RESUMO
Biotherapeutic modalities such as cell therapies, gene therapies, nucleic acids, and proteins are increasingly investigated as disease-modifying treatments for severe and life-threatening neurodegenerative disorders. Such diverse bio-derived test articles are fraught with unique and often unpredictable biological consequences, while guidance regarding nonclinical experimental design, neuropathology evaluation, and interpretation is often limited. This paper summarizes key messages offered during a half-day continuing education course on toxicologic neuropathology of neuro-targeted biotherapeutics. Topics included fundamental neurobiology concepts, pharmacology, frequent toxicological findings, and their interpretation including adversity decisions. Covered biotherapeutic classes included cell therapies, gene editing and gene therapy vectors, nucleic acids, and proteins. If agents are administered directly into the central nervous system, initial screening using hematoxylin and eosin (H&E)-stained sections of currently recommended neural organs (brain [7 levels], spinal cord [3 levels], and sciatic nerve) may need to expand to include other components (e.g., more brain levels, ganglia, and/or additional nerves) and/or special neurohistological procedures to characterize possible neural effects (e.g., cell type-specific markers for reactive glial cells). Scientists who evaluate the safety of novel biologics will find this paper to be a practical reference for preclinical safety testing and risk assessment.
Assuntos
Neuropatologia , Ácidos Nucleicos , Encéfalo , Medula Espinal , Nervo IsquiáticoRESUMO
Dorsal root ganglia (DRG), trigeminal ganglia (TG), other sensory ganglia, and autonomic ganglia may be injured by some test article classes, including anti-neoplastic chemotherapeutics, adeno-associated virus-based gene therapies, antisense oligonucleotides, nerve growth factor inhibitors, and aminoglycoside antibiotics. This article reviews ganglion anatomy, cytology, and pathology (emphasizing sensory ganglia) among common nonclinical species used in assessing product safety for such test articles (TAs). Principal histopathologic findings associated with sensory ganglion injury include neuron degeneration, necrosis, and/or loss; increased satellite glial cell and/or Schwann cell numbers; and leukocyte infiltration and/or inflammation. Secondary nerve fiber degeneration and/or glial reactions may occur in nerves, dorsal spinal nerve roots, spinal cord (dorsal and occasionally lateral funiculi), and sometimes the brainstem. Ganglion findings related to TA administration may result from TA exposure and/or trauma related to direct TA delivery into the central nervous system or ganglia. In some cases, TA-related effects may need to be differentiated from a spectrum of artifactual and/or spontaneous background changes.
Assuntos
Gânglios Espinais , Fibras Nervosas , Animais , Medula Espinal , BiologiaRESUMO
Nonclinical implantation studies are a common and often critical step for medical device safety assessment in the bench-to-market pathway. Nonclinical implanted medical devices or drug-device combination products require complex macroscopic and microscopic pathology evaluations due to the physical presence of the device itself and unique tissue responses to device materials. The Medical Device Implant Site Evaluation working group of the Society of Toxicologic Pathology's (STP) Scientific and Regulatory Policy Committee (SRPC) was tasked with reviewing scientific, technical, and regulatory considerations for these studies. Implant site evaluations require highly specialized methods and analytical schemes that should be designed on a case-by-case basis to address specific study objectives. Existing STP best practice recommendations can serve as a framework when performing nonclinical studies under Good Laboratory Practices and help mitigate limitations in standards and guidances for implant evaluations (e.g., those from the International Organization for Standardization [ISO], ASTM International). This article integrates standards referenced by sponsors and regulatory bodies with practical pathology evaluation methods for implantable medical devices and combination products. The goal is to ensure the maximum accuracy and scientific relevance of pathology data acquired during a medical device or combination drug-device implantation study.
Assuntos
PolíticasRESUMO
Borrelia burgdorferi is one of the few extracellular pathogens capable of establishing persistent infection in mammals. The mechanisms that sustain long-term survival of this bacterium are largely unknown. Here we report a unique innate immune evasion strategy of B. burgdorferi, orchestrated by a surface protein annotated as BBA57, through its modulation of multiple spirochete virulent determinants. BBA57 function is critical for early infection but largely redundant for later stages of spirochetal persistence, either in mammals or in ticks. The protein influences host IFN responses as well as suppresses multiple host microbicidal activities involving serum complement, neutrophils, and antimicrobial peptides. We also discovered a remarkable plasticity in BBA57-mediated spirochete immune evasion strategy because its loss, although resulting in near clearance of pathogens at the inoculum site, triggers nonheritable adaptive changes that exclude detectable nucleotide alterations in the genome but incorporate transcriptional reprograming events. Understanding the malleability in spirochetal immune evasion mechanisms that ensures their host persistence is critical for the development of novel therapeutic and preventive approaches to combat long-term infections like Lyme borreliosis.
Assuntos
Proteínas de Bactérias/fisiologia , Borrelia burgdorferi/imunologia , Evasão da Resposta Imune , Lipoproteínas/fisiologia , Proteínas de Membrana/fisiologia , Animais , Antígenos de Bactérias/imunologia , Peptídeos Catiônicos Antimicrobianos/biossíntese , Peptídeos Catiônicos Antimicrobianos/genética , Vetores Aracnídeos/microbiologia , Proteínas de Bactérias/genética , Borrelia burgdorferi/genética , Borrelia burgdorferi/patogenicidade , Células Cultivadas , Proteínas do Sistema Complemento/imunologia , Citocinas/biossíntese , Citocinas/genética , Feminino , Regulação Bacteriana da Expressão Gênica , Humanos , Ixodes/microbiologia , Lipoproteínas/genética , Doença de Lyme/imunologia , Doença de Lyme/microbiologia , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Camundongos SCID , Organismos Livres de Patógenos Específicos , VirulênciaRESUMO
Harmonization of diagnostic terminology used during the histopathologic analysis of rodent tissue sections from nonclinical toxicity studies will improve the consistency of data sets produced by laboratories located around the world. The INHAND Project (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) is a cooperative enterprise of 4 major societies of toxicologic pathology to develop a globally accepted standard vocabulary for proliferative and nonproliferative lesions in rodents. A prior manuscript (Toxicol Pathol 2012;40[4 Suppl]:87S-157S) defined multiple diagnostic terms for toxicant-induced lesions, common spontaneous and age-related changes, and principal confounding artifacts in the rat and mouse central nervous system (CNS) and peripheral nervous system (PNS). The current article defines 9 new diagnostic terms and updates 2 previous terms for findings in the rodent CNS and PNS, the need for which has become evident in the years since the publication of the initial INHAND nomenclature for findings in rodent neural tissues. The nomenclature presented in this document is also available electronically on the Internet at the goRENI website (http://www.goreni.org/).
Assuntos
Sistema Nervoso Periférico , Animais , Camundongos , RatosRESUMO
Thorough morphologic evaluations of medical devices placed in or near the nervous system depend on many factors. Pathologists interpreting a neurologic device study must be familiar with the regulatory framework affecting device development, biocompatibility and safety determinants impacting nervous tissue responses, and appropriate study design, including the use of appropriate animal models, group design, device localization, euthanasia time points, tissue examination, sampling and processing, histochemistry and immunohistochemistry, and reporting. This overview contextualizes these features of neurologic medical devices for pathologists engaged in device evaluations.
Assuntos
Desenho de Equipamento/normas , Segurança de Equipamentos/normas , Equipamentos e Provisões/normas , Sistema Nervoso/patologia , Patologistas , Animais , Materiais Biocompatíveis/normas , Humanos , Teste de Materiais/métodos , Legislação de Dispositivos MédicosRESUMO
Increased understanding of pediatric acute lymphoblastic leukemia (ALL) pathobiology has led to dramatic improvements in patient survival. However, there is still a need to develop targeted therapies to enable reduced chemotherapy intensity and to treat relapsed patients. The interleukin-7 receptor α (IL-7Rα) signaling pathways are prime therapeutic targets because these pathways harbor genetic aberrations in both T-cell ALL and B-cell precursor ALL. Therapeutic targeting of the IL-7Rα signaling pathways may lead to improved outcomes in a subset of patients.
Assuntos
Interleucina-7/antagonistas & inibidores , Proteínas de Neoplasias/antagonistas & inibidores , Leucemia-Linfoma Linfoblástico de Células Precursoras B/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Humanos , Interleucina-7/metabolismo , Proteínas de Neoplasias/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/metabolismoRESUMO
Transduced mouse immature thymocytes can be differentiated into T cells in vitro using the delta-like 4-expressing bone marrow stromal cell line co-culture system (OP9-DL4). As retroviral transduction requires dividing cells for transgene integration, OP9-DL4 provides a suitable in vitro environment for cultivating hematopoietic progenitor cells. This is particularly advantageous when studying the effects of the expression of a specific gene during normal T cell development and leukemogenesis, as it allows researchers to circumvent the time-consuming process of generating transgenic mice. To achieve successful outcomes, a series of coordinated steps involving the simultaneous manipulation of different types of cells must be carefully performed. Although these are very well-established procedures, the lack of a common source in the literature often means a series of optimizations are required, which can be time-consuming. This protocol has been shown to be efficient in transducing primary thymocytes followed by differentiation on OP9-DL4 cells. Detailed here is a protocol that can serve as a quick and optimized guide for the co-culture of retrovirally transduced thymocytes on OP9-DL4 stromal cells.
Assuntos
Leucemia de Células T , Timócitos , Camundongos , Animais , Timócitos/metabolismo , Técnicas de Cocultura , Diferenciação Celular/fisiologia , Células Estromais , Camundongos Transgênicos , Oncogenes , Leucemia de Células T/genética , Leucemia de Células T/metabolismoRESUMO
Riluzole is the only treatment known to improve survival in patients with Amyotrophic Lateral Sclerosis (ALS). However, oral riluzole efficacy is modest at best, further it is known to have large inter-individual variability of serum concentration and clearance, is formulated as an oral drug in a patient population plagued with dysphagia, and has known systemic side-effects like asthenia (limiting patient compliance) and elevated liver enzymes. In this context, we postulated that continuous intrathecal (IT) infusion of low doses of riluzole could provide consistent elevations of the drug spinal cord (SC) concentrations at or above those achieved with oral dosing, without increasing the risk for adverse events associated with systemic drug exposure or off-target side effects in the brain. We developed a formulation of riluzole for IT delivery and conducted our studies in purpose-bred hound dogs. Our non-GLP studies revealed that IT infusion alone was able to increase SC concentrations above those provided by oral administration, without increasing plasma concentrations. We then conducted two GLP studies that combined IT infusion with oral administration at human equivalent dose, to evaluate SC and brain concentrations of riluzole along with assessments of safety and tolerability. In the 6-week study, the highest IT dose (0.2 mg/hr) was well tolerated by the animals and increased SC concentrations above those achieved with oral riluzole alone, without increasing brain concentrations. In the 6-month study, the highest dose tested (0.4 mg/hr) was not tolerated and yielded SC significantly above those achieved in all previous studies. Our data show the feasibility and safety profile of continuous IT riluzole delivery to the spinal cord, without concurrent elevated liver enzymes, and minimal brain concentrations creating another potential therapeutic route of delivery to be used in isolation or in combination with other therapeutics."
Assuntos
Esclerose Lateral Amiotrófica , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos , Humanos , Animais , Cães , Esclerose Lateral Amiotrófica/tratamento farmacológico , Riluzol/uso terapêutico , Encéfalo , Administração OralRESUMO
The IL-7 pathway is required for normal T cell development and survival. In recent years the pathway has been shown to be a major driver of acute lymphoblastic leukemia (ALL), the most common cancer in children. Gain-of-function mutations in the alpha chain of the IL-7 receptor found in ALL patients clearly demonstrated that this pathway was a driver. However mutant IL-7R alone was insufficient to transform primary T cell progenitors, indicating that cooperating mutations were required. Here we review evidence for additional oncogenic mutations in the IL-7 pathway. We discuss several oncogenes, loss of tumor suppressor genes and epigenetic effects that can cooperate with mutant IL-7 receptor. These include NRas, HOXA, TLX3, Notch 1, Arf, PHF6, WT1, PRC, PTPN2 and CK2. As new therapeutics targeting the IL-7 pathway are developed, combination with agents directed to cooperating pathways offer hope for novel therapies for ALL.
Assuntos
Regulação Leucêmica da Expressão Gênica , Interleucina-7/genética , Mutação , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Receptores de Interleucina-7/genética , Transdução de Sinais/genética , Caseína Quinase II/genética , Caseína Quinase II/metabolismo , Criança , Epigênese Genética , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Interleucina-7/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Proteína Tirosina Fosfatase não Receptora Tipo 2/genética , Proteína Tirosina Fosfatase não Receptora Tipo 2/metabolismo , Receptor Notch1/genética , Receptor Notch1/metabolismo , Receptores de Interleucina-7/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Linfócitos T/metabolismo , Linfócitos T/patologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas WT1/genética , Proteínas WT1/metabolismoRESUMO
BACKGROUND: Determining mitotic index by counting mitotic figures (MFs) microscopically from tumor areas with most abundant MF (hotspots [HS]) produces a prognostically useful tumor grading biomarker. However, interobserver concordance identifying MF and HS can be poorly reproducible. Immunolabeling MF, coupled with computer-automated counting by image analysis, can improve reproducibility. A computational system for obtaining MF values across digitized whole-slide images (WSIs) was sought that would minimize impact of artifacts, generate values clinically relatable to counting ten high-power microscopic fields of view typical in conventional microscopy, and that would reproducibly map HS topography. MATERIALS AND METHODS: Relatively low-resolution WSI scans (0.50 µm/pixel) were imported in grid-tile format for feature-based MF segmentation, from naturally occurring canine melanomas providing a wide range of proliferative activity. MF feature extraction conformed to anti-phospho-histone H3-immunolabeled mitotic (M) phase cells. Computer vision image processing was established to subtract key artifacts, obtain MF counts, and employ rotationally invariant feature extraction to map MF topography. RESULTS: The automated topometric HS (TMHS) algorithm identified mitotic HS and mapped select tissue tiles with greatest MF counts back onto WSI thumbnail images to plot HS topographically. Influence of dye, pigment, and extraneous structure artifacts was minimized. TMHS diagnostic decision support included image overlay graphics of HS topography, as well as a spreadsheet and plot of tile-based MF count values. TMHS performance was validated examining both mitotic HS counting and mapping functions. Significantly correlated TMHS MF mapping and metrics were demonstrated using repeat analysis with WSI in different orientation (R 2 = 0.9916) and by agreement with a pathologist (R 2 = 0.8605) as well as through assessment of counting function using an independently tuned object counting algorithm (OCA) (R 2 = 0.9482). Limits of agreement analysis support method interchangeability. MF counts obtained led to accurate patient survival prediction in all (n = 30) except one case. By contrast, more variable performance was documented when several pathologists examined similar cases using microscopy (pair-wise correlations, rho range = 0.7597-0.9286). CONCLUSIONS: Automated TMHS MF segmentation and feature engineering performance were interchangeable with both observer and OCA in digital mode. Moreover, enhanced HS location accuracy and superior method reproducibility were achieved using the automated TMHS algorithm compared to the current practice employing clinical microscopy.
RESUMO
Validating digital pathology as substitute for conventional microscopy in diagnosis remains a priority to assure effectiveness. Intermodality concordance studies typically focus on achieving the same diagnosis by digital display of whole slide images and conventional microscopy. Assessment of discrete histological features in whole slide images, such as mitotic figures, has not been thoroughly evaluated in diagnostic practice. To further gauge the interchangeability of conventional microscopy with digital display for primary diagnosis, 12 pathologists examined 113 canine naturally occurring mucosal melanomas exhibiting a wide range of mitotic activity. Design reflected diverse diagnostic settings and investigated independent location, interpretation, and enumeration of mitotic figures. Intermodality agreement was assessed employing conventional microscopy (CM40×), and whole slide image specimens scanned at 20× (WSI20×) and at 40× (WSI40×) objective magnifications. An aggregate 1647 mitotic figure count observations were available from conventional microscopy and whole slide images for comparison. The intraobserver concordance rate of paired observations was 0.785 to 0.801; interobserver rate was 0.784 to 0.794. Correlation coefficients between the 2 digital modes, and as compared to conventional microscopy, were similar and suggest noninferiority among modalities, including whole slide image acquired at lower 20× resolution. As mitotic figure counts serve for prognostic grading of several tumor types, including melanoma, 6 of 8 pathologists retrospectively predicted survival prognosis using whole slide images, compared to 9 of 10 by conventional microscopy, a first evaluation of whole slide image for mitotic figure prognostic grading. This study demonstrated agreement of replicate reads obtained across conventional microscopy and whole slide images. Hence, quantifying mitotic figures served as surrogate histological feature with which to further credential the interchangeability of whole slide images for primary diagnosis.
RESUMO
Acute lymphoblastic leukemia (ALL) is the most common cancer in children. Current chemotherapy is quite toxic in growing children and more directed therapeutics are being sought. The IL-7R pathway is a major driver of ALL and here we evaluate two drugs directed to that pathway using a model of T cell ALL. Mutant gain-of-function IL-7Rα was transduced into an IL-7-dependent murine thymocyte line conferring ligand-independent survival and growth. JAK1 is associated with IL-7Rα and mediates signaling from the mutant receptor. In vitro, treating the transformed cell line with the JAK1/2 inhibitor ruxolitinib inhibited ligand-independent signaling and induced cell death. Transfer of the transformed cell line into mice resulted in aggressive leukemia and untreated mice succumbed in about three weeks. Treatment with ruxolitinib incorporated into chow showed a potent therapeutic benefit with reduction in leukemic burden and extension of survival. BCL-2 is an anti-apoptotic downstream mediator of the IL-7R survival mechanism. Venetoclax, an inhibitor of BCL-2, showed activity against the transformed cell line in vitro and could be combined with ruxolitinib in vivo. These findings support the therapeutic potential of treating T-ALL by targeting the IL-7R pathway.
Assuntos
Carcinoma de Células Escamosas/veterinária , Neoplasias Cardíacas/secundário , Doenças dos Cavalos/patologia , Neoplasias Pulmonares/secundário , Neoplasias Penianas/veterinária , Animais , Carcinoma de Células Escamosas/patologia , Cavalos , Linfonodos/patologia , Masculino , Neoplasias Penianas/patologiaAssuntos
Transformação Celular Neoplásica/patologia , Regulação Leucêmica da Expressão Gênica , Genes ras/genética , Mutação , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologia , Receptores de Interleucina-7/genética , Linfócitos T/patologia , Animais , Transformação Celular Neoplásica/genética , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Linfócitos T/metabolismo , Células Tumorais CultivadasRESUMO
A 7-year-old, intact male standard Poodle dog with hypothyroidism and atypical hyperadrenocorticism developed acute signs of lethargy, weakness, inappetence, vomiting, and diarrhea. Clinical signs progressed to hind limb proprioceptive deficits, aggressive behavior with obtundation, and an equivocal seizure. Necropsy revealed a mass in the sellar region that histologically consisted of multinucleated giant cells, macrophages, and hemosiderin-laden macrophages with fewer lymphocytes and plasma cells admixed with large regions of cholesterol cleft deposition, fibrin, and prominent Rosenthal fibers. Pituitary tissue was not identified on gross or histologic examination, but the mass was partially bordered by epithelial cells. The histologic characteristics are similar to changes described in the human medical literature as xanthogranuloma of the sellar region and xanthogranulomatous hypophysitis.
Assuntos
Neoplasias Encefálicas/veterinária , Doenças do Cão/patologia , Granuloma/veterinária , Sela Túrcica/patologia , Xantomatose/veterinária , Animais , Neoplasias Encefálicas/patologia , Cães , Evolução Fatal , Granuloma/patologia , Histocitoquímica/veterinária , Masculino , Xantomatose/patologiaRESUMO
Pseudorabies is caused by Suid herpesvirus 1, a member of the Alphaherpesvirinae subfamily. Although pigs are the natural host of Pseudorabies virus (PRV), the virus has a broad host range and may cause fatal encephalitis in many species. The United States obtained PRV-free status in 2004 after the virus was eradicated from domestic swineherds, but the virus is still present in feral swine populations. The current report describes PRV infection in 3 dogs that were used to hunt feral swine. The dogs developed clinical signs including facial pruritus with facial abrasions, dyspnea, vomiting, diarrhea, ataxia, muscle stiffness, and death. Two were euthanized, and 1 died within approximately 48 hr after onset of clinical signs. The salient histologic changes consisted of neutrophilic trigeminal ganglioneuritis with neuronophagia and equivocal intranuclear inclusion bodies. Pseudorabies virus was isolated from fresh tissues from 2 of the dogs, and immunohistochemistry detected the virus in the third dog. Virus sequencing and phylogeny, based upon available GenBank sequences, revealed that the virus was likely a field strain that was closely related to a cluster of PRV strains previously identified in Illinois. Though eradicated from domestic swine in the United States, PRV is present in populations of feral swine, and should therefore continue to be considered a possible cause of disease in dogs and other domestic animals with compatible clinical history and signs. Continued surveillance is necessary to prevent reintroduction of PRV into domestic swine.