Alpha1beta1 integrin is crucial for accumulation of epidermal T cells and the development of psoriasis.

Psoriasis is a common T cell-mediated autoimmune inflammatory disease. We show that blocking the interaction of alpha1beta1 integrin (VLA-1) with collagen prevented accumulation of epidermal T cells and immunopathology of psoriasis. Alpha1beta1 integrin, a major collagen-binding surface receptor, was exclusively expressed by epidermal but not dermal T cells. Alpha1beta1-positive T cells showed characteristic surface markers of effector memory cells and contained high levels of interferon-gamma but not interleukin-4. Blockade of alpha1beta1 inhibited migration of T cells into the epidermis in a clinically relevant xenotransplantation model. This was paralleled by a complete inhibition of psoriasis development, comparable to that caused by tumor necrosis factor-alpha blockers. These results define a crucial role for alpha1beta1 in controlling the accumulation of epidermal type 1 polarized effector memory T cells in a common human immunopathology and provide the basis for new strategies in psoriasis treatment focusing on T cell-extracellular matrix interactions.

Cell viability and noninvasive in vivo MRI tracking of 3D cell encapsulating self-assembled microcontainers.

Several molecular therapies require the implantation of cells that secrete biotherapeutic molecules and imaging the location and microenvironment of the cellular implant to ascertain its function. We demonstrate noninvasive in vivo magnetic resonance imaging (MRI) of self-assembled microcontainers that are capable of cell encapsulation. Negative contrast was obtained to discern the microcontainer with MRI; positive contrast was obtained in the complete absence of background signal. MRI on a clinical scanner highlights the translational nature of this research. The microcontainers were loaded with cells that were dispersed in an extracellular matrix, and implanted both subcutaneously and in human tumor xenografts in SCID mice. MRI was performed on the implants, and microcontainers retrieved postimplantation showed cell viability both within and proximal to the implant. The microcontainers are characterized by their small size, three dimensionality, controlled porosity, ease of parallel fabrication, chemical and mechanical stability, and noninvasive traceability in vivo.

Tumor targeting with newly designed biparatopic antibodies directed against two different epitopes of the carcinoembryonic antigen (CEA).

In an attempt to improve tumor targeting and tumor retention time of monoclonal antibodies (MAbs), we prepared biparatopic antibodies (BpAbs) having the capability of binding 2 different non-overlapping epitopes on the same target antigen molecule, namely, the carcinoembryonic antigen (CEA). Six BpAbs were constructed by coupling 2 different Fab' fragments from 4 different specific anti-CEA MAbs recognizing 4 CEA epitopes (Gold 1-4). Demonstration of the double paratopic binding of these antibodies for CEA was confirmed in vitro by inhibition radioimmunoassay and cross-inhibition analysis by surface plasmon resonance (SPR; BIACORE) technology. Using the latter technique, the affinity constants for CEA immobilized onto the sensor chip were found to range from 0.37 to 1.54 x 10(9) M(-1) for the 4 parental F(ab')2 fragments and from 1.88 to 10.14 x 10(9) M(-1) for the BpAbs, demonstrating the advantage of biparatopic binding over conventional F(ab')2 binding. The Ka improvement was particularly high for BpAb F6/35A7 and BpAb F6/B17 with a 9.5- and 8.1-fold increase, respectively, as compared with the parental F(ab')2. In vivo, the 6 BpAbs were compared with their 2 respective parental F(ab')2 by injection of 131I-BpAb/125I-F(ab')2 parental fragments into nude mice xenografted with the human colon carcinoma T380. Dissection 72 hr post-injection demonstrated that BpAb B17/CE25 and BpAb F6/B17 gave higher tumor uptake than that of their parental F(ab')2. This finding is particularly interesting for BpAb F6/B17, which compared favorably with the F6 F(ab')2, one of the best parental F(ab')2 fragments used in our study.

Effect of RasGAP N2 fragment-derived peptide on tumor growth in mice.

Peptides that interfere with the natural resistance of cancer cells to genotoxin-induced apoptosis may improve the efficacy of anticancer regimens. We have previously reported that a cell-permeable RasGAP-derived peptide (TAT-RasGAP(317-326)) specifically sensitizes tumor cells to genotoxin-induced apoptosis in vitro. Here, we examined the in vivo stability of a protease-resistant D-form of the peptide, RI.TAT-RasGAP(317-326), and its effect on tumor growth in nude mice bearing subcutaneous human colon cancer HCT116 xenograft tumors. After intraperitoneal injection, RI.TAT-RasGAP(317-326) persisted in the blood of nude mice for more than 1 hour and was detectable in various tissues and subcutaneous tumors. Tumor-bearing mice treated daily for 7 days with RI.TAT-RasGAP(317-326) (1.65 mg/kg body weight) and cisplatin (0.5 mg/kg body weight) or doxorubicin (0.25 mg/kg body weight) displayed reduced tumor growth compared with those treated with either genotoxin alone (n = 5-7 mice per group; P = .004 and P = .005, respectively; repeated measures analysis of variance [ANOVA, two-sided]). This ability of the RI.TAT-RasGAP(317-326) peptide to enhance the tumor growth inhibitory effect of cisplatin was still observed at peptide doses that were at least 150-fold lower than the dose lethal to 50% of mice. These findings provide the proof of principle that RI.TAT-RasGAP(317-326) may be useful for improving the efficacy of chemotherapy in patients.

Direct comparison of a radioiodinated intact chimeric anti-CEA MAb with its F(ab')2 fragment in nude mice bearing different human colon cancer xenografts.

Tumour localisation and tumour to normal tissue ratios of a chimeric anti-carcinoembryonic antigen (CEA) monoclonal antibody (MAb), in intact form and as an F(ab')2 fragment labelled with 125I and 131I, were compared in groups of nude mice bearing four different colon cancer xenografts, T380, Co112 or LoVo, of human origin, or a rat colon cancer transfected with human CEA cDNA, called '3G7'. For each tumour, three to four mice per time point were analysed 6, 12, 24, 48 and 96 h after MAb injection. In the different tumours, maximal localisation of intact MAb was obtained at 24 to 48 h, and of F(ab')2 fragment 12 to 24 h after injection. Among the different tumours, localisation was highest with colon cancer T380, with 64% of the injected dose per gram (% ID/g) for the intact MAb and 57% for its F(ab')2 fragment, while in the three other tumours, maximal localisation ranged from 14 to 22% ID g-1 for the intact MAb and was about 11% for the F(ab')2. Tumour to normal tissue ratios of intact MAb increased rapidly until 24 h after injection and remained stable or showed only a minor increase thereafter. In contrast, for the F(ab')2 fragment, the tumour to normal tissue ratios increased steadily up to 4 days after injection reaching markedly higher values than those obtained with intact MAb. For the four different xenografts, tumour to blood ratios of F(ab')2 were about 2, 3 and 5 to 16 times higher than those of intact antibodies at 12, 24 and 96 h after injection, respectively.

Tumor-necrosis factor can enhance radio-antibody uptake in human colon carcinoma xenografts by increasing vascular permeability.

Marked differences in the tumor uptake of a 125I-labeled monoclonal antibody (MAb) directed against carcinoembryonic antigen (CEA) were observed in 4 serially transplanted human colorectal carcinomas in nude mice. A comparative study showed that elevated values of measurable tumor vascular parameters, such as permeability, blood flow and blood volume, correlated better with high MAb tumor uptake than the concentration of target antigen in the tumor. In an attempt to modify the vascular parameters and to determine if this could increase antibody uptake by the tumor, rhTNF alpha (TNF) was injected i.t. or i.v. and antibody localization experiments were performed immediately thereafter. Results showed that the permeability of the tumor vessels increased 8 to 10 fold 1 hr after i.t. injection of TNF as compared to control tumors injected with saline. Tumor uptake of 125I-labeled anti-CEA MAb, was 3 times higher 2 hr after i.v. injection and still 27% higher 22 hr later, as compared to results from controls. Intravenous injection of TNF simultaneously with the 125I-labeled anti-CEA MAb also resulted in a 2-fold increase in tumor uptake 4 hr after injection, but the increase was no longer significant 24 hr after injection. Interestingly after i.v. injection of TNF, the MAb concentration in the blood and other normal tissues, such as liver, kidneys, lungs and heart was decreased, resulting in significantly higher ratios of tumor to normal tissue. Taken together the results demonstrate that injection of TNF can increase tumor vascular permeability and improve radio-antibody uptake. This raises the possibility of increasing the radiation dose delivered by antibody to the tumor in the course of radioimmunotherapy.

Repair of lamellar scleral lesions in dogs with preserved equine renal capsule: short report

OBJETIVO: Avaliar o uso da cápsula renal de eqüino preservada em glicerina 98% no reparo de lesões lamelares esclerais em cães. MÉTODOS: Foram utilizados 12 cães, machos e fêmeas, com peso médio de 12kg. Foram realizadas avaliações clínica e morfológica aos 1, 3, 7, 15, 30 e 60 dias de pós-operatório. Após anestesia geral e procedimentos padrões de preparo do campo operatório, foi realizada cantotomia temporal, seguida de incisão conjutival e escleral com área de 0,5x0,5 cm na posição de 1hora, próxima ao limbo. em seguida, um fragmento de mesma dimensão de cápsula renal de eqüino preservada em glicerina, previamente hidratado em solução salina, foi aplicado ao defeito escleral criado sendo fixado com pontos simples isolados com vicryl 7-0®. RESULTADOS: A avaliação clínica revelou blefaroespasmo/fotofobia até o sétimo dia de pós-operatório. Foi observado edema conjuntival até o quinto dia, acompanhado de secreção ocular mucóide, que persistiu até o décimo dia de pós-operatório. Não foram observados sinais clínicos de rejeição do enxerto em todos os animais, em todos os períodos avaliados. Os segmentos anterior e posterior do bulbo ocular não apresentaram sinais de inflamação. A análise morfológica revelou exsudação inflamatória aguda nos períodos precoces e intermediários da avaliação e inflamação crônica nos períodos tardios da observação. Houve incorporação do enxerto ao leito receptor. CONCLUSÃO: Os resultados sugerem que a cápsula renal de eqüino preservada pode ser mais uma alternativa de membrana biológica para o reparo de lesões esclerais lamelares em cães e no homem.

Resposta tecidual ao enxerto xenógeno inorgânico de osso bovino: avaliação histomorfológica e histomorfométrica

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Terapia de defeito crítico em crânio de ratos pela associação de xenoenxerto bovino e células derivadas de periósteo autógeno

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Progenitor cell therapies for traumatic brain injury: barriers and opportunities in translation.

Traumatic brain injury (TBI) directly affects nearly 1.5 million new patients per year in the USA, adding to the almost 6 million cases in patients who are permanently affected by the irreversible physical, cognitive and psychosocial deficits from a prior injury. Adult stem cell therapy has shown preliminary promise as an option for treatment, much of which is limited currently to supportive care. Preclinical research focused on cell therapy has grown significantly over the last decade. One of the challenges in the translation of this burgeoning field is interpretation of the promising experimental results obtained from a variety of cell types, injury models and techniques. Although these variables can become barriers to a collective understanding and to evidence-based translation, they provide crucial information that, when correctly placed, offers the opportunity for discovery. Here, we review the preclinical evidence that is currently guiding the translation of adult stem cell therapy for TBI.

Sinonasal Disorders In Hematopoietic Stem Cell Transplantation.

hematopoietic stem cell transplantation (HSCT) is associated with more respiratory infections due to immunosuppression. this study aimed to verify the frequency of rhinosinusitis after HSCT, and the association between rhinosinusitis and chronic graft vs. host disease (GVHD) and type of transplantation, clinical treatment, surgical treatment, and survival. this was a retrospective study in a tertiary university hospital. A total of 95 patients with hematological diseases undergoing HSCT between 1996 and 2011 were selected. chronic myeloid leukemia was the most prevalent disease. The type of transplant most often performed was the allogenic type (85.26%). The frequency of rhinosinusitis was 36%, with no difference between the autologous and the allogenic types. Chronic GVHD occurred in 30% of patients. Patients with GVHD had a higher frequency and recurrence of rhinosinusitis, in addition to more frequent need for endoscopic sinusectomy and decreased overall survival. there was a higher frequency of rhinosinusitis in HSCT and GVHD. The type of transplant does not appear to predispose to the occurrence of rhinosinusitis. GVHD seems to be an aggravating factor and requires a more stringent treatment.

Influence Of Cyclosporine On The Occurrence Of Nephrotoxicity After Allogeneic Hematopoietic Stem Cell Transplantation: A Systematic Review.

Cyclosporine, a drug used in immunosuppression protocols for hematopoietic stem cell transplantation that has a narrow therapeutic index, may cause various adverse reactions, including nephrotoxicity. This has a direct clinical impact on the patient. This study aims to summarize available evidence in the scientific literature on the use of cyclosporine in respect to its risk factor for the development of nephrotoxicity in patients submitted to hematopoietic stem cell transplantation. A systematic review was made with the following electronic databases: PubMed, Web of Science, Embase, Scopus, CINAHL, LILACS, SciELO and Cochrane BVS. The keywords used were: bone marrow transplantation OR stem cell transplantation OR grafting, bone marrow AND cyclosporine OR cyclosporin OR risk factors AND acute kidney injury OR acute kidney injuries OR acute renal failure OR acute renal failures OR nephrotoxicity. The level of scientific evidence of the studies was classified according to the Oxford Centre for Evidence Based Medicine. The final sample was composed of 19 studies, most of which (89.5%) had an observational design, evidence level 2B and pointed to an incidence of nephrotoxicity above 30%. The available evidence, considered as good quality and appropriate for the analyzed event, indicates that cyclosporine represents a risk factor for the occurrence of nephrotoxicity, particularly when combined with amphotericin B or aminoglycosides, agents commonly used in hematopoietic stem cell transplantation recipients.

Unusual Erythrocyte Split Chimerism In Pregnancy After Allogeneic Stem Cell Transplantation.

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Successful transplant of mesenchymal stem cells in induced osteonecrosis of the ovine femoral head: preliminary results

PURPOSE: Evaluate the bone tissue recovery following transplantation of ovine mesenchymal stem cells (MSC) from bone marrow and human immature dental-pulp stem cells (hIDPSC) in ovine model of induced osteonecrosis of femoral head (ONFH). METHODS: Eight sheep were divided in three experimental groups. First group was composed by four animals with ONFH induced by ethanol through central decompression (CD), for control group without any treatment. The second and third group were compose by two animals, six weeks after ONFH induction received transplantation of heterologous ovine MSC (CD + oMSC), and hIDPSC (CD + hIDPSC), respectively. In both experiments the cells were transplanted without application of any type of immunosupression protocol. RESULTS: Our data indicate that both cell types used in experiments were able to proliferate within injured site providing bone tissue recovery. The histological results obtained from CD+hIDPSC suggested that the bone regeneration in such animals was better than that observed in CD animals. CONCLUSION: Mesenchymal stem cell transplant in induced ovine osteonecrosis of femoral head by central decompression technique is safe, and apparently favors bone regeneration of damaged tissues.

Heterologous expression of glucose oxidase in the yeast Kluyveromyces marxianus

Background: In spite of its advantageous physiological properties for bioprocess applications, the use of the yeast Kluyveromyces marxianus as a host for heterologous protein production has been very limited, in constrast to its close relative Kluyveromyces lactis. In the present work, the model protein glucose oxidase (GOX) from Aspergillus niger was cloned into K. marxianus CBS 6556 and into K. lactis CBS 2359 using three different expression systems. We aimed at verifying how each expression system would affect protein expression, secretion/localization, post-translational modification, and biochemical properties. Results: The highest GOX expression levels (1552 units of secreted protein per gram dry cell weight) were achieved using an episomal system, in which the INU1 promoter and terminator were used to drive heterologous gene expression, together with the INU1 prepro sequence, which was employed to drive secretion of the enzyme. In all cases, GOX was mainly secreted, remaining either in the periplasmic space or in the culture supernatant. Whereas the use of genetic elements from Saccharomyces cerevisiae to drive heterologous protein expression led to higher expression levels in K. lactis than in K. marxianus, the use of INU1 genetic elements clearly led to the opposite result. The biochemical characterization of GOX confirmed the correct expression of the protein and showed that K. marxianus has a tendency to hyperglycosylate the protein, in a similar way as already observed for other yeasts, although this tendency seems to be smaller than the one of e. g. K. lactis and S. cerevisiae. Hyperglycosylation of GOX does not seem to affect its affinity for the substrate, nor its activity. Conclusions: Taken together, our results indicate that K. marxianus is indeed a good host for the expression of heterologous proteins, not only for its physiological properties, but also because it correctly secretes and folds these proteins.