Mesenchymal Stem Cells Genetically Modified by Lentivirus-Express Soluble TRAIL and Interleukin-12 Inhibit Growth and Reduced Metastasis-Relate Changes in Lymphoma Mice Model.

BACKGROUND: Cancer treatment has many side effects; therefore, more efficient treatments are needed. Mesenchymal stem cells (MSC) have immunoregulatory properties, tumor site migration and can be genetically modified. Some proteins, such as soluble TRAIL (sTRAIL) and interleukin-12 (IL-12), have shown antitumoral potential, thus its combination in solid tumors could increase their activity. MATERIALS AND METHODS: Lentiviral transduction of bone marrow MSC with green fluorescent protein (GFP) and transgenes (sTRAIL and IL-12) was confirmed by fluorescence microscopy and Western blot. Soluble TRAIL levels were quantified by ELISA. Lymphoma L5178Y cells express a reporter gene (GFP/mCherry), and TRAIL receptor (DR5). RESULTS: An in vivo model showed that combined treatment with MSC expressing sTRAIL+IL-12 or IL-12 alone significantly reduced tumor volume and increased survival in BALB/c mice (p < 0.05) with only one application. However, at the histological level, only MSC expressing IL-12 reduced tumor cell infiltration significantly in the right gastrocnemius compared with the control group (p < 0.05). It presented less tissue dysplasia confirmed by fluorescence and hematoxylin-eosin dye; nevertheless, treatment not inhibited hepatic metastasis. CONCLUSIONS: MSC expressing IL-12, is or combination with BM-MSC expressing sTRAIL represents an antitumor strategy for lymphoma tumors since they increase survival and reduce tumor development. However, the combination did not show significative additive effect. The localized application did not inhibit metastasis but reduced morphological alterations of tissue associated with liver metastasis.

A combined antitumor strategy of separately transduced mesenchymal stem cells with soluble TRAIL and IFNß produces a synergistic activity in the reduction of lymphoma and mice survival enlargement.

As the understanding of cancer grows, new therapies have been proposed to improve the well-known limitations of current therapies, whose efficiency relies mostly on early detection, surgery and chemotherapy. Mesenchymal stem cells (MSCs) have been introduced as a promissory and effective therapy. This fact is due to several useful features of MSCs, such as their accessibility and easy culture and expansion in vitro, and their remarkable ability for 'homing' towards tumors, allowing MSCs to exert their anticancer effects directly into tumors. Additionally, MSCs offer the practicability of being genetically engineered to carry anticancer genes, increasing their specificity and efficacy for fighting tumors. In the present study, the antitumoral efficacy and post-implant survival of mice bearing lymphomas implanted intratumorally were determined using mouse bone marrow-derived (BM)-MSCs transduced with soluble TRAIL (sTRAIL), full length TRAIL (flTRAIL), or interferon ß (IFNß), naïve BM-MSCs, or combinations of these. The percentage of surviving mice was determined once all not-implanted mice succumbed. It was found that the percentage of surviving mice implanted with the combination of MSCs-sTRAIL and MSCs-IFN-ß was 62.5%. Lymphoma model achieved 100% fatality in the non-treated group by day 41. On the other hand, the percentage of surviving mice implanted with MSCs-sTRAIL was 50% and with MSCs-INFß 25%. All the aforementioned differences were statistically significant (P<0.05). In conclusion, all implants exhibited tumor size reduction, growth delay, or apparent tumor clearance. MSCs proved to be effective anti-lymphoma agents; additionally, the combination of soluble TRAIL and IFN-ß resulted in the most effective antitumor and life enlarging treatment, showing an additive antitumoral effect compared with individual treatments.

Repair of ovine peripheral nerve injuries with xenogeneic human acellular sciatic nerves prerecellularized with allogeneic Schwann-like cells-an innovative and promising approach.

INTRODUCTION: The iatrogenic effects of repairing peripheral nerve injuries (PNIs) with autografts (AGTs) encouraged the present study to involve a new approach consisting of grafting xenogeneic prerecellularized allogeneic cells instead of AGTs. METHODS: We compared sheep's AGT regenerative and functional capacity with decellularized human nerves prerecellularized with allogeneic Schwann-like cell xenografts (onwards called xenografts). Mesenchymal stem cells were isolated from ovine adipose tissue and induced in vitro to differentiate into Schwann-like cells (SLCs). Xenografts were grafted in ovine sciatic nerves. Left sciatic nerves (20 mm) were excised from 10 sheep. Then, five sheep were grafted with 20 mm xenografts, and five were reimplanted with their nerve segment rotated 180° (AGT). RESULTS: All sheep treated with xenografts or AGT progressively recovered the strength, movement, and coordination of their intervened limb, which was still partial when the study was finished at sixth month postsurgery. At this time, numerous intrafascicular axons were observed in the distal and proximal graft extremes of both xenografts or AGTs, and submaximal nerve electrical conduction was observed. The xenografts and AGT-affected muscles appeared partially stunted. CONCLUSIONS: Xenografts and AGT were equally efficacious in starting PNI repair and justified further studies using longer observation times. The hallmarks from this study are that human xenogeneic acellular scaffolds were recellularized with allogenic SCL and were not rejected by the nonhuman receptors but were also as functional as AGT within a relatively short time postsurgery. Thus, this innovative approach promises to be more practical and accessible than AGT or allogenic allografts and safer than AGT for PNI repair.

Treatment of Chondral Knee Lesions with Autologous Chondrocytes Embedded in a Fibrin Scaffold. Clinical and Functional Assessment.

Objective The aim of our study is to analyze the clinical and functional results obtained using autologous chondrocytes embedded in a fibrin scaffold in knee joint injuries. Methods We included 56 patients, 36 men and 20 women, with a mean age 36 years. Six of the patients were professional athletes, with single knee injuries that were either chondral or osteochondral (43 chondral, 9 osteochondral, 2 cases of osteochondritis dissecans and 2 osteochondral fractures), 2 to 10 cm 2 in size and ≤ 10 mm deep, with no signs of osteoarthritis. The location of the injury was in the patella (8), the medial femoral condyle (40) and lateral femoral condyle (7) and one in the trochlea. The mean follow-up was 3 (range: 1-6) years. The clinical course was assessed using the Cincinnati and Knee Injury and Osteoarthritis Outcome (KOOS) scores, 6 and 12 months after surgery. The paired Student t-test was used to compare pre-and postoperative results. Results Six months after the implant, patients resumed their everyday activities. On the assessment scores, their condition was improving in comparison with their presurgical state ( p < 0.05). They were also able to carry out their sporting activities more easily than prior to surgery ( p < 0.05). Conclusion The seeding of chondrocytes in fibrin may provide a favorable micro-environment for the synthesis of extracellular matrix and improved the clinical condition and activity of the patients 1 year after surgery.

Chemosensitivity analysis and study of gene resistance on tumors and cancer stem cell isolates from patients with colorectal cancer.

Colorectal cancer (CRC) is one of the main causes of mortality. Recent studies suggest that cancer stem cells (CSCs) can survive after chemotherapy and promote tumor invasiveness and aggression. According to a higher hierarchy complexity of CSC, different protocols for isolation, expansion, and characterization have been used; however, there are no available resistance biomarkers that allow predicting the clinical response of treatment 5­fluorouracil (5FU) and oxaliplatin. Therefore, the primary aim of the present study was to analyze the expression of gene resistance on tumors and CSC­derived isolates from patients CRC. In the present study, adenocarcinomas of the colon and rectum (CRAC) were classified based on an in vitro adenosine triphosphate­based chemotherapy response assay, as sensitive and resistant and the percentage of CD24 and CD44 markers are evaluated by immunohistochemistry. To isolate resistant colon­CSC, adenocarcinoma tissues resistant to 5FU and oxaliplatin were evaluated. Finally, all samples were sequenced using a custom assay with chemoresistance­associated genes to find a candidate gene on resistance colon­CSC. Results showed that 59% of the CRC tissue analyzed was resistant and had a higher percentage of CD44 and CD24 markers. An association was found in the expression of some genes between the tumor­resistant tissue and CSC. Overall, isolates of the CSC population CD44+ resistant to 5FU and oxaliplatin demonstrated different expression profiles; however, the present study was able to identify overexpression of the KRT­18 gene, in most of the isolates. In conclusion, the results of the present study showed overexpression of KRT­18 in CD44+ cells is associated with chemoresistance to 5FU and oxaliplatin in CRAC.

Treatment of Chondral Knee Lesions with Autologous Chondrocytes Embedded in a Fibrin Scaffold. Clinical and Functional Assessment / Tratamento de lesões condrais no joelho com condrócitos autólogos embebidos em arcabouço de fibrina. Avaliação clínica e funcional

Abstract Objective The aim of our study is to analyze the clinical and functional results obtained using autologous chondrocytes embedded in a fibrin scaffold in knee joint injuries. Methods We included 56 patients, 36 men and 20 women, with a mean age 36 years. Six of the patients were professional athletes, with single knee injuries that were either chondral or osteochondral (43 chondral, 9 osteochondral, 2 cases of osteochondritis dissecans and 2 osteochondral fractures), 2 to 10 cm2 in size and ≤ 10 mm deep, with no signs of osteoarthritis. The location of the injury was in the patella (8), the medial femoral condyle (40) and lateral femoral condyle (7) and one in the trochlea. The mean follow-up was 3 (range: 1-6) years. The clinical course was assessed using the Cincinnati and Knee Injury and Osteoarthritis Outcome (KOOS) scores, 6 and 12 months after surgery. The paired Student t-test was used to compare pre-and postoperative results. Results Six months after the implant, patients resumed their everyday activities. On the assessment scores, their condition was improving in comparison with their presurgical state (p < 0.05). They were also able to carry out their sporting activities more easily than prior to surgery (p < 0.05). Conclusion The seeding of chondrocytes in fibrin may provide a favorable microenvironment for the synthesis of extracellular matrix and improved the clinical condition and activity of the patients 1 year after surgery.

Resumo Objetivo O objetivo do nosso estudo é analisar os resultados clínicos e funcionais do tratamento de lesões nas articulações do joelho com condrócitos autólogos embebidos em arcabouço de fibrina. Métodos O estudo foi realizado com 56 pacientes (36 homens e 20 mulheres) com idade média de 36 anos; 6 indivíduos eram atletas profissionais. Os pacientes apresentavam lesões únicas, condrais ou osteocondrais (43 condrais, nove osteocondrais, 2 casos de osteocondrite dissecante e duas fraturas osteocondrais) no joelho, com 2 a 10 cm2 de tamanho e ≤ 10 mm de profundidade, sem sinais de osteoartrite. As lesões estavam localizadas na patela (8), no côndilo femoral medial (40), no côndilo femoral lateral (7) e na tróclea (1). O período médio de acompanhamento foi de 3 anos (faixa de 1-6 anos). A evolução clínica foi avaliada pelos escores de Cincinnati e Knee Injury and Osteoarthritis Outcome (KOOS), 6 e 12 meses após a cirurgia. O teste t de Student pareado foi utilizado para comparação dos achados pré e pós-operatórios. Resultados Os pacientes retomaram suas atividades diárias 6 meses após o implante. Os escores avaliados demonstraram a melhora em comparação ao estado pré-cirúrgico (p < 0,05). Além disso, os pacientes conseguiram realizar suas atividades esportivas com mais facilidade do que antes da cirurgia (p < 0,05). Conclusão A cultura de condrócitos em fibrina pode proporcionar um microambiente favorável para a síntese de matriz extracelular e melhorar a condição clínica e a atividade dos pacientes 1 ano após a cirurgia

Updates and new medical treatments for vitiligo (Review).

Vitiligo is a multifactorial disease characterized by the loss of skin pigment, which results in achromic macules and patches. There are currently several medical treatments available, which aim to arrest progression and induce skin repigmentation. These treatments alone or combined have exhibited varying degrees of pigmentation, and the majority are safe and effective. All therapies for vitiligo are limited, and no known treatment can consistently produce repigmentation in all patients. Individualized treatment is appropriate according to the location, clinical presentation and the presence of disease activity. The present review summarizes the medical treatments available for vitiligo: Systemic and topic pharmacological therapies, physical and depigmentation treatments. Several treatments are still underway and have not yet been approved. However, due to the promising preliminary results, these are also mentioned in the present review.

Novel immunological and genetic factors associated with vitiligo: A review.

Vitiligo is a skin disorder characterized by depigmentation of the skin due to a lack of melanin. This condition affects men and woman of all ages and its incidence is not restricted by ethnicity or region. Vitiligo is a multifactorial disease, in which melanocytes, which serve important functions in skin pigmentation and immune processes, are impaired. There is sufficient evidence that immunological and genetic factors are primarily responsible for the destruction and dysfunction of melanocytes. Therefore, genetic DNA sequence variants that participate in skin homeostasis, pigmentation and immune response regulation, as well as altered expression patterns, may contribute to the risk of developing vitiligo. The current review presented an overview of the mechanism of pigmentation and of currently known factors involved in depigmentation, as well as the classification, epidemiology, associated comorbidities, risk factors, immunopathogenesis and several genetic and molecular changes associated with vitiligo.

Anti­inflammatory and anti­catabolic effect of non­animal stabilized hyaluronic acid and mesenchymal stem cell­conditioned medium in an osteoarthritis coculture model.

Previous clinical studies have reported the clinical effectiveness of non­animal stabilized hyaluronic acid (NASHA) and adipose­derived mesenchymal stromal/stem cells (MSC) in the treatment of knee osteoarthritis (OA). Unlike MSC secreted mediators, in vitro anti­inflammatory effects of NASHA have not been evaluated. We aimed to evaluate and compare the anti­inflammatory effect of NASHA and MSC conditioned medium (stem cell­conditioned medium; SC­CM), in an explant­based coculture model of OA. Cartilage and synovial membrane from seven patients undergoing total knee arthroplasty were used to create a coculture system. Recombinant IL­1ß was added to the cocultures to induce inflammation. Four experimental groups were generated: i) Basal; ii) IL­1ß; iii) NASHA (NASHA + IL­1ß); and iv) SC­CM (SC­CM + IL­1ß). Glycosaminoglycans (GAG) released in the culture medium and of nitric oxide (NO) production were quantified. Gene expression in cartilage and synovium of IL­1ß, matrix metallopeptidase 13 (MMP13), ADAM metallopeptidase with thrombospondin type 1 motif 5 (ADAMTS5) and tissue inhibitor of metalloproteinases 1 (TIMP1) was measured by reverse transcription­quantitative PCR. Media GAG concentration was decreased in cocultures with NASHA and SC­CM (48 h, P<0.05; 72 h, P<0.01) compared with IL­1ß. Production of NO was significantly lower only in SC­CM after 72 h (P<0.01). In cartilage, SC­CM inhibited the expression of IL­1ß, MMP13 and ADAMTS5, while NASHA had this effect only in MMP13 and ADAMTS5. In synovium, SC­CM decreased the expression level of MMP13 and ADAMTS5, while NASHA only decreased ADAMTS5 expression. Both NASHA and SC­CM increased TIMP1 expression in cartilage and synovium. Treatments with NASHA and SC­CM were shown to be a therapeutic option that may help counteract the catabolism produced by the inflammatory state in knee OA. The anti­inflammatory mediators produced by MSC promote a lower expression of inflammatory targets in our study model.

Selective efficacy and safety of first line and non front line antineoplasic agent combinations determined with individualized assays on colorectal cancer primary cultures.

PURPOSE: To evaluate the efficacy of first-line and not-conventional antineoplastic drug combinations in colorectal adenocarcinoma primary cultures (CRAC PCs). METHODS: The efficacy and safety of 21 drug combinations (DCs) were evaluated using a simplified adenosine triphosphate-based chemotherapy response assay (sATP-CRA). The efficacy of each DC was reported as the percentage of cell death (PCD) produced on each of 12 CRAC-PCs, and the safety of each DC was evaluated as a safety window (SW). The SW was calculated as the quotient of PCD-CRAC PC/PCD-hMSC-TA (human mesenchymal stem cells derived from adipose tissue). Nine DCs contained 5-fluorouracil and oxaliplatin, and 1-3 non-front line drugs (NFLDs [carboplatin, doxorubicin, cisplatin, aspirin, or 3,3' diindolylmethane]). The other 11 DCs only contained 2-4 NFLDs. RESULTS: The efficacy and safety each DC were highly variable and depended on each CRAC PC and DC. The usefulness of DCs was considered as a combination of PCD >20 and an SW >0.6: 13 /21 DCs (62%) met the requirements of efficacy and safety on 7/12 CRAC PCs (58.3%). CONCLUSIONS: The resistance to 5-fluorouracil/oxaliplatin of CRAC PCs and the usefulness of seven new DCs strongly suggest the convenience of performing ex vivo individualized assays to evaluate DCs, and implement new and more useful treatments, instead of submitting patients to standardized chemotherapies in a blinded manner. Approaches such as this and properly evaluated in clinical assays could increase the life expectancy of patients with cancer and improve their quality of life.

Magnetite Nanoparticles Coated with PEG 3350-Tween 80: In Vitro Characterization Using Primary Cell Cultures.

Some medical applications of magnetic nanoparticles require direct contact with healthy tissues and blood. If nanoparticles are not designed properly, they can cause several problems, such as cytotoxicity or hemolysis. A strategy for improvement the biological proprieties of magnetic nanoparticles is their functionalization with biocompatible polymers and nonionic surfactants. In this study we compared bare magnetite nanoparticles against magnetite nanoparticles coated with a combination of polyethylene glycol 3350 (PEG 3350) and polysorbate 80 (Tween 80). Physical characteristics of nanoparticles were evaluated. A primary culture of sheep adipose mesenchymal stem cells was developed to measure nanoparticle cytotoxicity. A sample of erythrocytes from a healthy donor was used for the hemolysis assay. Results showed the successful obtention of magnetite nanoparticles coated with PEG 3350-Tween 80, with a spherical shape, average size of 119.2 nm and a zeta potential of +5.61 mV. Interaction with mesenchymal stem cells showed a non-cytotoxic propriety at doses lower than 1000 µg/mL. Interaction with erythrocytes showed a non-hemolytic propriety at doses lower than 100 µg/mL. In vitro information obtained from this work concludes that the use of magnetite nanoparticles coated with PEG 3350-Tween 80 is safe for a biological system at low doses.

Schwann Cell Precursor Transplant in a Rat Spinal Cord Injury Model.

BACKGROUND: Differentiation of mesenchymal stem cells into Schwann cell precursors could reverse established lesions and sequelae of medullary transection. OBJECTIVE: The objective of this study was to study the clinical response of mesenchymal stem cell transplantation with Schwann precursor cell transplantation in a rat spinal cord injury model, using motor function and histopathologic studies. MATERIALS AND METHODS: A total of 28 Sprague-Dawley rats were randomly divided among four groups (n = 7 in each): sham group, control group, mesenchymal stem cell transplant group, and Schwann cell precursor transplant group. The surgical procedure was a laminectomy with transection of the spinal cord at the T11 level in the transplant groups and the injury control group. After 1 week, the transplant groups received stem cells directly in the injury site. Hind limb motor function was assessed using the locomotive scale of Basso, Beattie, and Bresnahan. 1 month after transplantation, all specimens were sacrificed to make a histopathologic description of sections taken from the site of injury and where stem cells were transplanted. Mean scores of mobility were compared using analysis of variance (ANOVA) of one factor with 95% reliability between groups and ANOVA of repetitive measures to evaluate evolution in the same group. RESULTS: We observed that the control group had statistically greater mobility than the other groups (p < 0.0001) and that the group with spinal injury without treatment had the lowest mean mobility. The mobility score values from the Schwann cell precursor group were statistically higher than the group treated with mesenchymal stem cells (p < 0.0001). CONCLUSION: Schwann precursor cells had a greater effect on locomotive function than mesenchymal stem cells.

The matrix synthesis and anti-inflammatory effect of autologous leukocyte-poor platelet rich plasma in human cartilage explants.

OBJECTIVE: To determine the effects of autologous leukocyte-poor platelet-rich plasma (LP-PRP) on the expression of markers involved in cartilage-extracellular matrix production and inflammation in cartilage explants bearing osteoarthritis. MATERIALS AND METHODS: Cartilage explants and LP-PRP were obtained from 10 patients who underwent total knee arthroplasty. The explants were cultured in spinner flasks for 28 days in the presence of interleukin (IL)-1ß and/or LP-PRP. The gene expression of catabolic (MMP13, ADAMTS5, and IL1ß) and anabolic factors (COL2A1, ACAN, and SOX9) was quantified. A histological assessment was performed according to a modified Mankin score, and quantification of type II and I collagen deposition. RESULTS: The gene expression of catabolic factors and the Mankin score were lower in LP-PRP- and LP-PRP/IL-1ß- than in IL-1ß-treated explants, suggesting less matrix degradation in explants cultured in the presence of LP-PRP. Higher expression of genes involved in cartilage matrix restoration was observed in LP-PRP and LP-PRP/IL-1ß- when compared to IL-1ß-treated explants. The explants treated with LP-PRP and LP-PRP/IL-1ß exhibited a higher deposition of type II collagen as well as a lower deposition of type I collagen and also better surface integrity and a significant increase in the number of chondrocytes. CONCLUSION: LP-PRP treatment favored restoration in early osteoarthritic cartilage and reduced the pro-inflammatory effect of IL-1ß. LP-PRP is a promising therapy for early osteoarthritis, as it promotes extracellular matrix repair, reduces inflammation, and slows cartilage degeneration.

Bone marrow mesenchymal stem cells: improving transgene expression level, transfection efficiency and cell viability.

PURPOSE: Advanced cancer is a catastrophic medical condition that is generally treated with surgery and conventional anticancer drugs, which are very toxic and often fail. A promising alternative is using genetically engineered mesenchymal stem cells. A popular method for genetically engineering mesenchymal stem cells (MSCs) is by employing transfection reagents. Nevertheless, a serious limitation of this procedure is its consistently low transfection efficiency. Therefore, the utility of transfection reagents in regenerative medicine - including cancer treatment - might increase strikingly by increasing their transfection efficiency and maintaining, to the greatest extent possible, cell viability and transgene expression levels. The purpose of this study was to analyze various effects on gene expression level, transfection efficiency, and cell viability by increasing the volume of transfection reagents and the plasmid DNA mass. METHODS: Mouse bone marrow MSCs were transfected with trademarked Xfect®, Turbofect® or Lipofectamine 3000® and the plasmid pTracer-EF-His-A® expressing the green fluorescent protein (GFP). Additionally, we tested a protocol modification recommended by the Xfect manufacturer. The GFP expression level, transfection efficiency, and cell viability were evaluated together using a performance index. RESULTS: By doubling the quantities recommended by the manufacturers (reagent volume), plasmid DNA mass or both variables and by following a modified Xfect method, the transfection efficiency improved to 70%, the cell viability did not diminish, and the performance index increased to 47.7% with respect to the values determined using the original Xfect protocol. CONCLUSION: Transgene expression levels, transfection efficiency, and cell viability may be strikingly improved, by increasing the volume of the transfectant agent, the plasmid DNA mass or both, beyond those recommended by transfection kit manufacturers.

How desirable and undesirable features of naïve or genetically reengineered mesenchymal stem cells are being considered in preclinical or clinical assays.

The implantation of adult mesenchymal stem cells (MSCs) has become a promising alternative in cancer treatments. Accordingly, in this article we revised the ultimate advances in the knowledge on the MSC-homing mechanism, the cancer cell and MSCs interactions and the microvesicles and exosomes used by malignant cells to transport and deliver pro-cancer cytokines or microRNA (miRNA), or by MSCs to favor or fight cancer progression. In addition, we analyzed the current knowledge generated by ongoing or terminated preclinical and clinical trials, using naive MSCs as natural anti-cancer living factors or gene-engineered MSCs as cytokine delivering vehicles, where anti-cancer cytokines were chosen and the pro-cancer factors were avoided. Finally, we present some concerns about the implantation of MSCs and anti-cancer therapies and hypothesize the MSC implantation combines with conventional or new therapies to treat cancer.

Remarkably higher efficacy and a wider safety window for nonfrontline over first-line drug combinations in the adenocarcinoma Colo 320DM cell line.

PURPOSE: To determine in vitro, the efficacy and safety window of not-front-line and first-line anti-colorectal (CRC) drug combinations. METHODS: The adenocarcinoma cell line Colo 320DM and normal human mesenchymal stem cells derived from adipose tissue were used respectively to determine the anti-CRC efficacy (% of Colo 320DM cell death [CD]) and safety window [SW] - % Colo 320DM percent cancer death (PCD)/% of mesenchymal stem cell's death) of drug combinations, using the adenosine triphosphate-based chemotherapy response assay (ATP-CRA). RESULTS: First-line anti-CRC drug combinations (5-fluorouracil [5FU]/oxaliplatin [oxa] and 5-FU/Oxa /leucovorin [Leuco]) produced 57.7% and 52.4% CD, and 1.38 and 2.44 SW, respectively. Combinations of 5-FU/Oxa and 1 to 3 non-front line drugs led to 56.3-99.8% CD and to 0.96-2.2 SW. The highest safety window corresponded to 5FU/Oxa/ carboplatin [Carbo] (93% CD and 1.4 SW) and to 5-FU/ Oxa/cisplatin [Cispl] (93.5% CD and 1.4 SW). In contrast, non-front line drugs led to 89.8-97.4% CD and to 1.1-78.2 SW. Outstandingly, those combinations containing Carbo/ Cispl/3,3'-diindolylmethane (DIM), aspirin (Asp), or 3,3'- DIM/ Asp showed a very high CD (91.9-96.9% [39.2-39.5 times higher than first-line-combined drugs]) and very wide SW (57.8-81.56 [66.6-40 times higher than the first-line drug combinations]). CONCLUSIONS: Human mesenchymal stem cells could be an excellent alternative to laboratory animals, when testing the safety profiles of drugs. The most promising combinations of non-frontline drugs to treat CRC are Carbo/Cispl/ Asp and Carbo/Cispl/DIM.

A New Gene Expression Signature for Triple Negative Breast Cancer Using Frozen Fresh Tissue before Neoadjuvant Chemotherapy.

Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer tumors. Comparisons between TNBC and non-triple negative breast cancer (nTNBC) may help to differentiate key components involved in TNBC neoplasms. The purpose of the study was to analyze the expression profile of TNBC versus nTNBC tumors in a homogeneous population from northeastern Mexico. A prospective study of 50 patients was conducted (25 TNBC and 25 nTNBC). Clinic parameters were equally distributed for TNBC and nTNBC: age at diagnosis (51 vs 47 years, p=0.1), glucose levels (107 mg/dl vs 104 mg/dl, p=0.64), and body mass index (28 vs 29, p=0.14), respectively. Core biopsies were collected for histopathological diagnosis and gene expression analyses. Total RNA was isolated and expression profiling was performed. 40 genes showed differential expression pattern in TNBC tumors. Among these, 9 over-expressed genes (PRKX/PRKY, UGT8, HMGA1, LPIN1, HAPLN3, and ANKRD11), and one under-expressed (ANX9) gene are involved in general metabolism. Based on this biochemical peculiarity, and the over-expression of BCL11A and FOXC1 (involved in tumor growth and metastasis, respectively) we validated by qPCR the expression profile of 7 genes out of the signature. In this report, a new gene signature for TNBC is proposed. To our knowledge, this is the first TNBC signature which describes genes involved in general metabolism. The findings may be pertinent for Mexican patients and require to be evaluated in further ethnic groups and populations.

Implant Composed of Demineralized Bone and Mesenchymal Stem Cells Genetically Modified with AdBMP2/AdBMP7 for the Regeneration of Bone Fractures in Ovis aries.

Adipose-derived mesenchymal stem cells (ADMSCs) are inducible to an osteogenic phenotype by the bone morphogenetic proteins (BMPs). This facilitates the generation of implants for bone tissue regeneration. This study evaluated the in vitro osteogenic differentiation of ADMSCs transduced individually and in combination with adenoviral vectors expressing BMP2 and BMP7. Moreover, the effectiveness of the implant containing ADMSCs transduced with the adenoviral vectors AdBMP2/AdBMP7 and embedded in demineralized bone matrix (DBM) was tested in a model of tibial fracture in sheep. This graft was compared to ewes implanted with untransduced ADMSCs embedded in the same matrix and with injured but untreated animals. In vivo results showed accelerated osteogenesis in the group treated with the AdBMP2/AdBMP7 transduced ADMSC graft, which also showed improved restoration of the normal bone morphology.

Thymidylate synthase polymorphism in Mexican patients with colon cancer treated with 5-fluorouracil.

PURPOSE: We analyzed the genotype and allele frequency of variable number tandem repeats (VNTR)-thymidylate synthase (TS) and its relationship with the disease evolution in colon cancer patients. METHODS: We selected 24 paraffin-embedded colon cancer tissue samples from Mexican patients who received a 5-fluorouracil (5-FU)-based chemotherapy regimen. Tumor tissue was digested with proteinase K and genomic DNA was isolated by the standard method with phenol-chloroform extraction. Polymerase chain reaction (PCR) was performed for TS genotyping of VNTR and the results were evaluated directly in a stained agarose gel. RESULTS: The allele frequency of 2 repeats (2R) was greater (0.66) than 3R (0.34) in metastatic colon cancer (x2=10.24; p=0.001)) however, no difference in allelic distribution between 2R (0.54) and 3R (0.46) in non metastatic patients was observed (x2=0.640; p=0.424). CONCLUSION: Our results suggest that Mexican patients with colon cancer present differences in the allelic distribution, the 2R allele being the most frequent.