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The toxicity of ATR-107, a human anti-interleukin-21 receptor (IL-21R) monoclonal antibody (mAb), was evaluated in CD-1 mice and cynomolgus monkeys after single-dose intravenous (IV) administration, and in Sprague-Dawley (SD) rats and cynomolgus monkeys after weekly IV and subcutaneous (SC) administration in 13-week toxicity studies that included recovery. Adverse liver necrosis, diffuse bridging fibrosis, and higher liver enzymes occurred in rats in the low-dose IV group (10 mg/kg), but not at 50 or 250 mg/kg IV, and not following SC administration despite overlapping systemic ATR-107 exposures. Similar findings were not seen in mice or cynomolgus monkeys. A series of investigative rat toxicity studies showed liver findings only occurred after administration of at least 3 weekly doses, only occurred in rats that developed anti-drug antibodies (ADAs), and the incidence was associated with higher ADAs titers. However, the presence of ADAs did not always result in liver injury. Liver findings did not occur in nude rats, which had high ATR-107 exposures and no ADAs. These findings suggest an adaptive immune response with formation of ADAs was necessary for development of ATR-107-related liver findings, and that liver injury can occur in rats secondary to development of ADAs following repeated administration of a human therapeutic mAb.
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PF-07209960 is a novel bispecific fusion protein composed of an anti-PD-1 antibody and engineered IL-15 cytokine mutein with reduced binding affinity to its receptors. The pharmacokinetics (PK), pharmacodynamics (PD), and toxicity of PF-07209960 were evaluated following once every other week subcutaneous (SC) or intravenous (IV) administration to cynomolgus monkeys in a repeat-dose PKPD (0.01-0.3 mg/kg/dose) and GLP toxicity study (0.1-3 mg/kg/dose). PF-07209960 showed dose dependent pharmacokinetics with a terminal T1/2 of 8 and 13 hours following IV administration at 0.03 and 0.1 mg/kg, respectively. The clearance is faster than a typical IgG1 antibody. Slightly faster clearance was also observed following the second dose, likely due to increased target pool and formation of anti-drug antibodies (ADA). Despite a high incidence rate of ADA (92%) observed in GLP toxicity study, PD-1 receptor occupancy, IL-15 signaling (STAT5 phosphorylation) and T cell expansion were comparable following the first and second doses. Activation and proliferation of T cells were observed with largest increase in cell numbers found in gamma delta T cells, followed by CD4+ and CD8+ T cells, and then NK cells. Release of cytokines IL-6, IFNγ, and IL-10 were detected, which peaked at 72 hours postdose. There was PF-07209960-related mortality at ≥1 mg/kg. At scheduled necropsy, microscopic findings were generalized mononuclear infiltration in various tissues. Both the no observed adverse effect level (NOAEL) and the highest non severely toxic dose (HNSTD) were determined to be 0.3 mg/kg/dose, which corresponded to mean Cmax and AUC48 values of 1.15 µg/mL and 37.9 µg*h/mL, respectively.
Assuntos
Anticorpos Monoclonais , Receptor de Morte Celular Programada 1 , Animais , Macaca fascicularis , Interleucina-15 , Administração Intravenosa , Citocinas , Inibidores de Checkpoint ImunológicoRESUMO
Purpose: Bevacizumab-bvzr (Zirabev®), a recombinant humanized monoclonal antibody targeting vascular endothelial growth factor and a biosimilar to bevacizumab, is approved for intravenous administration for various indications worldwide. The objectives of this study were to evaluate the ocular toxicity, systemic tolerability, and toxicokinetics (TKs) of bevacizumab-bvzr following repeat intravitreal (IVT) injection to cynomolgus monkeys. Methods: Male monkeys were administered saline, vehicle, or bevacizumab-bvzr at 1.25 mg/eye/dose once every 2 weeks (3 doses total) for 1 month by bilateral IVT injection, followed by a 4-week recovery phase to evaluate the reversibility of any findings. Local and systemic safety was assessed. Ocular safety assessments included in-life ophthalmic examinations, tonometry (intraocular pressure, IOP), electroretinograms (ERGs), and histopathology. In addition, concentrations of bevacizumab-bvzr were measured in serum and in ocular tissues (vitreous humor, retina, and choroid/retinal pigment epithelium) and ocular concentration-time profiles and serum TKs were evaluated. Results: Bevacizumab-bvzr was tolerated locally and systemically, with an ocular safety profile comparable to the saline or vehicle control group. Bevacizumab-bvzr was observed in both serum and in the evaluated ocular tissues. There were no bevacizumab-bvzr-related microscopic changes or effects on IOP or ERGs. Bevacizumab-bvzr-related trace pigment or cells in vitreous humor (in 4 of 12 animals; commonly associated with IVT injection) and transient, nonadverse, mild ocular inflammation (in 1 of 12 animals) were noted upon ophthalmic examination and fully reversed during the recovery phase. Conclusions: Bevacizumab-bvzr was well tolerated via biweekly IVT administration in healthy monkeys, with an ocular safety profile comparable to saline or its vehicle control.
Assuntos
Medicamentos Biossimilares , Animais , Masculino , Bevacizumab/farmacologia , Macaca fascicularis , Fator A de Crescimento do Endotélio Vascular , Injeções Intravítreas , Toxicocinética , Retina , Inibidores da AngiogêneseRESUMO
Bococizumab is an anti-PCSK9 monoclonal antibody that was intended for the treatment of hypercholesterolemia. After reviewing the 6-month rat toxicity study data, in which there was a low spontaneous tumor incidence, unrelated to bococizumab administration, the U.S. FDA granted a carcinogenicity waiver request based on a weight-of-evidence assessment of low carcinogenic risk. Subsequently, after reviewing 6-month rat toxicity study data from another anti-PCSK9 antibody, RN317, with a similar low tumor incidence (unrelated to RN317), the U.S. FDA rescinded the bococizumab carcinogenicity study waiver and requested a full 2-year rat carcinogenicity study be conducted. The resulting 2-year carcinogenicity study demonstrated no bococizumab-related increase in tumors, confirming the weight-of-evidence evaluation and alleviating concerns regarding the carcinogenic potential. Here we report the scientific and regulatory background that led to the request for a rat carcinogenicity study, the feedback on the design of the carcinogenicity study, and the results from this study which affirmed the original weight-of-evidence assessment of low carcinogenic risk.
Assuntos
Carcinógenos , Hipercolesterolemia , Animais , Anticorpos Monoclonais/toxicidade , Testes de Carcinogenicidade , Carcinógenos/toxicidade , LDL-Colesterol , Pró-Proteína Convertase 9 , RatosRESUMO
BACKGROUND: CD40 is a compelling target for cancer immunotherapy, however, attempts to successfully target this pathway have consistently been hampered by dose-limiting toxicity issues in the clinic that prevents the administration of efficacious doses. METHODS: Here, using cytokine and cytokine receptor depletion strategies in conjunction with a potent CD40 agonist, we investigated mechanisms underlying the two primary sources of CD40 agonist-associated toxicity, hepatotoxicity and cytokine release syndrome (CRS). RESULTS: We demonstrate that CD40 agonist -induced hepatotoxicity and CRS are mechanistically independent. Historical data have supported a role for interleukin-6 (IL-6) in CRS-associated wasting, however, our findings instead show that an inflammatory cytokine network involving TNF, IL-12p40, and IFNγ underlie this process. Deficiency of TNF or IFNγ did not influence CD40-induced hepatitis however loss of IL-12p40 significantly decreased circulating concentrations of liver enzymes and reduced the frequency of activated CD14+MHCII+ myeloid cells in the liver, indicating a role for IL-12p40 in liver pathology. CONCLUSIONS: As clinical research programs aim to circumnavigate toxicity concerns while maintaining antitumor efficacy it will be essential to understand which features of CD40 biology mediate antitumor function to develop both safe and efficacious agonists.
Assuntos
Doença Hepática Induzida por Substâncias e Drogas/diagnóstico , Imunoterapia/métodos , Subunidade p40 da Interleucina-12/efeitos adversos , Animais , Feminino , Humanos , CamundongosRESUMO
PURPOSE: Gastrointestinal cancers remain areas of high unmet need despite advances in targeted and immunotherapies. Here, we demonstrate potent, tumor-selective efficacy with PF-07062119, a T-cell engaging CD3 bispecific targeting tumors expressing Guanylyl Cyclase C (GUCY2C), which is expressed widely across colorectal cancer and other gastrointestinal malignancies. In addition, to address immune evasion mechanisms, we explore combinations with immune checkpoint blockade agents and with antiangiogenesis therapy. EXPERIMENTAL DESIGN: PF-07062119 activity was evaluated in vitro in multiple tumor cell lines, and in vivo in established subcutaneous and orthotopic human colorectal cancer xenograft tumors with adoptive transfer of human T cells. Efficacy was also evaluated in mouse syngeneic tumors using human CD3ε transgenic mice. IHC and mass cytometry were performed to demonstrate drug biodistribution, recruitment of activated T cells, and to identify markers of immune evasion. Combination studies were performed with anti-PD-1/PD-L1 and anti-VEGF antibodies. Toxicity and pharmacokinetic studies were done in cynomolgus macaque. RESULTS: We demonstrate that GUCY2C-positive tumors can be targeted with an anti-GUCY2C/anti-CD3ε bispecific, with selective drug biodistribution to tumors. PF-07062119 showed potent T-cell-mediated in vitro activity and in vivo efficacy in multiple colorectal cancer human xenograft tumor models, including KRAS- and BRAF-mutant tumors, as well as in the immunocompetent mouse syngeneic tumor model. PF-07062119 activity was further enhanced when combined with anti-PD-1/PD-L1 treatment or in combination with antiangiogenic therapy. Toxicity studies in cynomolgus indicated a monitorable and manageable toxicity profile. CONCLUSIONS: These data highlight the potential for PF-07062119 to demonstrate efficacy and improve patient outcomes in colorectal cancer and other gastrointestinal malignancies.
Assuntos
Anticorpos Biespecíficos/administração & dosagem , Complexo CD3/imunologia , Neoplasias Colorretais/terapia , Neoplasias Gastrointestinais/terapia , Imunoterapia/métodos , Receptores de Enterotoxina/imunologia , Linfócitos T/imunologia , Transferência Adotiva/métodos , Animais , Anticorpos Biespecíficos/farmacocinética , Linhagem Celular Tumoral , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/metabolismo , Modelos Animais de Doenças , Feminino , Neoplasias Gastrointestinais/imunologia , Neoplasias Gastrointestinais/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Distribuição TecidualRESUMO
The restricted expression pattern of B-cell maturation antigen (BCMA) makes it an ideal tumor-associated antigen (TAA) for the treatment of myeloma. BCMA has been targeted by both CD3 bispecific antibody and antibody-drug conjugate (ADC) modalities, but a true comparison of modalities has yet to be performed. Here we utilized a single BCMA antibody to develop and characterize both a CD3 bispecific and 2 ADC formats (cleavable and noncleavable) and compared activity both in vitro and in vivo with the aim of generating an optimal therapeutic. Antibody affinity, but not epitope was influential in drug activity and hence a high-affinity BCMA antibody was selected. Both the bispecific and ADCs were potent in vitro and in vivo, causing dose-dependent cell killing of myeloma cell lines and tumor regression in orthotopic myeloma xenograft models. Primary patient cells were effectively lysed by both CD3 bispecific and ADCs, with the bispecific demonstrating improved potency, maximal cell killing, and consistency across patients. Safety was evaluated in cynomolgus monkey toxicity studies and both modalities were active based on on-target elimination of B lineage cells. Distinct nonclinical toxicity profiles were seen for the bispecific and ADC modalities. When taken together, results from this comparison of BCMA CD3 bispecific and ADC modalities suggest better efficacy and an improved toxicity profile might be achieved with the bispecific modality. This led to the advancement of a bispecific candidate into phase I clinical trials.
Assuntos
Anticorpos Biespecíficos/administração & dosagem , Antígeno de Maturação de Linfócitos B/metabolismo , Complexo CD3/imunologia , Imunoconjugados/administração & dosagem , Mieloma Múltiplo/tratamento farmacológico , Animais , Anticorpos Biespecíficos/efeitos adversos , Anticorpos Biespecíficos/farmacologia , Afinidade de Anticorpos , Antígeno de Maturação de Linfócitos B/antagonistas & inibidores , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Imunoconjugados/efeitos adversos , Imunoconjugados/farmacologia , Camundongos , Mieloma Múltiplo/metabolismo , Transdução de Sinais/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: Immune checkpoint inhibitors (ICI) targeting PD1, PDL1, or CTLA4 are associated with immune-related adverse events (irAE) in multiple organ systems including myocarditis. The pathogenesis and early diagnostic markers for ICI-induced myocarditis are poorly understood, and there is currently a lack of laboratory animal model to enhance our understanding. We aimed to develop such a model using cynomolgus monkeys. EXPERIMENTAL DESIGN: Chinese-origin cynomolgus monkeys were dosed intravenously with vehicle or nivolumab 20 mg/kg plus ipilimumab 15 mg/kg once weekly and euthanized on day 29. RESULTS: Multiple organ toxicities were observed in cynomolgus monkeys, and were characterized by loose feces, lymphadenopathy, and mononuclear cell infiltrations of varying severity in heart, colon, kidneys, liver, salivary glands, and endocrine organs. Increased proliferation of CD4+ and CD8+ T lymphocytes as well as an increase in activated T cells and central memory T cells in the blood, spleen, and lymph nodes, were observed. Transcriptomic analysis suggested increased migration and activation of T cells and increased phagocytosis and antigen presentation in the heart. Mononuclear cell infiltration in myocardium was comprised primarily of T cells, with lower numbers of macrophages and occasional B cells, and was associated with minimal cardiomyocyte degeneration as well as increases in cardiac troponin-I and NT-pro-BNP. Morphologically, cardiac lesions in our monkey model are similar to the reported ICI myocarditis in humans. CONCLUSIONS: We have developed a monkey model characterized by multiple organ toxicities including myocarditis. This model may provide insight into the immune mechanisms and facilitate biomarker identification for ICI-associated irAEs.
Assuntos
Antineoplásicos Imunológicos/toxicidade , Fatores Imunológicos/toxicidade , Inflamação/etiologia , Linfócitos do Interstício Tumoral/imunologia , Miocardite/induzido quimicamente , Neoplasias/tratamento farmacológico , Animais , Modelos Animais de Doenças , Feminino , Inflamação/patologia , Ipilimumab/toxicidade , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Macaca fascicularis , Miocardite/imunologia , Miocardite/patologia , Neoplasias/imunologia , Neoplasias/patologia , Nivolumabe/toxicidadeRESUMO
Antibody-drug conjugates (ADCs) are promising therapies for haematological cancers. Historically, their therapeutic benefit is due to ADC targeting of lineage-restricted antigens. The C-X-C motif chemokine receptor 4 (CXCR4) is attractive for targeted therapy of haematological cancers, given its expression in multiple tumour types and role in cancer "homing" to bone marrow. However, CXCR4 is also expressed in haematopoietic cells and other normal tissues, raising safety challenges to the development of anti-CXCR4 ADCs for cancer treatment. Here, we designed the first anti-CXCR4 ADC with favourable therapeutic index, effective in xenografts of haematopoietic cancers resistant to standard of care and anti-CXCR4 antibodies. We screened multiple ADC configurations, by varying type of linker-payload, drug-to-antibody ratio (DAR), affinity and Fc format. The optimal ADC bears a non-cleavable linker, auristatin as payload at DAR = 4 and a low affinity antibody with effector-reduced Fc. Contrary to other drugs targeting CXCR4, anti-CXCR4 ADCs effectively eliminated cancer cells as monotherapy, while minimizing leucocytosis. The optimal ADC selectively eliminated CXCR4+ cancer cells in solid tumours, but showed limited toxicity to normal CXCR4+ tissues, sparing haematopoietic stem cells and progenitors. Our work provides proof-of-concept that through empirical ADC design, it is possible to target proteins with broad normal tissue expression.
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Antineoplásicos Imunológicos , Desenho de Fármacos , Imunoconjugados , Receptores CXCR4/imunologia , Animais , Antineoplásicos Imunológicos/administração & dosagem , Antineoplásicos Imunológicos/efeitos adversos , Antineoplásicos Imunológicos/síntese química , Antineoplásicos Imunológicos/química , Células CHO , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Células Cultivadas , Cricetinae , Cricetulus , Desoxicitidina/administração & dosagem , Desoxicitidina/efeitos adversos , Desoxicitidina/análogos & derivados , Feminino , Humanos , Imunoconjugados/administração & dosagem , Imunoconjugados/efeitos adversos , Imunoconjugados/química , Fragmentos Fab das Imunoglobulinas/administração & dosagem , Fragmentos Fab das Imunoglobulinas/efeitos adversos , Fragmentos Fab das Imunoglobulinas/química , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos SCID , Camundongos Transgênicos , Modelos Moleculares , Estrutura Terciária de Proteína , Receptores CXCR4/antagonistas & inibidores , Resultado do Tratamento , Ensaios Antitumorais Modelo de Xenoenxerto , GencitabinaRESUMO
To test the diagnostic approach described in part 1 of this article, 2 exercises were completed by pathologists from multiple companies/agencies. Pathologist's examination of whole slide image (WSI) heart sections from rats using personal diagnostic approaches (exercise #1) corroborated conclusions from study #1. Using the diagnostic approach described in part 1, these pathologists examined the same WSI heart sections (exercise #2) to determine whether that approach increased consistency of diagnosis of rodent progressive cardiomyopathy (PCM) lesions. In exercise #2, there was improved consistency of categorization of small borderline morphologies and mild lesions, but a decrement in consistency of categorizing minimal lesions. Exercises 1 and 2 suggest the described diagnostic approach is representative of that in use by the majority of toxicologic pathologists across companies/agencies and that application by all may improve diagnostic consistency of PCM/like lesions. Additionally, a criterion of approximately 5% heart section involvement is suggested for separating mild from moderate or greater severity. While evidence is not absolute, until further investigation shows otherwise, microscopic changes resembling PCM, but located in the epicardial and subepicardial region of the right ventricle, may be considered as part of the spectrum of PCM.
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Cardiomiopatias/patologia , Diagnóstico por Imagem/métodos , Ventrículos do Coração/patologia , Ratos Sprague-Dawley , Doenças dos Roedores/patologia , Testes de Toxicidade/métodos , Animais , Cardiomiopatias/veterinária , Cardiotoxicidade/patologia , Cardiotoxicidade/veterinária , Simulação por Computador , Diagnóstico por Imagem/normas , Diagnóstico por Imagem/veterinária , Progressão da Doença , Masculino , Testes de Toxicidade/veterináriaRESUMO
Spontaneous rodent progressive cardiomyopathy (PCM) in the Sprague Dawley rat may confound identification and/or interpretation of potential test article (TA)-related cardiotoxicity. Pathologists apply diagnostic term(s) and thresholds for diagnosing and assigning severity grades for PCM and/or PCM-like (PCM/like) lesions consistently within a study, which is necessary to identify and interpret TA-related findings. Due to differences in training and/or experiences, diagnostic terms and thresholds may vary between pathologists. Harmonized terminology and thresholds across studies will generate better historical control data, will likely enhance interpretation of study data, and may further enhance our understanding of the spontaneous change. An assessment of the diagnostic approaches of a group of 37 pathologists identified an approach that is relatively easily applied; and if adopted, it could enhance diagnostic consistency across studies. This approach uses the single "slash" term "necrosis/inflammatory cell infiltrate (NICI)" as the diagnosis for the spectrum of lesions seen in younger rats, uses no threshold for diagnosis (e.g., diagnose all lesions clearly identifiable as PCM/like), and uses aggregate lesion size of approximately ≥45% of the field of view (FOV) using a 10×/22 eyepiece and the 40× objective or approximately ≥100% of the FOV using the 60× objective as the criterion separating minimal from mild severities.
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Cardiomiopatias/patologia , Diagnóstico por Imagem/métodos , Ratos Sprague-Dawley , Doenças dos Roedores/patologia , Testes de Toxicidade/veterinária , Animais , Cardiomiopatias/veterinária , Cardiotoxicidade/patologia , Cardiotoxicidade/veterinária , Simulação por Computador , Diagnóstico por Imagem/normas , Diagnóstico por Imagem/veterinária , Progressão da Doença , Masculino , Necrose , Índice de Gravidade de DoençaRESUMO
A sporadic, diffuse, interstitial mixed cell epididymitis of unknown etiology was noted in the epididymal cauda and distal corpus of young control Sprague-Dawley (SD) rats. Rats from 2 different suppliers were examined as part of routine toxicology studies. The incidence of this finding was 5/5 (study 1), 2/7 (study 2), and 2/7 (study 3). Although 2 of these studies partially overlapped temporally, none of the affected animals from any study was maintained concurrently with affected animals from any of the other 2 studies, and infectious causes, control article toxicity, or autoimmune processes were considered unlikely etiologies. Inflammation similar to that noted in the epididymides of these young rats was not present in other tissues and was not noted in study cohorts sacrificed at ages older than approximately 11 weeks or in rats of similar age from other concurrent studies. Similar findings were noted sporadically in historical control data, and consequently an age-related finding of unknown etiology and occurring in sporadic clusters is reported in SD rats ≤11 weeks old.
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Epididimite/epidemiologia , Epididimite/patologia , Animais , Animais de Laboratório , Doenças Autoimunes/patologia , Edema/patologia , Epididimo/patologia , Achados Incidentais , Masculino , Tamanho do Órgão , Ratos , Ratos Sprague-DawleyRESUMO
Intracardiac injection of human tumor cells into anesthetized nude mice is an established model of bone metastasis. However, intracardiac injection of some human tumor cell lines cause acute neurologic signs and high mortality, making some potentially relevant tumor cell lines unusable for investigation. We showed that intracardiac injection of tumor cells can induce a hypercoagulable state leading to platelet consumption and thromboemboli formation and that pretreatment with intravenous injection of low-molecular-weight heparin (LMWH; enoxaparin) blocks this state. In addition, intravenous injection of enoxaparin before intracardiac injection with 2 different small-cell lung carcinoma lines, H1975 and H2126, dramatically decreased mouse mortality while still generating bone metastases. Therefore, reduction of mortality by pretreatment with LMWH increases the types of cells that can be studied in this metastasis model and decreases the number of animals used.
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Anticoagulantes/farmacologia , Heparina de Baixo Peso Molecular/farmacologia , Longevidade/efeitos dos fármacos , Transplante Heterólogo , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Neoplasias Ósseas/secundário , Carcinoma Pulmonar de Células não Pequenas/secundário , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Humanos , Injeções/efeitos adversos , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Nus , Metástase Neoplásica , Tromboembolia/etiologia , Tromboembolia/mortalidade , Tromboembolia/prevenção & controleRESUMO
INTRODUCTION: Methods to determine infarct size following ischemia-reperfusion injury include gross staining with triphenyltetrazolium chloride (TTC) and perfusion of colored dyes to demarcate the non-ischemic zone. Infarcted tissue (INF) can typically appear a mottled tan to brownish color, making a border between INF and TTC-positive tissue difficult to discern. Previous work in our lab indicated that following TTC staining, prolonged washing of thick sections dramatically sharpened this boundary. METHODS: Adult rats underwent 30 min ischemia via LAD ligation and reperfusion/recovery over 24 h. Hearts were then harvested, thick-sectioned, and stained with TTC. Stained sections were stored in PBS at 4 degrees C for up to 3 weeks. RESULTS: Histology on thin sections from infarcted hearts fixed directly after harvest revealed extensive hemorrhage within the INF. However, this hemorrhage is washed out when hearts are stored in PBS for 3 weeks. SDS-PAGE of PBS samples taken at 1, 2, and 3 weeks showed a low molecular weight band appearing over time. Peptide sequencing revealed the presence of several proteins including the heme-containing proteins (HCPs) hemoglobin, cytochrome c, and myoglobin. The loss of HCPs from thick sections to PBS corresponded with the blanching of the previously mottled INF within each section. HPLC analysis of these samples confirmed the loss of HCPs contributes to INF whitening. Further, analysis of infarct size values derived from heart slices with or without HCPs showed a significant decrease in measurement error when values were derived from slices without HCPs. DISCUSSION: These data suggest that HCPs in the heart tissue contribute to the non-uniform and discolored appearance of the INF, and that washout of these proteins produces an INF more easily distinguished from neighboring non-infarcted tissue. This method greatly reduces the error associated with infarct measurements and improves the analysis of the effects of drug treatments and other interventions designed to impact ischemia reperfusion injury.
Assuntos
Artefatos , Hemeproteínas/metabolismo , Infarto do Miocárdio/patologia , Traumatismo por Reperfusão Miocárdica/patologia , Coloração e Rotulagem/métodos , Animais , Cromatografia Líquida de Alta Pressão , Corantes/química , Eletroforese em Gel de Poliacrilamida , Hemeproteínas/química , Hemorragia/etiologia , Hemorragia/metabolismo , Hemorragia/patologia , Infarto do Miocárdio/complicações , Infarto do Miocárdio/metabolismo , Traumatismo por Reperfusão Miocárdica/complicações , Traumatismo por Reperfusão Miocárdica/metabolismo , Miocárdio/química , Miocárdio/metabolismo , Miocárdio/patologia , Ratos , Ratos Sprague-Dawley , Análise de Sequência de Proteína , Sais de Tetrazólio/químicaRESUMO
The carotid artery shows a common response to many forms of injury, including a rapid activation of smooth muscle cell (SMC) proliferation in the media and migration of SMCs into the intima to form a neointima. Platelet-derived growth factor (PDGF) is believed to play a role in this response to injury, but it has proven difficult to distinguish whether it is stimulating cell migration or cell proliferation, and whether the action is direct or indirect. To determine this, we created chimeric mice composed of both wild-type (WT) and marked PDGF receptor beta (PDGFRbeta)-deficient cells, and determined the consequences of PDGFRbeta expression for SMC participation in response to ligation of the left common carotid artery. The proportion of PDGFRbeta-/- SMCs increased 4.5-fold in the media and decreased 1.8-fold during formation of the neointima, consistent with migration of WT SMCs out of the media and into the intima, leaving the PDGFRbeta-/- cells behind. The fibrotic reaction in the adventitia, which does not involve cell migration, did not result in any change in relative abundance of WT and PDGFRbeta-deficient fibroblasts. We conclude that the most significant direct role of PDGFRbeta is to mediate responses that involve cell migration rather than proliferation.
Assuntos
Lesões das Artérias Carótidas/fisiopatologia , Quimiotaxia , Músculo Liso Vascular/fisiopatologia , Miócitos de Músculo Liso , Receptor beta de Fator de Crescimento Derivado de Plaquetas/metabolismo , Animais , Lesões das Artérias Carótidas/patologia , Artéria Carótida Primitiva , Agregação Celular , Movimento Celular , Quimera , Fibroblastos , Fibrose , Cinética , Camundongos , Camundongos Endogâmicos C57BL , Músculo Liso Vascular/patologia , Túnica Íntima , Túnica MédiaRESUMO
The retinoic acid receptor beta2 (RARbeta2) protein is a putative tumor suppressor that inhibits proliferation and can induce apoptosis when introduced into breast, cervical, lung, and pancreatic cancer cell lines. To determine if RARbeta2 suppresses proliferation of mammary-derived cancer cells in vivo, we transduced MDA-MB-435 breast cancer cells with the LXSN retroviral vector containing RARbeta2 and implanted LXSN vector- or RARbeta2-transduced cells into the mammary fat pads of nude and severe combined immune deficiency (SCID) mice. We analyzed the xenografts for several tumor parameters, including tumor size, inflammation, vascularity, mitoses, tumor recurrence at the primary site following resection, and metastases. We found that 19 of 52 mice inoculated with vector-transduced cells developed metastases in multiple organs while only one of 55 mice receiving RARbeta2-transduced cells displayed evidence of metastases (p < 0.000001, combined experiments, two-tailed Fisher's exact test). Moreover, RARbeta2-tumor cell recipient mice had a lower incidence of post-resection tumor recurrence (8/55 vs. 25/52, p = 0.0004), 34% less necrosis (in three of four experiments, p = 0.001), and 39% fewer mitoses in tumor tissue (p < 0.000001). Our findings suggest that RARbeta2 may play a role in inhibiting the metastatic cascade in a mouse mammary gland xenograft tumor model and is a potential candidate for therapeutic intervention in human breast cancer.
Assuntos
Neoplasias da Mama/patologia , Neoplasias Mamárias Experimentais/patologia , Receptores do Ácido Retinoico/fisiologia , Animais , Northern Blotting , Western Blotting , Feminino , Regulação Neoplásica da Expressão Gênica , Hormônios , Humanos , Imuno-Histoquímica , Luciferases , Pulmão/patologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Camundongos SCID , Metástase Neoplásica , Transplante de Neoplasias , Molécula-1 de Adesão Celular Endotelial a Plaquetas/imunologia , Receptores do Ácido Retinoico/análise , Receptores do Ácido Retinoico/genética , Organismos Livres de Patógenos Específicos , Transdução Genética , Transplante HeterólogoRESUMO
After 30 Sim: (NCR) nu/nu mice were anesthetized with intraperitoneal tribromoethanol, they were injected with 106 human breast cancer cells or phosphate buffered saline (PBS) into the mammary fat pad through a surgical incision. Over the next six days, 24 of 30 mice became ill or died. Necropsies showed peritonitis, and a variety of bacteria were isolated from the peritoneal cavities of the mice. The tribromoethanol solution, PBS solution, and neoplastic cell line were all cultured and were microbiologically negative. Three immunocompetent mice were subsequently injected intraperitoneally with the same tribromoethanol solution used for the 30 nude mice. All three of the immunocompetent mice showed histologic evidence of peritonitis, but only one yielded positive peritoneal bacterial cultures and showed gross evidence of peritonitis. The tribromoethanol solution, previously reported to cause peritonitis, likely induced a chemical peritonitis in these mice, and the bacteria isolated were a secondary infection from normal gastrointestinal flora. We conclude that adequate mixing and storage of tribromoethanol are essential and that it is a safe and effective anesthetic when properly used.