity and incidence were efficiently reduced when CIA mice were treated with MFK12 at 30 mg/kg/day compared with the control. Immunohistochemical staining of joint tissues in MFK12 treated mice exhibited reduced angiogenesis. In treated mice, expression of transcripts regarding inflammatory mediators was markedly suppressed and immunoblotting of ICAM-1 and RANKL from whole extract of hind paws also showed a significant reduction. CONCLUSION: This study shows that MFK12 is effective in treating RA, although further study is warranted to improve the therapeutic efficacy.

beta ig-h3-Mediated Adhesion of Fibroblast-Like Synoviocytes in Rheumatoid Arthritis / 대한류마티스학회지

OBJECTIVE: beta ig-h3 is an extracellular matrix protein, which is overexpressed in synovial tissues of rheumatoid arthritis (RA) similar to adhesive glycoproteins. We sought to evaluate the compensatory role of beta ig-h3 with adhesive glycoproteins in mediating the adhesion of fibroblast- like synoviocytes (FLS) and to confirm the inhibitory effect of YH18 peptide of the 2nd fas-1 domain in beta ig-h3-mediated adhesion. METHODS: The adhesion of FLS isolated from synovial tissues of RA, was evaluated in 96 well microtiter plate coated with matrix proteins. Inhibitory effect of YH18 peptides from the 2nd and 4th fas-1 domains was estimated in beta ig-h3-mediated adhesion of FLS. RESULTS: The adhesion of FLS on beta ig-h3 was weaker than that of fibronectin and vitronectin. The beta ig-h3-mediated adhesion was enhanced by the stimulation with phorbol myristate acetate (PMA), but not by cytokines and growth factors. Combination of fibronectin with beta ig-h3 synergistically enhanced the adhesion of FLS, in contrast to the additive effect of vitronectin combined with beta ig-h3. YH18 peptide of the 2nd fas-1 domain did not block the beta ig-h3-mediated adhesion of FLS. CONCLUSION: Our results reveal that beta ig-h3 may regulate the adhesion of FLS through the interaction with adhesive glycoproteins and confirm that the essential motifs mediating adhesion on beta ig-h3 are different according to the type of cells.

Expression patterns of beta ig-h3 in chondrocyte differentiation during endochondral ossification

beta ig-h3 is a TGF-beta-induced extracellular matrix protein which is expressed in many tissues including bones and cartilages. In previous reports, we showed that beta ig-h3 mediates cell adhesion and migration and, especially in bones, negatively regulates the mineralization in the end stage of endochondral ossification. Here, to elucidate the expression pattern and role of beta ig-h3 in chondrocyte differentiation, ATDC5 chondrocytes and embryonic and postnatal mice were used for in vitro differentiation studies and in vivo studies, respectively. beta ig-h3 was strongly induced by the treatment of TGF-beta1 and the expression level of beta ig-h3 mRNA and protein were highly expressed in the early stages of differentiation but decreased in the late stages in ATDC5. Furthermore, the patterns of TGF-beta1, -beta2, and -beta3 mRNA expression were concurrent with beta ig-h3 in ATDC5. beta ig-h3 was deeply stained in perichondrium (PC), periosteum (PO), and prehypertrophic chondrocytes (PH) through the entire period of endochondral ossification in mice. beta ig-h3 was mainly expressed in PC and PH at embryonic days and obviously in PH in postnatal days. These results suggest that beta ig-h3 may play a critical role as a regulator of chondrogenic differentiation in endochondral ossification.

T-CAM, a fastatin-FIII 9-10 fusion protein, potently enhances anti-angiogenic and anti-tumor activity via alphavbeta3 and alpha5beta1 integrins

We made fusion protein of fastatin and FIII 9-10, termed tetra-cell adhesion molecule (T-CAM) that can interact simultaneously with alphavbeta3 and alpha5beta1 integrins, both playing important roles in tumor angiogenesis. T-CAM can serve as a cell adhesion substrate mediating adhesion and migration of endothelial cells in alphavbeta3 and alpha5beta1 integrin-dependent manner. T-CAM showed pronounced anti-angiogenic activities such as inhibition of endothelial cell tube formation, endothelial cell proliferation, and induction of endothelial cell apoptosis. T-CAM also inhibited angiogenesis and tumor growth in mouse xenograft model. The anti-angiogenic and anti-tumoral activity of molecule like fastatin could be improved by fusing it with integrin-recognizing cell adhesion domain from other distinct proteins. The strategy of combining two distinct anti-angiogenic molecules or cell adhesion domains could facilitate designing improved anticancer agent of therapeutic value.

Recombinant tetra-cell adhesion motifs supports adhesion, migration and proliferation of keratinocytes/fibroblasts, and promotes wound healing

An extracellular matrix protein plays an important role in skin wound healing. In the present study, we engineered a recombinant protein encompassing the 9th and 10th type III domains of fibronectin, and 4th FAS1 domain of beta ig-h3. This recombinant protein, in total, harbors four known-cell adhesion motifs for integrins: Pro-His-Ser-Arg-Asn (PHSRN) and Arg-Gly-Asp (RGD) in 9th and 10th type III domains of fibronectin, respectively, and Glu-Pro-Asp-Ile-Met (EPDIM) and Try-His (YH) in 4th FAS1 domain of big-h3, were designated to tetra-cell adhesion motifs (T-CAM). In vitro studies showed T-CAM supporting adhesion, migration and proliferation of different cell types including keratinocytes and fibroblasts. In an animal model of full-thickness skin wound, T-CAM exhibited excellent wound healing effects, superior to both 4th FAS1 domain of beta ig-h3 or 9th and 10th type III domains of fibronectin. Based on these results, T-CAM can be applied where enhancement of cell adhesion, migration and proliferation are desired, and it could be developed into novel wound healing drug.

Identification of TGF-beta-induced Gene Product, betaig-h3 in Ischemic Acute Renal Failure / 대한신장학회지

PURPOSE: Acute renal failure remains a potentially devastating clinical problem. This study aimed to examine whether the expression of TGF-beta-induced gene product, betaig-h3, is altered in ischemia- reperfusion (I/R) injury and urinary excretion of betaig-h3 is changed in I/R injury. METHODS: I/R injury was performed by clamping both renal arteries. Daily urine output, serum creatinine and urinary TGF-beta and betaig-h3 were measured after I/R injury. Also, the renal expression of betaig-h3 by western blotting and immunohistochemistry were investigated. In the second step, urinary betaig-h3 was measured at 4, 10, 16, and 24 hours after I/R injury to investigate whether it could be used as an early and sensitive marker for detecting I/R injury. RESULTS: Urinary betaig-h3 was significantly elevated at 24 hours and maintained higher than the controls until 2 days after I/R injury. In contrast, western blotting did not reveal any changes of betaig-h3 expression. Immunohistochemistry showed that labeling of betaig-h3 was seen at the basement membranes of proximal tubule cells mainly located at the medullary ray (S3 segment) in both groups. Following I/R injury, the labeling was also seen in the basement membrane of injured or regenerated proximal tubular epithelial cells. Within 24 hours, urinary betaig-h3 was significantly increased at 4 hours after I/R injury. Importantly, the urinary appearance of betaig-h3 preceded that of N-acetyl-beta-D-glucosaminidase. CONCLUSION: These results suggest that endogenous renal betaig-h3 may serve to promote tissue regeneration in I/R injury and urinary betaig-h3 could be used as an early and sensitive marker demonstrating I/R injury.

betaig-h3 triggers signaling pathways mediating adhesion and migration of vascular smooth muscle cells through alphavbeta5 integrin

Adhesion and migration of vascular smooth muscle cells (VSMCs) play an important role in the pathogenesis of atherosclerosis. These processes involve the interaction of VSMCs with extracellular matrix proteins. Here, we investigated integrin isoforms and signaling pathways mediating the adhesion and migration of VSMCs on betaig-h3, a transforming growth factor (TGF)-beta-inducible extracellular matrix protein that is elevated in atherosclerotic plaques. Adhesion assays showed that the alphavbeta5 integrin is a functional receptor for the adhesion of aortic VSMCs to betaig-h3. An YH18 motif containing amino acids between 563 and 580 of betaig-h3 was an essential motif for the adhesion and growth of VSMCs. Interaction between the YH18 motif and the alphavbeta5 integrin was responsible for the migration of VSMCs on betaig-h3. Inhibitors of phosphatidylinositide 3-kinase, extracellular signal-regulated kinase (ERK), and Src kinase reduced the adhesion and migration of VSMCs on betaig-h3. betaig-h3 triggered phosphorylation and activation of AKT, ERK, focal adhesion kinase, and paxillin mediating the adhesion and migration of VSMCs. Taken together, these results suggest that betaig-h3 and alphavbeta5 integrin play a role in the adhesion and migration of VSMCs during the pathogenesis of atherosclerosis.

Regulation of betaig-h3 Production in Rheumatoid Synovitis by Inflammatory Mediators / 대한류마티스학회지

OBJECTIVE: To investigate the expression pattern of transforming growth factor-beta-inducible gene-h3 (betaig-h3) within rheumatoid synovial tissue and the regulation of betaig-h3 synthesis in fibroblast-like synoviocyte (FLS). METHODS: Synovial tissues obtained from patients with rheumatoid arthritis and osteoarthritis were obtained during joint replacement surgery. betaig-h3 expression was evaluated with immunohistochemical stain. FLS was isolated from synovial tissues and stimulated with cytokines including TGF-beta, TNF-alpha, IL-1beta, IFN-gamma, IL-6, IL-4, and IL-10. betaig-h3 synthesis was measured using semiquantitative RT-PCR, ELISA, immunofluorescence stain, and flow cytometry. RESULTS: Expression of betaig-h3 was diffuse and abundant in both lining and sublining layers of rheumatoid synovium, which was more prominent than those of osteoarthritis. Production of betaig-h3 in FLS was regulated by TGF-beta1 in a dose-dependent manner and was highest at 5 ng/mL of TGF-beta1. TNF-alpha and IL-1beta upregulated the production of betaig-h3 from FLS synergistically with TGF-beta1 but other cytokines such as IL-4, IL-6, IL-10 did not affect. betaig-h3 synthesis was efficiently inhibited by dexamethasone at higher dose (100 nM) but not by cyclosporine-A. CONCLUSION: Production of betaig-h3, which is highly upregulated in rheumatoid synovitis, is differentially regulated by inflammatory cytokines.

The Bone Regenerative Effect of Calcium Sulfate- chitosan Complex Powder and Chitosan Microsphere Encapsulating Growth Hormone on Bone Formation in Bone Defect of Rabbits

This study was carried out on 32 New Zealand white rabbits, each weighing 3-3.5kg. Eight rabbits were allocated into each of four groups. The groups were a control group(I), hyaluronic acid group(II), chitosan microsphere encapsulating growth hormone group(III), calcium sulfate-chitosan powder group(IV). After a 1cm sized ostectomy was made on the tibial body with the periosteum preserved, artificial bone substitutes were implanted. Except group I, 1ml of hyaluronic acid were implanted in group II, 1ml of chitosan microsphere encapsulating growth hormone in group III, 1ml of manufactured calcium sulfate-chitosan complex powder in group IV. Results were evaluated using radiographic study every week, bone mineral density test and histologic examination at 2, 4, 6 weeks and three point bending test at 6 weeks after implantation. In the radiographic study, the formation and corticalization of callus were seen similarly in group III, IV and much more and earlier than group I, II. In the bone mineral density test and three point bending test to contralateral normal tibia in 6 weeks, the values in groups III and IV were statistically significantly higher than in group I and II(p<0.05). In histologic examination, group III and IV have more abundant and faster new boner formation than group I and II. In conclusion, calcium sulfate-chitosan complex powder and chitosan microsphere encapsulating growth hormone facilitates the formation of new bone. They will be used effectively as a bone substitute on defected bone in clinical situations.

Effect of beta-Ig H3 on Chondrogenesis by Human Bone Marrow Mesenchymal Stem Cells / 대한정형외과학회잡지

PURPOSE: This study was undertaken to compare the activity of beta-Ig H3 (BigH3) and transforming growth factor (TGF)-beta on chondrogenesis by mesenchymal stem cells (MSCs). MATERIALS AND METHODS: MSCs in human bone marrow aspirated from 20 healthy donors were isolated by density gradient Ficoll-hypaque separation and expanded in culture. MSC-alginate beads were prepared and incubated for 28 days in the presence of 0.5 or 10 ng/mL of TGF-beta 1, TGF-beta 1+TGF-beta 3 or BigH3. Cellular viability, total collagen and glycosaminoglycan (GAG) contents were measured and compared. SPSS version 9.0 was used for the statistical analysis. RESULTS: TGF-beta 3 significantly enhanced cell viability in beads by day 21 (p=0.029). No significant differences were found in terms of cell viability (p=0.197) or in total GAG content (p=0.253) between 10 ng/mL of TGF-beta 1+3 and 10 ng/mL of BigH3. Total collagen content was higher in the BigH3 added group on day 21 (p=0.041). CONCLUSION: The replacement of BigH3 instead of TGF-beta produced appropriate external signals indicating the chondrogenic differentiation of human bone marrow MSCs.

Comparison of Wound Healing Effect of Different Forms of Chitosan

This study is to compare the effect of wound healing using three different types of chitin, which include the shapes of sponge, velvet, thick non-woven fabrics, and thin non-woven fabrics. The sponge type had more capacity to absorb the first discharge of a wound than the velvet type and the two non-woven fabrics types. Instead of absorbing the discharge effectively, the velvet type showed a difficulty to take off the dressing stuff from a wound since it was solidly stuck to the wound. The sponge type showed less infiltration of inflammatory cells, producing angiogenesis and fibroblast faster than any other types. Next, the thick non-woven fabrics type was a little more effective than the thin non-woven fabrics type: However, there was no difference between two types. The velvet type sustained the infiltration of inflammatory cells for the longest duration, producing slower angiogenesis and fibroblast. In wound contraction and wound healing, the sponge type was most effective with statistical significance than any other types(p0.05). In conclusion, the sponge type showed the best effectiveness to absorb the early discharge, facilitating the progress of inflammatory phase to increase the healing rate. It induced an early healing of wound caused by wound contraction rather than by wound epithelization.

Effect of Injectable Chitosan Bead Encapsulating Calcium Sulfate on Bony Consolidation in Distraction Osteogenesis

The purpose of this project was to study the effect of injectable chitosan bead encapsulating calcium sulfate, which makes sustained release of chitosan and calcium sulfate after implantation, on early bony consolidation in distraction osteogenesis of a dog model. Forty five dogs were used for this study. An external distraction device was applied to the mandibular body after vertical osteotomy and the mandibular distraction was started five days after the operation at a rate of 1mm per day up to a 10mm distraction. The experimental group was divided into a control group(I), hyaluronic acid group(II), chitosan group(III), calcium sulfate group(IV), and injectable chitosan bead encapsulating calcium sulfate group(V). Normal saline was injected in the group I. In the group II, a 1-ml volume of hyaluronic acid solution was injected into the distracted area. In the group III, a 1-ml of injectable solution of chitosan mixed with hyaluronic acid was implanted. In the group IV, a 1-ml of injectable solution of calcium sulfate mixed with hyaluronic acid was implanted. In the group V, injectable form of powders of chitosan bead encapsulating calcium sulfate mixed with a 1-ml volume of hyaluronic acid was implanted. Bone mineral density was measured in each group at third and sixth week. The mean three point failure load was measured in each group. In histological findings, new bone was generated in all groups. In the group IV and V, the formation of active woven bone was observed throughout the distracted area at sixth week. The amount of new bone formation in the distracted zone was in the order of the group IV and V, group III and group II, and control group. In conclusion, these findings suggest that injectable chitosan bead encapsulating calcium sulfate appears to be effective in early bony consolidation in distraction osteogenesis.

Effect of Calcium Sulfate-Chitosan Composite Pellet on Bone Formation in Bone Defect of Rabbits

This study was carried out on 80 New Zealand white rabbits, each weighing 3-3.5kg. Twenty rabbits were allocated into each of the four groups. After a 1cm sized ostectomy was made on the tibial body with the periosteum preserved, artificial bone substitutes were implanted. Except for control group(I), manufactured chitosan pellets were implanted in group II, Osteoset(R)(calcium sulfate) in group III and manufactured calcium sulfate-chitosan composite pellets in group IV. Results were evaluated using radiographic study, bone mineral density test and histologic examination in 2, 4, 6 weeks and three point bending test in 6 weeks after implantation. In the radiographic study, the formation and corticalization of callus were similar in groups III, IV and this was much earlier than in groups I, II. In the bone mineral density test and three point bending test to contralateral normal tibia in 6 weeks, the values in groups III and IV were statistically significantly higher than in groups I and II(p<0.05). In histologic examination, groups III and IV have more abundant and faster new bone formation than groups I and II. In conclusion, the synergistic effect between calcium sulfate and chitosan in group IV is considered to facilitate new bone formation as effectively as Osteoset(R) does.

The Relationship between the Histological Findings and beta ig-h3 in IgA Nephropathy / 대한신장학회잡지

BACKGROUND: TGF-beta is involved in the pathogenesis of various kidney diseases characterized by glomerulosclerosis and tubulointerstitial fibrosis. It is reported that urinary TGF-beta reflects the grade of interstitial fibrosis in glomerular disease. Here, we evaluated the relationship between the histological findings and beta ig-h3 in IgA nephropathy. METHODS: In patients with IgA nephropathy, we measured blood pressure (BP), serum creatinine, 24-hour urinary protein excretion (UTp), creatinine clearance (Ccr), serum and urine beta ig-h3 levels, and urine TGF-beta levels at the time of renal biopsy. Histologic findings were semiquantitively scored according to the extent of glomerulosclerosis (GG), tubulointerstitial fibrosis (TIG) and hyaline arteriolosclerosis (HA) by the criteria suggested by To. Semiquantitive scoring of immunohistochemistry for beta ig-h3 was done. RESULTS: Mean BP 95.4+/-14.5 mmHg, serum creatinine 1.06+/-0.35 mg/dL, 24-hour UTp 1, 423+/-1, 439 mg/day, and Ccr was 97.84+/-59.73 mL/min. The number of patients that showed GG 3 were 5, GG 2 was 1, GG 1 were 12. And, the number of patients that showed TIG 3 were 2, TIG 2 were 5, TIG 1 were 11. HA was shown in 4 patients. beta ig-h3 immunostaining was observed in glomerular Bowman's capsules and basement membrane of proximal tubules. The degree of beta ig-h3 immunostaining was positively correlated with the degree of glomerulosclerosis (r=0.72, p<0.001), interstitial fibrosis (r=0.91, p<0.001), serum creatinine (r=0.592, p<0.05) and Ccr (r=-0.626, p<0.05), but not with 24-hour UTp. Serum and urine beta ig-h3 levels did not correlate with any of these parameters. CONCLUSION: Renal beta ig-h3 expression in patients with IgA nephropathy may be related to glomerulosclerosis and interstitial fibrosis. However, urinary beta ig-h3 levels did not represent the pathologic changes of IgA nephropathy. Long-term study to measure renal beta ig-h3 expression and urinary beta ig-h3 is required to elucidate the roles of beta ig-h3 in IgA nephropathy.

Beta ig-h3 promotes renal proximal tubular epithelial cell adhesion, migration and proliferation through the interaction with alpha3beta1 integrin

Betaig-h3 (betaig-h3) is a secretory protein composed of fasciclin I-like repeats containing sequences that allows binding of integrins and glycosaminoglycans in vivo. Expression of betaig-h3 is responsive to TGF-beta and the protein is found to be associated with extracellular matrix (ECM) molecules, implicating betaig-h3 as an ECM adhesive protein of developmental processes. We previously observed predominant expression of betaig-h3 expression in the basement membrane of proximal tubules of kidney. In this study, the physiological relevance of such localized expression of betaig-h3 was examined in the renal proximal tubular epithelial cells (RPTEC). RPTEC constitutively expressed betaig-h3 and the expression was dramatically induced by exogenous TGF-beta1 treatment. betaig-h3 and its second and fourth FAS1 domain were able to mediate RPTEC adhesion, spreading and migration. Two known alpha3beta1 integrin-interaction motifs including aspartatic acid and isoleucine residues, NKDIL and EPDIM in betaig-h3 were responsible to mediate RPTEC adhesion, spreading, and migration. By using specific antibodies against integrins, we confirmed that alpha3beta1 integrin mediates the adhesion and migration of RPTECs on betaig-h3. In addition, it also enhanced proliferation of RPTECs through NKDIL and EPDIM. These results indicate that betaig-h3 mediates adhesion, spreading, migration and proliferation of RPTECs through the interaction with alpha3beta1 integrin and is intimately involved in the maintenance and the regeneration of renal proximal tubular epithelium.

Wound Healing Effect of the Chitosan Sponges Containing the T-CAM or Regenin

Tetra cell adhesion molecule(T-CAM) is a new recombinant mixture of fibronectin and ig-h3. Fibronectin and ig-h3 are extracellular matriprotein involved in each phase of wound healing, and the combination of these materials may generate a synergistic effect in wound healing. Regenin is easily attainable from protein recombination. It can be developed as wound healing material, and also it has a good effect in cell adhesion and proliferation. We combined the chitosan with regenin or T-CAM at different concentration, which are gene recombination material. They were applied to the artificial wound of white rabbit to compare the healing effect in each group. Round full thickness skin defects, 3 cm in diameter, were made bilaterally on the dorsolateral aspect of New Zealand white rabbit. Experimental group was divided into 6 groups, according to concentration of T-CAM and regenin with chitosan-based dressing materials as followings; Group C: control group - oint material dressing, Group Ch: chitosan base only, Group T1: chitosan base in combination with 25 microgram/ml of T-CAM, Group T2: chitosan base in combination with 625 microgram/ml of T-CAM, Group R1: chitosan base in combination with 25 microgram/ml of Regenin, Group R2: chitosan base in combination with 625 microgram/ml of Regenin. Gross findings by means of percentage of wound contraction, percentage of wound epithelization and percentage of total wound healed area were compared with surface tracing of the remained wound area at the time of 7, 14, 21 days after wound formation. Wound biopsy were performed at the time of 7, 14, 21 days after wound formation. T1, T2 group and R1, R2 groups have less infiltration of inflammtory cell, fast appearance of new vessels, fibroblast, increased volume of collagen fiber comparing to C and Ch group. there's more statistical significance between T1 and T2 group. The same results were shown in Regenin group. In conclusion, our results suggest that T-CAM and Regenin have good effect in wound healing and higher concentration of T-CAM and Regenin is more effective in wound healing than lower concentration. In addition, comparision of same concentration of T-CAM and Regenin group presented almost same results.

The GSTT1 Genotype as A Marker for Susceptibility to Lung Cancer in Korean Female Never-Smokers / 결핵

BACKGROUND: Most previous studies regarding the role of GSTMl and GSTT1 on lung cancer risk have been focused mainly on male smokers. However, epidemiological characteristics, histologic types and risk factors are different in female and male lung cancers, we investigated the association between these genotypes and lung cancer risk in males and females separately. MATERIALS AND METHODS: The study population consisted of 253 lung cancer (153 males and 100 females) and 243 controls (140 males and 103 females). GSTM1 and GSTT1 genotypes were determined by a multiplex PCR. RESULTS: In the male population, neither GSTM1 nor GSTT1 null genotype showed significant difference between cases and controls. In the female population, the frequencies of GSTM1 null genotype showed no significant difference between cases and controls. However, the frequencies of GSTT1 null genotype was significantly higher in cases (70.3%) than controls (55.3%, odds ratio (OR)=2.18; 95% confidence interval (CI=l.21-3.93). When the female population was stratified by age and smoking status, the ORs for GSTT1 null genotype were significantly higher in subgroups of ≤60 years (OR=4.82; 95% CI=l.61-14.4) and never-smokers (OR=4.29; 95% CI=1.94-9.48) but not in subgroups of >60 years or smokers. When stratifying the female never-smokers by age, the ORs for GSTT1 null genotype were significantly higher in both age groups of ≤60 years (OR=7.64; 95% CI=2.00-29.2) and >60 years (OR=2.89; 95% CI=1.05-7.94). CONCLUSION: We found that GSTT1 null genotype was associated with an increased risk of lung cancer in Korean female never-smokers. This result suggests that GSTT1 null genotype could be used as a biomarker for genetic susceptibility to lung cancer in Korean female never-smokers.

The Effects of New Recombinant Protein of Fibronectin and beta ig-h3, T-CAM(Tetra Cell Adhesion Molecule) on Wound Healing

T-CAM(Tetra cell adhesion molecule) is new recombinant mixture of fibronectin and beta ig-h3. Fibronectin and beta ig-h3 are extracellular matrix protein involved in each phase of wound healing and sum of these materials may play synergistic effect on the wound healing. In order to evaluate wound healing effect of T-CAM on open wound in rabbit, we made four round full thickness skin defects, 3 cm in diameter, bilaterally on the dorsolateral aspect of New Zealand white rabbit's trunk. We divided the wound into four groups, according to the content of topocally applied onitments: Group A treated with ointment base only; Group B with ointment containing fibronectin microgram/ml; Group C with ointment containing beta ig-h3 300 microgram/ml; Group D with ointment containing T-CAM 300 microgram/ml. These ointments were applied daily on the wound. We compared each group with gross findings by means of percentage of wound contraction, percentage of wound epithelization and percentage of total wound healed area with surface tracing the remained wound area on the 0, 6,th 12th, 18th day after wound formation and wound biopsy were performed on the 3rd, 6th, 10th, 14th, and 21st day after wound formation.The T-CAM group shows statistically significant (p < 0.05 ANOVA test and Scheffe's test) in wound contraction and totalwound healed area than other groups on the 6th day after wound formation, and equally significant on the 12th and 18th day after wound formation except in group C. In histological examination, T-CAM group shows less inflammatory cell infiltration, faster angiogenesis and marked fibroblast proliferation than other groups in early inflammatory period, and more matured, thickened reepithelization and regularly aligned collagen formation on the 14th and 21st day. This study suggest recombinant T-CAM shows synergistic effect on wound healing, and is expected as new potent material for treatment of wound healing.

Effect of Growth Hormone on Early Bony Regeneration in Distraction Osteogenesis

The purpose of this project was to study the effect of growth hormone on early bony consolidation in distraction osteogenesis of a dog mandible. Sixteen dogs were used for this study. An external distraction device was applied to the mandibular body and the mandibular distraction was started five days after the operation at a rate of 1 mm per day up to a 10-mm distraction. Dogs in the growth hormone group received a daily subcutaneous injection of 1 IU of recombinant human growth hormone per kilogram of body weight per week. Normal saline was injected in the control group. Bone mineral density was higher in the growth hormone group than the control group in the whole period. Bone mechanical strength was 300% higher in the growth hormone group than that in the control group. However, results were more suggestive than conclusive. Upon histological examination, the formation of a substantial amount of active woven bone was observed throughout the distracted zone in 6 weeks in the growth hormone group. In the control group, new bone was generated from the edge to the center of the distracted zone. But, the most central area of the distracted zone was filled with fibrous tissue in 6 weeks. In conclusion, growth hormone appears to be effective in early bony consolidation in distraction osteogenesis.