Implant cement spacer-a cost-effective solution for reconstruction of proximal humerus defects after tumor resection.

The proximal humerus is a common location for both primary benign and malignant bone tumors and may require sacrificing deltoid muscles, axillary nerve and/or rotator cuff along with proximal humerus resection. Thus, post operatively shoulder movements are restricted. The main goals of reconstruction are to maintain a stable shoulder so that the function of elbow and hand can be optimized. Various reconstruction options are available after proximal humerus resection. We present our experience in using implant-cement spacers as a primary reconstruction option for limb salvage in the primary tumors of proximal humerus. All cases were retrieved from our prospectively maintained surgical database. 142 patients (96 males and 46 females) with a median age of 17.5 years (3-70 years) were operated with implant cement spacer between January 2006 and April 2019. Median follow up was 34 months (1-174 months). Functional outcome of the surgery was assessed in survivors by Musculoskeletal Tumor Society score (MSTS). Implant survival was assessed by Kaplan Meier analysis and competing risk analysis. On last follow up, out of 142 cases, 81 patients had died, 54 are alive and seven were lost to follow up. 18(13%) patients underwent revision surgery for symptomatic proximal migration, implant failure or infection. Four (2.8%) patients underwent forequarter amputation for local recurrence. The five years implant survival (IS) by Kaplan Meier analysis was 79.6% and as per competing risk analysis, the chances of implant revision are 12% and 18% at five and ten years respectively. Mean MSTS score in survivors was 71% (60-80%). Implant cement spacer is a cost-effective alternative for reconstruction of proximal humerus with revision rates and function comparable to other reconstructions in cases where deltoid, axillary nerve and/or rotator cuff are excised.

Orthopedic manifestation as the presenting symptom of acute lymphoblastic leukemia.

INTRODUCTION: The diagnosis of Acute lymphoblastic leukemia (ALL) is delayed due to vague presentation and normal hematological investigations. OBJECTIVE: The objectives were to identify the frequency of ALL cases presented to the orthopedic department and with normal hematological investigations. MATERIAL AND METHODS: 250 consecutive ALL cases were retrospectively evaluated to identify cases with musculoskeletal manifestations, and laboratory investigations. RESULTS: Twenty-two patients (4- vertebral compression fractures, 12- joint pain, 6- bone pain), presented primarily to the orthopedic department. Six patients had a normal peripheral smear. CONCLUSION: The primary physician should maintain a high index of suspicion despite a normal peripheral smear.

Evaluation of effectiveness of wavelet based denoising schemes using ANN and SVM for bearing condition classification.

The wavelet based denoising has proven its ability to denoise the bearing vibration signals by improving the signal-to-noise ratio (SNR) and reducing the root-mean-square error (RMSE). In this paper seven wavelet based denoising schemes have been evaluated based on the performance of the Artificial Neural Network (ANN) and the Support Vector Machine (SVM), for the bearing condition classification. The work consists of two parts, the first part in which a synthetic signal simulating the defective bearing vibration signal with Gaussian noise was subjected to these denoising schemes. The best scheme based on the SNR and the RMSE was identified. In the second part, the vibration signals collected from a customized Rolling Element Bearing (REB) test rig for four bearing conditions were subjected to these denoising schemes. Several time and frequency domain features were extracted from the denoised signals, out of which a few sensitive features were selected using the Fisher's Criterion (FC). Extracted features were used to train and test the ANN and the SVM. The best denoising scheme identified, based on the classification performances of the ANN and the SVM, was found to be the same as the one obtained using the synthetic signal.

Three-dimensional culture regulates Raf-1 expression to modulate fibronectin matrix assembly.

Oncogenic transformation has been associated with decreased fibronectin (FN) matrix assembly. For example, both the HT-1080 fibrosarcoma and MAT-LyLu cell lines fail to assemble a FN matrix when grown in monolayer culture (2-dimensional [2D] system). In this study, we show that these cells regain the ability to assemble a FN matrix when they are grown as aggregates (3-dimensional [3D] system). FN matrix assembly in 3D correlates with decreased Raf-1 protein expression compared with cells grown in monolayer culture. This effect is associated with reduced Raf-1 mRNA levels as determined by quantitative RT-PCR and not proteasome-mediated degradation of endogenous Raf-1. Interestingly, transient expression of a Raf-1 promoter-reporter construct demonstrates increased Raf-1 promoter activity in 3D, suggesting that the transition to 3D culture may modulate Raf-1 mRNA stability. Finally, to confirm that decreased Raf-1 expression results in increased FN matrix assembly, we used both pharmacological and small interfering RNA knockdown of Raf-1. This restored the ability of cells in 2D culture to assemble a FN matrix. Moreover, overexpression of Raf-1 prevented FN matrix assembly by cells cultured in 3D, resulting in decreased aggregate compaction. This work provides new insight into how the cell microenvironment may influence Raf-1 expression to modulate cell-FN interactions in 3D.

Relationship of glycosyltransferases and mRNA levels to ganglioside expression in neuroblastoma and melanoma cells.

Most human neuroblastoma tumors are characterized by the high expression of GD2 (or GD2 and/or GM2) gangliosides, whereas melanomas characteristically express GD3 ganglioside. The molecular basis for these patterns was investigated by examining the relationship between ganglioside levels, glycosyltransferase (GM2/GD2 synthase and GD3 synthase) activity, and corresponding mRNA levels in a panel of human neuroblastoma and melanoma cell lines. In general, the ganglioside patterns could be explained by the levels of the transferases and their mRNA, indicating control at the level of transcription. A key role was noted for GD3 synthase. Notably, it was found that neuroblastoma cell lines with high GD2 ganglioside levels had low levels of GD3, its synthase, and mRNA for the enzyme even though this step provides the substrate for GD2 synthesis. The key role for GD3 synthase was also examined by stably transfecting GD3 synthase cDNA into a neuroblastoma cell line (SH-SY5Y) not expressing GD3 and GD2. The resulting cell line had high levels of GD2 ganglioside and altered morphology and growth characteristics.

Human melanoma cell lines deficient in GD3 ganglioside expression exhibit altered growth and tumorigenic characteristics.

We have selected GD3-deficient human melanoma cell lines, in order to investigate the function of GD3 ganglioside. This was done by treating SK-MEL-28 cells with anti-GD3 antibody (R24) and rabbit complement and subsequent subcloning of the surviving cells, resulting in the derivation of two cell lines deficient in the cell surface expression of GD3. Neither cell line (designated SK-MEL-28-N1 and SK-MEL-28-N2) had detectable cell surface expression of GD3 as analyzed with monoclonal antibody R24, and no GD3 was detectable in either cell line by glycolipid isolation, thin-layer chromatography, or resorcinol-HC1 spray, but thin-layer chromatography immunostaining with monoclonal antibody R24 showed the presence of low amounts of GD3 in both N1 and N2 (1/40 of the amount in the parent cell line in N1 and 1/500 in N2). In SK-MEL-28-N1, the residual GD3 was shown by immunofluorescence assays on permeabilized cells to be present in discrete intracellular organelles, suggesting that these cells have a defect in the transport of GD3 as well as in its synthesis. Both SK-MEL-28-N1 and -N2 had an increase in detectable GM3 expression. The mutant cell lines had altered cell morphology in comparison to the parent cell line and both had slower growth rates in vitro and lower tumorgenicity in nu/nu mice. These results indicate that GD3 ganglioside plays an important role in proliferation and growth of melanoma cells.

Analysis of melanoma cells stably transfected with beta 1,4GalNAc transferase (GM2/GD2 synthase) cDNA: relative glycosyltransferase levels play a dominant role in determining ganglioside expression.

Previous studies (Y. Nagata, S. Yamashiro, J. Yodoi, K. O. Lloyd, H. Shiku, and K. Furukawa (1992) J. Biol. Chem. 267, 12082-12089) had isolated a putative cDNA coding for beta 1,4 N-acetylgalactosaminyltransferase (GM2/GD2 synthase) and demonstrated the presence of GM2 and/or GD2 gangliosides in melanoma cell lines stably transfected with this gene. We have now measured the levels of glycosyltransferase activities and mRNA levels in five transfected cell lines in comparison with their parent cell lines (mouse melanoma B16 and human melanoma MeWo). Membrane preparations from the transfected cell lines demonstrated de novo synthesis of GM2 or GD2 in in vitro assays using GM3 or GD3, respectively, as ganglioside acceptors. The enzyme levels, however, varied considerably among the different transfectants, as did the mRNA levels for the beta 1,4 Gal-NAc-transferase. The effect of the transfected gene on levels of preexisting glycosyltransferases involved in ganglioside biosynthesis was also measured and the ganglioside composition of the cell lines was determined. The level of beta 1,4 GalNAc-transferase expressed in the different cell lines was found to dramatically influence the overall ganglioside composition of the cell. In the transfected cell line with the highest levels of the transferase, for example, biosynthesis was almost completely redirected away from the b pathway to the a pathway with the resulting expression of only GM2. These data from this family of related cell lines clearly demonstrate the primary roles that relative glycosyltransferase levels play in determining the ganglioside composition of cells.

Fibroblast activation protein: purification, epitope mapping and induction by growth factors.

The human fibroblast activation protein (FAP) defined by monoclonal antibody (MAb) F19 is a cell surface antigen expressed in reactive stromal fibroblasts of breast, colorectal, lung and other epithelial cancers. In contrast to its stroma-specific localization in epithelial neoplasms, FAP is expressed in the malignant mesenchymal cells of bone and soft tissue sarcomas. FAP is transiently expressed in some fetal mesenchymal tissues but is absent or expressed at low levels in most adult tissues. FAP is induced in cultured fibroblasts and, in these cells, consists of a M(r) 95,000 subunit (FAP alpha) carrying the F19 epitope and a non-covalently bound M(r) 105,000 subunit (FAP beta) lacking the F19 epitope. Using MAb F19 and 5 newly derived MAbs, we identify 3 distinct epitopes on FAP alpha and tentatively assign one epitope to FAP beta. Analysis of detergent extracts of a FAP alpha high beta- sarcoma cell line by size exclusion-high performance liquid chromatography (HPLC) revealed that FAP alpha does not elute as a M(r) 95,000 species but as part of a high-molecular weight complex (M(r) > 400,000) that dissociates into M(r) 95,000 subunits in SDS gels. Immunoaffinity purification of FAP alpha followed by tryptic digestion, reversed-phase HPLC and microsequencing identified 3 unique FAP alpha peptides, with 2 showing sequence similarity (23/38 identical amino acids) to segments of CD26, a T-cell activation antigen. CD26 is a membrane-bound enzyme (dipeptidyl aminopeptidase IV), but immunopurified FAP alpha has little if any dipeptidase activity with typical CD26 substrates. Finally, studies with FAPlow leptomeningeal fibroblasts revealed that transforming growth factor-beta, 12-O-tetradecanoyl phorbol-13-acetate and retinoids can upregulate FAP expression, whereas serum and several other factors had no or little effect on FAP levels. FAP and CD26 may belong to a family of structurally related but functionally distinct activation proteins that are expressed on different cell types and show unique modes of regulation in normal and malignant cells.

Molecular cloning of fibroblast activation protein alpha, a member of the serine protease family selectively expressed in stromal fibroblasts of epithelial cancers.

The human fibroblast activation protein alpha (FAP alpha) is a M(r) 95,000 cell surface antigen selectively expressed in reactive stromal fibroblasts of epithelial cancers, granulation tissue of healing wounds, and malignant cells of bone and soft tissue sarcomas. Normal adult tissues are generally FAP alpha-, but some fetal mesenchymal tissues transiently express the molecule. Because of its restricted normal tissue distribution and abundant expression in the stroma of over 90% of breast, colorectal, and lung carcinomas, FAP alpha is under clinical evaluation as a target for immunodetection and immunotherapy of epithelial cancers. In the present study, we have isolated a full-length cDNA for FAP alpha through expression cloning in COS-1 cells. The FAP alpha cDNA codes for a type II integral membrane protein with a large extracellular domain, transmembrane segment, and short cytoplasmic tail. FAP alpha shows 48% amino acid sequence identity to the T-cell activation antigen CD26, a membrane-bound protein with dipeptidyl peptidase IV (DPPIV) activity; however, unlike FAP alpha, CD26 is widely expressed in normal tissues. Three catalytic domains shared by DPPIV homologues in different species and by other serine proteases are conserved in FAP alpha. Immunochemical analysis of COS-1 cells coexpressing FAP alpha and CD26 revealed that the two molecules form heteromeric cell surface complexes, suggesting that a previously identified FAP alpha-associated M(r) 105,000 protein of cultured fibroblasts and growth factor-stimulated melanocytes, FAP beta, is identical to CD26. In vivo coexpression of FAP alpha and CD26 is found in reactive fibroblasts of healing wounds but not in tumor stromal fibroblasts or sarcomas (FAP alpha +/CD26-). The putative serine protease activity of FAP alpha and its in vivo induction pattern may indicate a role for this molecule in the control of fibroblast growth or epithelial-mesenchymal interactions during development, tissue repair, and epithelial carcinogenesis.

Polymorphism of esterase D and haptoglobin in the Madras City population.

Blood and serum samples from random individuals of Madras City, South India, were screened for ESD and HP polymorphisms using mixed starch agarose gel electrophoresis and polyacrylamide slab gel electrophoresis respectively. The allelic frequencies for HP*1 and HP*2 in Madras are 0.1240 and 0.8760 respectively, while ESD*1 and ESD*2 gene frequency estimates were 0.7680 and 0.2320 respectively. The results are compared with those available for other Indian and world populations.

Studies of association between some genetic systems (ABO, MN, EsD, GLO, C3 and Hp).

Using random population data on the ABO, MN, esterase D (EsD), glyoxalase I, complement (C3) and haptoglobin markers in the population of Madras City associations were studied between these genetic systems. Out of a total of fifteen comparisons one significant association (chi 2 = 11.0; d.f. 4; 0.05 greater than p greater than 0.02) was found between the EsD and C3 phenotypes.

C3 polymorphism in Kotas and Badagas of Nilgiri Hills, south India.

Blood serum samples from Kota (n = 95) and Badaga (n = 113) groups of the Nilgiri Hills, South India, were screened for C3 polymorphism using polyacrylamide slab gel electrophoresis. The distribution of the three common phenotypes F, FS and S and a variant phenotype is reported. Two deficient individuals, one per subsample, have been found. The allelic frequencies for C3*F and C3*S in Kotas and Badagas are 0.1075, 0.8925 and 0.0982, 0.9018 respectively. The results are discussed in the light of available literature on C3 polymorphism in other Indian populations.

Glyoxalase-I and esterase D polymorphisms in Kotas and Badagas of Nilgiri Hills, south India.

Blood samples from 217 unrelated individuals belonging to two endogamous populations, Kotas and Badagas of the Nilgiri Hills, South India, were screened for glyoxalase I and esterase D polymorphisms using mixed starch-agarose gel electrophoresis. The GLO1*1 gene frequency estimates were 0.1887 and 0.1982 for Kotas and Badagas. The ESD*1 gene frequency estimates were 0.7123 and 0.7568 for Kotas and Badagas, respectively. The results are compared with those available for other Indian populations.

Distribution of HLA antigens in Kotas and Badagas of the Nilgiri Hills, South India.

Blood samples from 103 Kotas and 58 Badagas residing in the Nilgiri Hills, South India, were examined for HLA-A and -B antigen profiles. The Kota group was characterized by fairly high frequencies of A2 and B7 antigens as well as the haplotype A2-B7. The frequencies of Aw19, A28, and Bw22 were found to be higher in Badagas than in Kotas. The results are compared with the literature available on other Indian populations.