Experimental Evaluation of a New Tissue Factor-Based Topical Hemostat (TT-173) for Treatment of Hepatic Bleeding.

Background: Excessive blood loss is a relevant complication of partial liver resection. Topical hemostatic agents have proven useful to improve the control of the bleeding in this among other surgical indications. Until now all of these products have been based on the action of thrombin. In contrast TT-173 is a new topical hemostatic agent based on recombinant tissue factor naturally incorporated into membrane vesicles. This work sought to assess the efficacy and toxicity of TT-173 in an animal model of liver resection.Materials and Methods: Procoagulant activity of 0.15, 0.41, and 1 mg of TT-173 was evaluated in pigs subjected the resection of hepatic lobe margins. The most effective of these doses was also compared against thrombin. In addition, the toxicity, local tolerance, systemic absorption, and immunogenicity of the product were investigated in rats subjected to liver biopsy lesion.Results: The three doses of TT-173 evaluated significantly reduced the bleeding time in liver lesions. The highest dose of product was significantly more effective than the others and thrombin. Application of high doses of TT-173 in rats did not cause any local or systemic alterations. Absorption into blood stream was negligible and no immunogenic reaction against the product was detected.Conclusions: TT-173 shows favorable pharmacodynamic properties for improving hemostasis in partial liver resection which support further investigation of the product in this surgical indication.

The mitochondrial inhibitor oligomycin induces an inflammatory response in the rat knee joint.

BACKGROUND: Recent findings support a connection between mitochondrial dysfunction and activation of inflammatory pathways in articular cells. This study investigates in vivo in an acute model whether intra-articular administration of oligomycin, an inhibitor of mitochondrial function, induces an oxidative and inflammatory response in rat knee joints. METHODS: Oligomycin was injected into the rat left knee joint on days 0, 2, and 5 before joint tissues were obtained on day 6. The right knee joint served as control. Results were evaluated by macroscopy and histopathology and by measuring cellular and mitochondrial reactive oxygen species (ROS), 4-hydroxy-2-nonenal (4-HNE, a marker of lipid peroxidation), nuclear factor erythroid 2-related factor 2 (Nrf2), and CD68 (macrophages) and chemokine levels. The marker of mitochondrial mass COX-IV was also evaluated. RESULTS: The macroscopic findings showed significantly greater swelling in oligomycin-injected knees than in control knees. Likewise, the histological score of synovial damage was also increased significantly. Immunohistochemical studies showed high expression of IL-8, coinciding with a marked infiltration of polymorphonuclears and CD68+ cells in the synovium. Mitochondrial mass was increased in the synovium of oligomycin-injected joints, as well as cellular and mitochondrial ROS production, and 4-HNE. Relatedly, expression of the oxidative stress-related transcription factor Nrf2 was also increased. As expected, no histological differences were observed in the cartilage; however, cytokine-induced neutrophil chemoattractant-1 mRNA and protein expression were up-regulated in this tissue. CONCLUSIONS: Mitochondrial failure in the joint is able to reproduce the oxidative and inflammatory status observed in arthritic joints.

Nonclinical Evaluation of the New Topical Hemostatic Agent TT-173 for Skin Grafting Procedures.

Blood loss during grafting surgery represents a major concern of this procedure and the development of hemostatic agents for this indication is highly desirable. TT-173 is the first biologically active treatment based on tissue factor instead of thrombin. This study sought to investigate the efficacy, systemic absorption, and toxicology of TT-173 in animal models to support clinical evaluation of the product in donor sites of patients subjected to skin grafting. Procoagulant efficacy of 148 µg of TT-173 was evaluated in pigs in presence and absence of anticoagulant treatment with unfractioned heparin. Systemic absorption was quantified and characterized in rats subjected to severe skin lesions with affectation of muscular plane using TT-173 radiolabeled with I. The same animal model was used to test the toxicology of a dose of 80 µg of the product. Application of TT-173 significantly reduced the bleeding time of donor sites, even under anticoagulant treatment. Systemic absorption was low; it was excreted through urine and did not concentrate in organs such as liver, lung, or spleen suggesting that the absorbed dose could correspond to degradation fragments without procoagulant activity. Finally, a dose of 80 µg of TT-173 did not cause analytical disturbances suggestive of intravascular coagulation or any other adverse reaction. Nonclinical data obtained suggest that TT-173 could be useful to reduce the blood loss associated to burns treatment and support the clinical evaluation of the product in donor sites of patients subjected to skin grafting.

Gene expression profiles following intracoronary injection of mesenchymal stromal cells using a porcine model of chronic myocardial infarction.

BACKGROUND AIMS: We evaluated the therapeutic potential of injection of in vitro differentiated bone marrow mesenchymal stromal cells (MSC) using a swine model. METHODS AND RESULTS: Myocardial infarction was induced by coronary occlusion. Three groups (n = 5 each) were analyzed: one group received an injection of 17.8 ± 9.3 × 10(6) 5-azacytidine-treated allogeneic MSC 1 month after infarction; a placebo group received an injection of medium; and controls were kept untreated. After 4 weeks, heart samples were taken from three infarcted areas, interventricular septa, ventricles and atria. Gene expression profiles of genes related to contractility (Serca2a), fibrosis (Col1a1), cardiomyogenesis (Mef2c, Gata4 and Nkx2.5) and mobilization of stem cells (Sdf1, Cxcr4 and c-kit) were compared by quantitative real-time PCR (qRT-PCR). Gene expression profiles varied in different heart areas. Thus Serca2a expression was reduced in infarcted groups in all heart regions except for the left ventricles, where Col1a1 was overexpressed. The expression of genes related to cardiomyogenesis decreased in the infarcted zones and left atria compared with healthy hearts. Interestingly, increased expression of Cxcr4 was detected in infarcted regions of MSC-treated pigs compared with the placebo group. CONCLUSIONS: Infarction induced changes in expression of genes involved in various biologic processes. Genes involved in cardiomyogenesis were downregulated in the left atrium. The intracoronary injection of MSC resulted in localized changes in the expression of Cxcr4.

Segmental heterogeneity in Bcl-2, Bcl-xL and Bax expression in rat tubular epithelium after ischemia-reperfusion.

AIMS: Studies in rats with bilateral clamping of renal arteries showed transient Bcl-2, Bcl-xL and Bax expression in renal tubular epithelium following ischemia-reperfusion. However, current data on the preferential localization of specific mRNAs or proteins are limited because gene expression was not analysed at segmental level. This study analyses the mRNA expression of Bcl-2, Bcl-xL and Bax in four segments of proximal and distal tubules localized in the renal cortex and outer medulla in rat kidneys with bilateral renal clamping for 30 min and seven reperfusion times versus control animals without clamp. METHODS: Proximal convoluted tubule (PCT), distal convoluted tubule (DCT), proximal straight tubule (PST) and medullary thick ascending limb (MTAL) were obtained by manual microdissection. RT-PCR was used to analyse mRNA expression at segmental level. RESULTS: Proximal convoluted tubule and MTAL showed early, persistent and balanced up-regulation of Bcl-2, Bcl-xL and Bax, while PST and DCT revealed only Bcl-2 and Bcl-xL, when only Bax was detected in PST. DCT expressed Bcl-xL initially, and persistent Bcl-2 later. CONCLUSION: These patterns suggest a heterogeneous apoptosis regulatory response in rat renal tubules after ischemia-reperfusion, independently of cortical or medullary location. This heterogeneity of the expression patterns of Bcl-2 genes could explain the different susceptibility to undergo apoptosis, the different threshold to ischemic damage and the different adaptive capacity to injury among these tubular segments.

Design and creation of an experimental program of advanced training in reconstructive microsurgery.

In this study, we design an experimental protocol for the purpose of enhancing performance in training in microsurgery. It is based on five free tissue transfer exercises in rat (epigastric cutaneous flap, saphenous fasciocutaneous flap, epigastric neurovascular flap, saphenous muscular flap, and hindlimb replantation), which simulate the principal clinical procedures of reconstructive microsurgery. The first part of the study consists of an anatomical review of the flaps of 5 rats and in the second part we have carried out the free transfer of flaps on 25 rats divided into 5 groups. To differentiate between them, we have created a mathematical function, referred to as difficulty in a microsurgical exercise, which has enabled us to establish a scale of progression for training, ranging form the easiest to the most difficult. As a conclusion, we believe that this protocol is a useful instrument as it allows for a more precise assessment of microsurgical capacity due to enhanced accuracy in the reproduction of global procedures and the fact that the quantification of progress in training is based on clinical monitoring after 7 days.

Differential atrial versus ventricular ANKRD1 gene expression is oppositely regulated at diastolic heart failure.

Diastolic heart failure (DHF) was produced in 6-day-old piglets by intravenous administration of Doxorubicin, and ANKRD1 protein and mRNA levels were determined in atrial (A) and ventricular (V) chambers of failing vs control hearts. In controls, ANKRD1 showed a left-right (L-R) asymmetric distribution with protein levels 2-fold higher in the LA as compared to the RA, and 8-fold higher in the LV than the RV. In failing hearts, ANKRD1 levels were augmented about 2-fold in each ventricle but equally reduced in both atria as compared to controls. ANKRD1 downregulation in atria is discussed as a process associated with advanced DHF.

Lack of cross-species transmission of porcine endogenous retrovirus in pig-to-baboon xenotransplantation with sustained depletion of anti-alphagal antibodies.

BACKGROUND: Nonhuman primates are potential permissive animals for studying the risk of in vivo infection with porcine endogenous retrovirus (PERV). Anti-alphaGal natural antibodies are considered one of the barriers for preventing PERV infection, and it has been postulated that reduction of these antibodies could increase the risk of this infection. The aim of this study was to investigate the role of GAS 914, which depletes anti-alphaGal antibodies, in the potential in vivo transfer of PERV after pig-to-baboon organ xenotransplantation. METHODS: Twenty-seven baboons underwent xenotransplantation with hDAF or hMCP/hDAF transgenic pig organs, including heterotopic heart (n = 14) and kidney (n = 13) transplants. All of them received GAS 914 along with different immunosuppression protocols. PERV sequences were investigated by reverse-transcriptase polymerase chain reaction and by polymerase chain reaction assays in samples obtained at autopsy. The presence of PERV-specific antibodies and/or pig xenomicrochimerism was also evaluated. RESULTS: PERV RNA was not detected in any baboon plasma sample. In addition, all plasma samples were negative for PERV antibodies. However, PERV DNA sequences were detected in peripheral blood mononuclear cells from 6 of 14 (43%) animals investigated. Porcine mitochondrial DNA was also found in all of these positive samples and in six of the eight (75%) samples with negative PERV DNA, indicating that the detection of PERV sequences was attributable to xenochimerism. PERV-positive cells as a result of xenochimerism were also found in eight of nine (89%) spleen and lymph node tissue samples tested. CONCLUSIONS: Sustained depletion of anti-alphaGal antibodies does not augment the risk of PERV infection in pig-to-baboon organ transplantation.

Left-right asymmetric ventricular expression of CARP in the piglet heart: regional response to experimental heart failure.

BACKGROUND AND AIM: Cardiac ankyrin repeat protein (CARP), whose expression is down-regulated in response to doxorubicin (Dox) in vitro, has been proposed to be a marker of experimentally-induced cardiac hypertrophy in rodent models. In piglets, the rapid hypertrophy rate of the left ventricle (LV) as compared to that of the right ventricle (RV) represents a natural model of asymmetric ventricular enlargement. We tested whether CARP expression correlates with postnatal ventricular hypertrophy and to what extent CARP can be sensitive to Dox treatment in vivo. METHODS: CARP mRNA and protein levels were quantified (by Northern blot hybridization, semi-quantitative RT-PCR and Western blot) in the piglet heart, both during early postnatal development and upon Dox-induced cardiomyopathy (Dox-CM). RESULTS: The study revealed: (1) significantly augmented CARP mRNA and protein levels in the LV compared to the RV resulting in left vs. right asymmetry in ventricular CARP expression throughout early postnatal development; (2) dose- and chamber-dependent CARP mRNA and protein enrichment in ventricular myocardium in response to Dox; and (3) abolishment of asymmetric patterns of ventricular CARP expression at heart failure resulting from Dox-CM. CONCLUSIONS: (1) CARP is differentially regulated in the LV and RV during both postnatal development and disease; and (2) monitoring of ventricular CARP expression patterns can be used for further analysis of transition from compensated to overt heart failure.

Investigaçäo de genotoxicidade em pessoas profissionalmente expostas ao mercúrio em garimpos da Amazônia Legal: II. Resultados dosimétricos e genotóxicos / Investigation on mercury genotoxicity on profissionally exposed persons in prospection areas of Legal Amazonic Region: II. Dosimetric and genotoxic results

Referenciando-se a literatura técnica pertinente e a comunicaçäo anterior em que se relataram exploratoriamente dados clínicos e dosimétricos obtidos em programa de pesquisa sobre o hidrargirismo em garimpo aurífero da Amazônia Legal, constata-se que as informaçöes clínicas a respeito vêm-se revelando controversas na área. Nesta linha de investigaçäo, o presente projeto procede a exploraçäo de resultados dosimétricos de mercúrio em sangue, urina e cabelo, concomitantemente aos dos indicadores adotados para quantificaçäo da genotoxicidade em amostras de células esfoliadas da mucosa oral e linfócitos de sangue periférico em cultura temporária de garimpeiros, familiares e controles da cidade de Porto Nacional, Estado do Tocantins. Os primeiros foram obtidos pelo método analítico de determinaçäo baseado na transformaçäo rápida de compostos e os segundos consistiram na contagem da frequência relativa de micronúcleos e acidentes cromatídicos e cromossômicos (quebras e gaps). A análise quantitativa foi efetuada pela aplicaçäo do coeficiente de Spermann, admitindo-se o nível de significância de 5%. Observou-se correlaçäo (negativa) significante entre os valores do metal no cabelo e a frequência de quebras cromatídicas (rs=-0,393 e t=2,341). A discussäo travada aprofunda a problematizaçäo de tais resultados para, ao final, destacar a gravidade da questäo desvelada e a complexidade das respectivas medidas de controle: constata-se a necessidade da açäo setorial vigorosa e competente, mas que demanda, necessariamente, organizaçäo dos diferentes segmentos sociais envolvidos, trabalhadores, empresários, intelectuais, veículos de comunicaçäo social, aparelho de Estado e organizaçöes näo-governamentais aí se destacando.

Investigaçäo de genotoxicidade em pessoas profissionalmente expostas ao mercúrio em garimpos da Amazônia Legal: I. Resultados clínicos e dosimétricos / Investigation on mercury genotoxicity on profissinally exposed persons in prospection areas of Legal Amazonia Region: I. Clinical and dosimetric results

Dado o conhecido emprego do mercúrio no processo produtivo do extrativismo do ouro em nosso meio, admite-se que os garimpeiros estejam expostos a um risco adicional específico de hidrargirismo: além da contaminaçäo, compartilhada com a populaçäo local, pela ingestäo de peixes portadores da forma organificada, aspiram diretamente o vapor do metal, o que envolve mecanismos e consequências de conhecimento ainda impreciso, mesmo a nível internacional. Tal constataçäo levou à investigaçäo de diferentes condiçöes clínicas da afecçäo entre 41 pessoas de categorias profissionais diversas, em regiäo de prospecçäo da Amazônia Legal, localizada na cidade de Porto Nacional, Estado de Tocantins, bem como a que se procedesse às respectivas dosimetrias em sangue, urina e cabelo, pelo método analítico de determinaçäo baseado na transformaçäo rápida de compostos. Procedeu-se à análise estatística pelo emprego do teste näo-paramétrico de Kruskal-Wallis do teste de Goodman entre populaçöes multinominais e do coeficienten de correlaçäo de Spearmann. Os resultados obtidos revelam diferenças significantes entre os grupos estudados apenas em relaçäo aos valores urinários, fato que sinaliza para a adoçäo de procedimentos distintos e complementares discutidos a seguir. Trata-se, de um lado, de aplicarem-se, na busca de caracterizaçäo do potencial clastogênico aí envolvido, técnicas laboratoriais mais discriminativas, como a pesquisa de quebras e gaps cromossômicas. Intervençöes profiláticas, de outra parte, encontram pronta indicaçäo, face as alteraçöes se revelarem ainda inconspícuas.