Hypoxic Gene Signature of Primary and Metastatic Melanoma Cell Lines: Focusing on HIF-1ß and NDRG-1

Background: Hypoxia is an important microenvironmental factor significantly affecting tumor proliferation and progression. The importance of hypoxia is, however, not well known in oncogenesis of malignant melanoma. Aims: To evaluate the difference of hypoxic gene expression signatures in primary melanoma cell lines and metastatic melanoma cell lines and to find the expression changes of hypoxia-related genes in primary melanoma cell lines at experimental hypoxic conditions. Study Design: Cell study. Methods: The mRNA expression levels of hypoxia-related genes in primary melanoma cell lines and metastatic melanoma cell lines and at experimental hypoxic conditions in primary melanoma cell lines were evaluated by using real-time polymerase chain reaction. Depending on the experimental data, we focused on two genes/proteins, the hypoxia-inducible factor-1 beta and the N-myc downstream regulated gene-1. The expression levels of the two proteins were investigated by immunohistochemistry methods in 16 primary and metastatic melanomas, 10 intradermal nevi, and a commercial tissue array comprised of 208 cores including 192 primary and metastatic malignant melanomas. Results: The real-time polymerase chain reaction study showed that hypoxic gene expression signature was different between metastatic melanoma cell lines and primary melanoma cell lines. Hypoxic experimental conditions significantly affected the hypoxic gene expression signature. In immunohistochemical study, N-myc downstream regulated gene-1 expression was found to be lower in primary cutaneous melanoma compared to in intradermal nevi (p=0.001). In contrast, the cytoplasmic expression of hypoxia-inducible factor-1 beta was higher in primary cutaneous melanoma than in intradermal nevi (p=0.001). We also detected medium/strong significant correlations between the two proteins studied in the study groups. Conclusion: Hypoxic response consists of closely related proteins in more complex pathways. These findings will shed light on hypoxic processes in melanoma and unlock a Pandora's box for development of new therapeutic strategies.

Stem cells combined 3D electrospun nanofibrous and macrochannelled matrices: a preliminary approach in repair of rat cranial bones.

Repair of cranial bone defects is an important problem in the clinical area. The use of scaffolds combined with stem cells has become a focus in the reconstruction of critical-sized bone defects. Electrospinning became a very attracting method in the preparation of tissue engineering scaffolds in the last decade, due to the unique nanofibrous structure of the electrospun matrices. However, they have a limitation for three dimensional (3D) applications, due to their two-dimensional structure and pore size which is smaller than a cellular diameter which cannot allow cell migration within the structure. In this study, electrospun poly(ε-caprolactone) (PCL) membranes were spirally wounded to prepare 3D matrices composed of nanofibers and macrochannels. Mesenchymal stromal/stem cells were injected inside the scaffolds after the constructs were implanted in the cranial bone defects in rats. New bone formation, vascularisation and intramembranous ossification of the critical size calvarial defect were accelerated by using mesenchymal stem cells combined 3D spiral-wounded electrospun matrices.

Cranial bone regeneration via BMP-2 encoding mesenchymal stem cells.

Cranial bone repair and regeneration via tissue engineering principles has attracted a great deal of interest from researchers during last decade. Here, within this study, 6 mm critical-sized bone defect regeneration via genetically modified mesenchymal stem cells (MSC) were monitored up to 4 months. Cranial bone repair and new bone formations were evaluated by histological staining and real time PCR analysis in five different groups including autograft and bone morphogenetic protein-2 (BMP-2) transfected MSC groups. Results presented here indicate a proper cranial regeneration in autograft groups and a prospering regeneration for hBMP-2 encoding mesenchymal stem cells.

The effect of low-dose methotrexate on autologous fat graft survival.

BACKGROUND/AIM: The survival of autologous fat graft tissue is dependent on various factors such as vascularization and inflammation. The aim of the present study was to evaluate the possible beneficial effects of low-dose methotrexate (LD-MTX) on fat graft volume and survival. MATERIALS AND METHODS: A total of 13 male Wistar albino rats were divided into two groups, a control group and an LD-MTX group. An autologous fat graft obtained from the inguinal region of each rat was transferred to its back. LD-MTX was administered intraperitoneally in the LD-MTX group once a week for 4 weeks after the surgical procedure. The control group underwent surgery but was not administered MTX. Fat grafts were harvested for analyses. RESULTS: The results showed that 2 months postoperatively the fat graft weights of the control and LD-MTX groups were not significantly different. In addition, the vascularity of the grafts was higher in the LD-MTX group than it was in the control group. The mean lipid peroxidation levels were essentially the same in the two groups, but myeloperoxidation was significantly lower in the LD-MTX group than it was in the other group. CONCLUSION: The results showed that LD-MTX administration may not preserve the quality and volume of transplanted fat tissue in rats.

Metastasis suppressor proteins in cutaneous squamous cell carcinoma.

Cutaneous squamous cell carcinomas (cSCCs) are common human carcinomas. Despite having metastasizing capacities, they usually show less aggressive progression compared to squamous cell carcinoma (SCC) of other organs. Metastasis suppressor proteins (MSPs) are a group of proteins that control and slow-down the metastatic process. In this study, we established the importance of seven well-defined MSPs including NDRG1, NM23-H1, RhoGDI2, E-cadherin, CD82/KAI1, MKK4, and AKAP12 in cSCCs. Protein expression levels of the selected MSPs were detected in 32 cSCCs, 6 in situ SCCs, and two skin cell lines (HaCaT, A-431) by immunohistochemistry. The results were evaluated semi-quantitatively using the HSCORE system. In addition, mRNA expression levels were detected by qRT-PCR in the cell lines. The HSCOREs of NM23-H1 were similar in cSCCs and normal skin tissues, while RGHOGDI2, E-cadherin and AKAP12 were significantly downregulated in cSCCs compared to normal skin. The levels of MKK4, NDRG1 and CD82 were partially conserved in cSCCs. In stage I SCCs, nuclear staining of NM23-H1 (NM23-H1nuc) was significantly lower than in stage II/III SCCs. Only nuclear staining of MKK4 (MKK4nuc) showed significantly higher scores in in situ carcinomas compared to invasive SCCs. In conclusion, similar to other human tumors, we have demonstrated complex differential expression patterns for the MSPs in in-situ and invasive cSCCs. This complex MSP signature warrants further biological and experimental pathway research.

Effectiveness of Dimethylsulfoxide on the Survival and Volume Preservation of Autologous Fat Graft Tissue: A Preliminary Study.

BACKGROUND: The survival of autologous fat graft tissue is dependent on various factors, such as vascularization and inflammation. OBJECTIVE: This study aims to evaluate the possible beneficial effects of dimethylsulfoxide (DMSO) on fat graft volume and survival. METHODS: Eighteen male Wistar albino rats were divided randomly into three groups. An autologous fat graft obtained from the inguinal region of each rat was transferred to its back. DMSO was administered intraperitoneally (IP) in the DMSO-IP group and cutaneously (C) in the DMSO-C group once daily for 15 days after the surgical procedure. The control group underwent surgery but was not administered with DMSO. Two months after surgery, the grafted fatty tissues were harvested for histopathological and biochemical analyses. RESULTS: The results showed that 2 months postoperatively, fat grafts of the DMSO-C and DMSO-IP groups weighed significantly more than the grafts of the control group. Moreover, the vascularity of the grafts was higher in the DMSO-C group than in the control group, and no significant difference was found between the two DMSO groups. The mean lipid peroxidation levels were the same in the three groups, but myeloperoxidation was significantly lower in the DMSO-C group than in the other two groups. CONCLUSIONS: The study results showed that cutaneous rather than intraperitoneal DMSO administration could preserve the quality and volume of transplanted fat tissue in rats by enhancing vascularity and decreasing inflammation.

Differential expression patterns of metastasis suppressor proteins in basal cell carcinoma.

BACKGROUND: Basal cell carcinomas (BCCs) are common malignant skin tumors. Despite having a significant invasion capacity, they metastasize only rarely. Our aim in this study was to detect the expression patterns of the NM23-H1, NDRG1, E-cadherin, RHOGDI2, CD82/KAI1, MKK4, and AKAP12 metastasis suppressor proteins in BCCs. METHODS: A total of 96 BCC and 10 normal skin samples were included for the immunohistochemical study. Eleven frozen BCC samples were also studied by quantitative real time polymerase chain reaction (qRT-PCR) to detect the gene expression profile. RESULTS: NM23-H1 was strongly and diffusely expressed in all types of BCC. Significant cytoplasmic expression of NDRG1 and E-cadherin was also detected. However, AKAP12 and CD82/KAI1 expression was significantly decreased. The expressions of the other proteins were somewhere between the two extremes. Similarly, qRT-PCR analysis showed down-regulation of AKAP12 and up-regulation of NM23-H1 and NDRG1 in BCC. Morphologically aggressive BCCs showed significantly higher cytoplasmic NDRG1 expression scores and lower CD82/KAI1 scores than non-aggressive BCCs. CONCLUSION: The relatively preserved levels of NM23-H1, NDRG1, and E-cadherin proteins may have a positive effect on the non-metastasizing features of these tumors.

Application of C-Shaped Osteotomy and Distraction Osteogenesis for Correction of Radial Angulation Deformities of the Hand in Children With Apert Syndrome: Review of 10 Years of Experience.

Apert syndrome is characterized by short, radially deviated thumbs, leading to difficulties in daily life such as holding a fork or a spoon and buttoning up. The main goal of surgery is to achieve thumb to index finger pinch to overcome these difficulties. Seven patients (14 extremities) followed up with Apert syndrome underwent distraction after a C-shaped osteotomy to simultaneously correct the brachydactyly and the angulation deformity of the bilateral thumbs. The patients ranged in age from 4 to 7 years at the distraction operation, with a mean (SD) of 4.7 (1.7) years, and the mean (SD) length of follow-up was 100.6 (14.95) months. The mean (SD) length of the phalanges at the beginning of distraction was 19.1 (3.26) mm, and the mean (SD) length of the distracted phalanx at long-term follow-up visit was 26.2 (5.63) mm. The mean (SD) correction of radial angulation was calculated as 42.6 (9.95) degrees, and the difference was considered as being statistically significant (P < 0.001). Minor complications such as pin loosening were observed in 6 extremities of 4 patients, and 2 patients were treated for pin tract infection. Index-to-thumb pinching was acquired by all the patients after distraction and correction of angulation. The hand features of Apert syndrome are as noteworthy as the craniofacial features and thus may lead to functional impairment and limitations in daily life. Distraction with a C-shaped osteotomy seems to be a promising method to correct both the shortness and the radial angulation of the thumb to achieve functional results.

Clear cell sarcoma.

Malignant melanoma (MM) of soft tissue, also called clear cell sarcoma (CCS) of tendons and aponeuroses, derives from the neural crest. CCS is similar morphologically to MM but has no precursor skin lesion, and instead, has a characteristic chromosomal translocation. Prognosis is related to the tumor size. Early recognition and initial radical surgery is the key to a favorable outcome. The tumor has to be differentiated from other benign and malignant lesions of the soft tissues, such as fibrosarcoma. The demonstration of melanin and a positive immunohistochemical reaction for S-100 protein and HMB-45 can assist in the differential diagnosis. We report the case of a 58-year-old woman with CCS arising from the soft tissue of her little finger.

Stem cell suspension injected HEMA-lactate-dextran cryogels for regeneration of critical sized bone defects.

HEMA-Lactate-Dextran cryogel scaffolds were produced by cryogelation. Mesencyhmal stem cells (MSC) were isolated from rat bone marrow. Critical sized cranial bone defects were created in rat cranium. Stem cells were injected inside the macropores of the cryogel scaffolds prepared from HEMA-Lactate-Dextran possessing the same dimensions with the defect and placed in the cranial bone. The cryogels placed in the defect without stem cells served as control. After selected time intervals the experimental sites were removed from the animals and new bone formation and tissue integration were investigated by histological analysis. The in vivo results exhibited osseous tissue integration within the implant and mineralized functionally stable bone restoration of the cranial defects. Tissue formation started in the macrospores of the scaffold starting from periphery to the center. A significant ingrowth of connective tissue cells and new blood vessels allowed new bone formation. Histological data demonstrated that new bone per total defect area ratio, were not significantly different in "scaffold-stem cells" group compared to that of "scaffold only" group on all time points. However, the blood vessel density was significantly higher in "scaffold-stem cells" group comparing to that of the "scaffold only" group on day 30. "Scaffold-stem cells" given group gave better tissue response score when compared to "scaffold only" group on day 180.

Meridian pedicle-based breast shaping in reduction mammaplasty: a technical modification.

BACKGROUND: We present a technical modification of vertical reduction mammaplasty which provides a reliable pedicle that can be used in large and highly ptotic breasts with confidence when compared to vertical mammaplasty techniques without sacrificing conical breast shape and projection, in contrast to Wise pattern reduction techniques. METHODS: Thirty-two patients under general anesthesia were operated on using this modification between 2008 and 2012. The surgical technique is as follows: after general anesthesia induction and local anesthetic infiltration, skin incisions are made according to preoperative drawings. The breast meridian is prepared by superior and inferior plication of the vertical pedicle, including two dermal and one central attachment to the chest wall. Lateral and medial tissue resections are performed, thus preparing medial and lateral pillars after skin undermining. The pillars are sutured to the meridian to reconstruct a projectile conical breast shape. Inverted-T scar (87.5%, n=28) and vertical scar (12.5%, n=4) were used for closure. RESULTS: All patients were satisfied with the outcome regarding breast projection, shape, and size at 12 (n=30) and 24 months (n=15) after surgery except for 12 cases that needed reoperations: 2 cases for bottoming out and lower pole deformity, 2 cases needed more reduction by liposuction or re-excision, and 8 scar revisions. CONCLUSION: Early and late results (up to 2 years) regarding breast shape and projection were found to be satisfactory while providing a reliable pedicle with less postoperative drainage. LEVEL OF EVIDENCE V: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Intrascrotal extratesticular neurofibroma as a possible cause of failed descent in ipsilateral testis.

Intrascrotal extratesticular neurofibromas (IEN) often originate from genitofemoral nerve (GFN) and present as a paratesticular mass. Synchronous presence of IEN and undescended testis has not been reported previously. A 12-year-old boy with neurocutaneous syndrome and congenital giant melanocytic nevi along with IEN and ipsilateral undescended testis is presented, to discuss the underlying pathophysiology of failed testicular descent in the presence of IEN.

Management of gastrostomy to prevent perforation in acute severe corrosive esophagitis and gastritis: an experimental study.

BACKGROUND/AIMS: Symptomatic treatment is still the most commonly preferred treatment modality for acute severe esophagitis and gastritis. Clinical results of this treatment range from pathologies like stricture formation to loss of life. In our study, we aimed to demonstrate the effect of immediate gastrostomy in preventing perforation due to corrosive trauma. METHODS: We used 32 rats in two study groups. In Group I (n: 16 rats), 1 ml of corrosive agent (10% NaOH solution) was administered and immediate gastrostomies were performed within 2 hours. In Group II (n: 16 rats), 1 ml corrosive agent (10% NaOH solution) was administered and the rats were treated symptomatically; no operation was performed. RESULTS: Acute death was observed in 5 rats just after the corrosive agent was administered at the beginning of the study. Three rats from Group II died due to esophageal and gastric perforation within one week (25%). Necrosis was reported in 5 non-gastrostomized rats; however, no necrosis was observed in the gastrostomized group (p=0.037). CONCLUSIONS: Severe acute corrosive esophagitis and gastritis may be fatal. Furthermore, survivors may suffer from lifelong associated problems. From this study, we concluded that immediate gastrostomy in acute corrosive esophagitis and gastritis may play an important role in preventing necrosis and perforation risk.

Reconstruction of coup de sabre deformity (linear localized scleroderma) by using galeal frontalis muscle flap and demineralized bone matrix combination.

In this clinical report, we are presenting the combination of demineralized bone matrix combined with bilateral galea frontalis flaps. Based on our 6-month results, this seems to be a reasonable combination to accomplish long-lasting restoration of forehead defects related to en coup de sabre linear localized scleroderma.

Simultaneous reconstruction of medial canthal area and both eyelids with a single transverse split forehead island flap.

In this report, we are presenting a case in which we have split the paramedian forehead flap, thus providing 2 axially perfused skin flaps for simultaneous reconstruction of the upper and lower lid structures after resection of basal cell carcinoma from the left medial canthal area. We found that split forehead flap seems to be a favorable option for simultaneous reconstruction of the upper and lower eyelid defects by enabling nicely vascularized and abundant amount of regional skin.

Topographical anatomy of the dorsal branch of the ulnar nerve and artery: a cadaver study.

PURPOSE: The surgical anatomy of the dorsal branch of the ulnar nerve and artery on the dorsal aspect of the hand is important in design of neurocutaneous flaps for reconstructive surgery and serves as a donor site for nerve grafts. In this study, the course, location, and diameter of the dorsal branches of the ulnar nerve and artery were studied from anatomical and reconstructive perspectives. METHODS: Upper limbs of 14 (7 left and 7 right) and 22 formalin-preserved adult cadavers (15 left and 7 right) were dissected in two different centers. RESULTS: The diameters of the ulnar nerve, artery, and their dorsal branches were measured at selected reference points. The distances to specific anatomical landmarks were also measured, during their courses from the proximal forearm towards the middle phalanges of the 4th and 5th fingers. CONCLUSIONS: Our data may facilitate the design of neurocutaneous flaps nourished from the dorsal branches of the ulnar nerve and artery, and may aid in the harvesting of nerve grafts from the dorsal branch of the ulnar nerve, and provide a safe surgical approach to the dorsum of the hand.

Tolerance of prefabricated skin flaps to ischaemia and reperfusion: experimental study.

Custom prefabrication of tissues allows the surgeon to build what is required for the reconstruction and has enabled the surgeon to reinforce new blood supply into selected blocks of tissue without vessel anastomosis. However, prefabricated flaps have several drawbacks and characteristics that differentiate them from conventional flaps. The objective of this study was to test the tolerance of prefabricated flaps to ischaemia/reperfusion injury in rats. In the first stage, the unilateral-inferior-epigastric pedicle was ligated and divided, and then a skin flap was fabricated by implantation of distally ligated femoral arteriovenous pedicle beneath the abdominal skin. The femoral vessels were implanted either in skeletonised or in muscle-cuffed fashion beneath the abdominal skin, a portion of which was raised as an island flap, based on these vessels. Prefabricated flaps (3×6 cm) were raised 6 weeks after, and were subjected to 10 h ischaemia and followed by 12 h reperfusion. Flap survival and histological changes at the pedicle-skin junction were evaluated at 7 days. Flap necrosis in the sham group was 0%, whereas the control group (conventional) had 47.27±13.50% necrosis. Flaps prefabricated with skeletonised femoral pedicle demonstrated an insignificant pattern with 63.74±10.62% necrosis when compared with prefabricated flaps with muscle-cuffed pedicle with the percentage of necrosis of 64.51±11.24. The area of necrosis was significantly increased when both the prefabricated flaps were compared with the control group or with the sham-prefabricated group (p<0.05). Skin flaps prefabricated with either pedicle-alone or pedicles with muscle cuff are more susceptible to ischaemia and following reperfusion in comparison with the normal flaps.