RESUMO
BACKGROUND: Treatment for the debilitating disease hidradenitis suppurativa (HS) is inadequate in many patients. Despite an incidence of approximately 1%, HS is often under-recognized and underdiagnosed, and is associated with a high morbidity and poor quality of life. OBJECTIVES: To gain a better understanding of the pathogenesis of HS, in order to design new therapeutic strategies. METHODS: We employed single-cell RNA sequencing to analyse gene expression in immune cells isolated from involved HS skin vs. healthy skin. Flow cytometry was used to quantify the absolute numbers of the main immune populations. The secretion of inflammatory mediators from skin explant cultures was measured using multiplex and enzyme-linked immunosorbent assays. RESULTS: Single-cell RNA sequencing analysis identified a significant enrichment in the frequency of plasma cells, T helper (Th) 17 cells and dendritic cell subsets in HS skin, and the immune transcriptome was distinct and more heterogeneous than healthy skin. Flow cytometry revealed significantly increased numbers of T cells, B cells, neutrophils, dermal macrophages and dendritic cells in HS skin. Genes and pathways associated with Th17 cells, interleukin (IL)-17, IL-1ß and the NLRP3 inflammasome were enhanced in HS skin, particularly in samples with a high inflammatory load. Inflammasome constituent genes principally mapped to Langerhans cells and a subpopulation of dendritic cells. The secretome of HS skin explants contained significantly increased concentrations of inflammatory mediators, including IL-1ß and IL-17A, and culture with an NLRP3 inflammasome inhibitor significantly reduced the secretion of these, as well as other, key mediators of inflammation. CONCLUSIONS: These data provide a rationale for targeting the NLRP3 inflammasome in HS using small-molecule inhibitors that are currently being tested for other indications.
Assuntos
Hidradenite Supurativa , Humanos , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Qualidade de Vida , Pele/patologia , Inflamação , Mediadores da Inflamação/metabolismo , Mediadores da Inflamação/uso terapêuticoRESUMO
Psoriasis vulgaris is an inflammatory skin disease that affects 2%-3% of the population worldwide. One of the major challenges in discovering novel therapies is the poor translatability of animal models to human disease. Therefore, it is imperative to develop human preclinical models of psoriasis that are amenable to pharmacological intervention. Here, we report a 3-D reconstituted human epidermis (RHE) culture system treated with cytokines commonly associated with psoriasis (TNFα, IL-17A and IL-22) that reproduced some key features of the human disease. The effects on epidermal morphology, gene transcription and cytokine production, which are dysregulated in psoriasis were assessed. Certain morphological features of psoriatic epidermis were evident in cytokine-stimulated RHEs, including hypogranulosis and parakeratosis. In addition, RHEs responded to a cytokine mix in a dose-dependent manner by expressing genes and proteins associated with impaired keratinocyte differentiation (keratin 10/K10, loricrin), innate immune responses (S100A7, DEFB4, elafin) and inflammation (IL-1α, IL-6, IL-8, IL-10, IL-12/23p40, IL-36γ, GM-CSF and IFNγ) typical of psoriasis. These disease-relevant changes in morphology, gene transcription and cytokine production were robustly attenuated by pharmacologically blocking TNFα/IL-17A-induced NF-κB activation with IKK-2 inhibitor IV. Conversely, inhibition of IL-22-induced JAK1 signalling with ABT-317 strongly attenuated morphological features of the disease but had no effect on NFκB-dependent cytokine production, suggesting distinct mechanisms of action by the cytokines driving psoriasis. These data support the use of cytokine-induced RHE models for identifying and targeting keratinocyte signalling pathways important for disease progression and may provide translational insights into novel keratinocyte mechanisms for novel psoriasis therapies.
Assuntos
Interleucina-17 , Psoríase , Animais , Humanos , Interleucina-17/metabolismo , Queratinócitos/metabolismo , NF-kappa B/metabolismo , Psoríase/metabolismo , Pele/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Since first recognized in 1839, the pathogenesis of acne inversa (AI) has undergone repeated revisions. Although there is agreement that AI involves occlusion of hair follicles with subsequent inflammation and the formation of tracts, the histologic progression of this disease still requires refinement. The objective of this study was to examine the histologic progression of AI based on the examination of a large cohort of punch biopsies and excisional samples that were examined first by hematoxylin and eosin staining. The most informative of these samples were step-sectioned and stained by immunohistochemistry for epithelial and inflammatory markers. Based on this examination, the following observations were made: 1) AI arises from the epithelium of the infundibulum of terminal and vellus hairs; 2) These form cysts and epithelial tendrils that extend into soft tissue; 3) Immunohistochemical staining demonstrates the epithelium of AI is disordered with infundibular and isthmic differentiation and de novo expression of stem cell markers; 4) The inflammatory response in AI is heterogeneous and largely due to cyst rupture. The conclusions of this investigation were that AI is an epithelial-driven disease caused by infiltrative, cyst forming tendrils and most of the inflammation is due to cyst rupture and release of cornified debris and bacteria. Cyst rupture often occurs below the depths of punch biopsy samples indicating their use for analysis may give an incomplete picture of the disease. Finally, our data suggest that unless therapies inhibit tendril development, it is unlikely they will cause prolonged treatment-induced remission in AI.
Assuntos
Acne Vulgar/patologia , Progressão da Doença , Hidradenite Supurativa/patologia , Folículo Piloso/patologia , Humanos , Inflamação/patologiaRESUMO
A morphology-based assay such as immunohistochemistry (IHC) should be a highly effective means to define the expression of a target molecule of interest, especially if the target is a protein. However, over the past decade, IHC as a platform for biomarkers has been challenged by more quantitative molecular assays with reference standards but that lack morphologic context. For IHC to be considered a "top-tier" biomarker assay, it must provide truly quantitative data on par with non-morphologic assays, which means it needs to be run with reference standards. However, creating such standards for IHC will require optimizing all aspects of tissue collection, fixation, section thickness, morphologic criteria for assessment, staining processes, digitization of images, and image analysis. This will also require anatomic pathology to evolve from a discipline that is descriptive to one that is quantitative. A major step in this transformation will be replacing traditional ocular microscopes with computer monitors and whole slide images, for without digitization, there can be no accurate quantitation; without quantitation, there can be no standardization; and without standardization, the value of morphology-based IHC assays will not be realized.
Assuntos
Biomarcadores/análise , Imuno-Histoquímica/métodos , Animais , Expressão Gênica/fisiologia , Técnicas de Preparação Histocitológica , Humanos , Processamento de Imagem Assistida por Computador/métodos , Imuno-Histoquímica/normas , Microscopia , Padrões de Referência , Reprodutibilidade dos TestesRESUMO
Myelin is composed primarily of lipids and diseases affecting myelin are associated with alterations in its lipid composition. However, correlation of the spatial (in situ) distribution of lipids with the disease-associated compositional and morphological changes is not well defined. Herein we applied high resolution matrix-assisted laser desorption ionization imaging mass spectrometry (MALDI-IMS), immunohistochemistry (IHC), and liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI-MS) to evaluate brain lipid alterations in the dysmyelinating shiverer (Shi) mouse and cuprizone (Cz) mouse model of reversible demyelination. MALDI-IMS revealed a decrease in the spatial distribution of sulfatide (SHexCer) species, SHexCer (d42:2), and a phosphatidylcholine (PC) species, PC (36:1), in white matter regions like corpus callosum (CC) both in the Shi mouse and Cz mouse model. Changes in these lipid species were restored albeit not entirely upon spontaneous remyelination after demyelination in the Cz mouse model. Lipid distribution changes correlated with the local morphological changes as confirmed by IHC. LC-ESI-MS analyses of CC extracts confirmed the MALDI-IMS derived reductions in SHexCer and PC species. These findings highlight the role of SHexCer and PC in preserving the normal myelin architecture and our experimental approaches provide a morphological basis to define lipid abnormalities relevant to myelin diseases.
Assuntos
Ceramidas/metabolismo , Doenças Desmielinizantes/metabolismo , Bainha de Mielina/metabolismo , Fosfatidilcolinas/metabolismo , Sulfoglicoesfingolipídeos/metabolismo , Animais , Corpo Caloso/metabolismo , Corpo Caloso/ultraestrutura , Cuprizona/administração & dosagem , Doenças Desmielinizantes/induzido quimicamente , Doenças Desmielinizantes/genética , Doenças Desmielinizantes/patologia , Modelos Animais de Doenças , Imuno-Histoquímica , Metabolismo dos Lipídeos/efeitos dos fármacos , Masculino , Camundongos , Camundongos Transgênicos , Bainha de Mielina/ultraestrutura , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Substância Branca/metabolismo , Substância Branca/ultraestruturaRESUMO
Age- and gender-related lymphoid tissue variability in control male and female monkeys of various ages (under three years; three to six years; seven to fifteen years) was characterized. Spleen and thymus organ weights, organ-to-body and organ-to-brain ratios, morphology by light microscopy, and B- and T-cell immunohistochemistry (IHC) were evaluated. Splenic weights and ratios were not significantly different between various age groups or genders, except males and females in the three-to-six-years age group, who exhibited statistically significant changes from the under-three-years age group. No differences in the number of primary follicles, secondary follicles with germinal centers, B-cell follicles, and periarterial lymphoid sheath were seen between age groups or genders, and no trends were noted in the spleen. By IHC, no differences were observed in B- and T-cell splenic densities. Several age- and gender-related changes in weights and ratios were noted in the thymus. The thymus had a trend toward increased interlobular fat infiltration with increasing age in both males and females. Thymic delineation of the cortex and medulla was significantly decreased in the seven-to-fifteen-years age group for males only. The cortex-to-medulla ratio was significantly lower only in males in the seven-to-fifteen-years age group. B- and T-cell cellular density did not change across various ages.
Assuntos
Envelhecimento/fisiologia , Grupos Controle , Baço/citologia , Timo/citologia , Testes de Toxicidade/métodos , Animais , Feminino , Imuno-Histoquímica , Macaca fascicularis , Masculino , Tamanho do Órgão , Fatores SexuaisRESUMO
CI-1033 (canertinib) is an irreversible inhibitor of the erbB family of transmembrane tyrosine kinase receptors, including the epidermal growth factor (EGF) receptor. Various inhibitors of the EGF receptor, including CI-1033, have resulted in cutaneous toxicity in humans as a common adverse event. In a chronic toxicity study in rats, CI-1033 produced cutaneous lesions with morphologic characteristics similar to that reported in man. Here the authors describe in detail the dermal changes observed, along with other noteworthy findings of that study. Male and female Wistar rats (15/sex/group) were administered CI-1033 for 27 weeks at 2.5, 5, or 10 mg/kg (15, 30, or 60 mg/m(2), respectively) by gavage. Control animals (15/sex) received vehicle alone (aqueous 0.5% methylcellulose) in a dose volume of 5 mL/kg. Six animals/sex/dose were included for toxicokinetic evaluations. Skin lesions were the primary drug-related toxicity and occurred at > or = 2.5 mg/kg in a dose-dependent fashion. The major gross lesions were papules that evolved into crusts and scales that were first observed in weeks 1 and 3, respectively. Alopecia developed in conjunction with the papular eruptions. Skin changes were most pronounced in females, possibly due to higher drug levels. In week 13, CI-1033 plasma AUC(0-24) values were 527 to 1980 ng.h/mL in males and 844 to 2920 ng x h/mL in females at 2.5 to 10 mg/kg. Microscopic changes could be described as 3 patterns that affected the tail and body (haired skin). Pattern 1 consisted of epidermal changes that started as a superficial, perivascular spongiotic dermatitis with evolving epidermal hyperplasia, scale-crusts, and areas of ulceration. Areas of hyperplasia on the tail were often associated with the development of new hair follicles. Pattern 2 was characterized by a suppurative to pyogranulomatous infundibular folliculitis. Pattern 3 consisted of abnormally oriented hair follicles with malformed hair shafts that were associated with a deeper (isthmic) folliculitis; this correlated with alopecia. Elevations in bone marrow myeloid counts correlated with a peripheral leukocytosis, consistent with inflammatory changes in the dermis. In addition, hepatic cholestasis and epithelial atrophy in the gastrointestinal tract and vagina occurred at > or = 2.5 mg/kg. In conclusion, CI-1033 produced cutaneous lesions involving the epidermis and hair follicle, and the morphologic characteristics were similar to that reported in clinical studies with various inhibitors of the EGF receptor. These changes are consistent with pharmacologic inhibition of the EGF receptor in these tissues and demonstrate that the rat can serve as an animal model for investigating the mechanisms for this toxicity.
Assuntos
Receptores ErbB/antagonistas & inibidores , Foliculite/induzido quimicamente , Morfolinas/toxicidade , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Dermatopatias Papuloescamosas/induzido quimicamente , Pele/efeitos dos fármacos , Administração Oral , Alopecia/induzido quimicamente , Alopecia/patologia , Animais , Área Sob a Curva , Relação Dose-Resposta a Droga , Feminino , Foliculite/patologia , Longevidade/efeitos dos fármacos , Masculino , Morfolinas/farmacocinética , Ratos , Ratos Wistar , Pele/patologia , Dermatopatias Papuloescamosas/patologia , Testes de ToxicidadeRESUMO
Mouse models lupus nephritis (LN) have provided important insights into disease pathogenesis, although none have been able to recapitulate all features of the human disease. Using comprehensive longitudinal analyses, we characterized a novel accelerated mouse model of lupus using pristane treatment in SNF1 (SWR X NZB F1) lupus prone mice (pristane-SNF1 mice). Pristane treatment in SNF1 mice accelerated the onset and progression of proteinuria, autoantibody production, immune complex deposition and development of renal lesions. At week 14, the pristane-SNF1 model recapitulated kidney disease parameters and molecular signatures seen in spontaneous disease in 36 week-old SNF1 mice and in a traditional IFNα-accelerated NZB X NZW F1 (BWF1) model. Blood transcriptome analysis revealed interferon, plasma cell, neutrophil, T-cell and protein synthesis signatures in the pristane-SNF1 model, all known to be present in the human disease. The pristane-SNF1 model appears to be particularly useful for preclinical research, robustly exhibiting many characteristics reminiscent of human disease. These include i) a stronger upregulation of the cytosolic nucleic acid sensing pathway, which is thought to be key component of the pathogenesis of the human disease, and ii) more prominent kidney interstitial inflammation and fibrosis, which have been both associated with poor prognosis in human LN. To our knowledge, this is the only accelerated model of LN that exhibits a robust tubulointerstitial inflammatory and fibrosis response. Taken together our data show that the pristane-SNF1 model is a novel accelerated model of LN with key features similar to human disease.
Assuntos
Túbulos Renais/efeitos dos fármacos , Túbulos Renais/patologia , Nefrite Lúpica/patologia , Terpenos/farmacologia , Animais , Autoanticorpos/biossíntese , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Feminino , Fibrose , Glomerulonefrite/induzido quimicamente , Glomerulonefrite/complicações , Humanos , Hipergamaglobulinemia/induzido quimicamente , Hipergamaglobulinemia/complicações , Inflamação/induzido quimicamente , Inflamação/complicações , Nefrite Lúpica/complicações , Nefrite Lúpica/imunologia , Nefrite Lúpica/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Transcrição Gênica/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacosRESUMO
Over half of adults are seropositive for JC polyomavirus (JCV), but rare individuals develop progressive multifocal leukoencephalopathy (PML), a demyelinating JCV infection of the central nervous system. Previously, PML was primarily seen in immunosuppressed patients with AIDS or certain cancers, but it has recently emerged as a drug safety issue through its association with diverse immunomodulatory therapies. To better understand the relationship between the JCV life cycle and PML pathology, we studied autopsy brain tissue from a 70-year-old psoriasis patient on the integrin alpha-L inhibitor efalizumab following a ~2 month clinical course of PML. Sequence analysis of lesional brain tissue identified PML-associated viral mutations in regulatory (non-coding control region) DNA, capsid protein VP1, and the regulatory agnoprotein, as well as 9 novel mutations in capsid protein VP2, indicating rampant viral evolution. Nine samples, including three gross PML lesions and normal-appearing adjacent tissues, were characterized by histopathology and subject to quantitative genomic, proteomic, and molecular localization analyses. We observed a striking correlation between the spatial extent of demyelination, axonal destruction, and dispersion of JCV along white matter myelin sheath. Our observations in this case, as well as in a case of PML-like disease in an immunocompromised rhesus macaque, suggest that long-range spread of polyomavirus and axonal destruction in PML might involve extracellular association between virus and the white matter myelin sheath.
Assuntos
Encéfalo/virologia , Vírus JC/patogenicidade , Leucoencefalopatia Multifocal Progressiva/virologia , Bainha de Mielina/metabolismo , Replicação Viral , Idoso , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Feminino , Humanos , Vírus JC/genética , Vírus JC/fisiologia , Macaca mulatta , Masculino , Mutação , Bainha de Mielina/patologia , Bainha de Mielina/virologia , Proteínas Virais de Fusão/genética , Proteínas Virais Reguladoras e Acessórias/genética , Virulência/genéticaRESUMO
Reducing the production of larger aggregation-prone amyloid ß-peptides (Aß) remains an untested therapeutic approach for reducing the appearance and growth of Aß plaques in the brain, which are a hallmark pathological feature of Alzheimer's disease. γ-Secretase modulators (GSMs) are therapeutics that impact γ-secretase-dependent cleavage of amyloid precursor protein to promote the production of shorter Aß peptides that are less prone to aggregation and plaque deposition. This is accomplished without inhibiting overall γ-secretase function and cleavage of other substrates, which is believed to be a source of deleterious side effects. Here, we report the pharmacokinetic and pharmacodynamic properties of BIIB042, a novel bioavailable and brain-penetrant GSM. In cell-based assays, BIIB042 reduced the levels of Aß42, increased the levels of Aß38 and had little effect on the levels of Aß40, the most abundant Aß species. Similar pharmacodynamic properties were confirmed in the central nervous system and in plasma of mice and rats, and also in plasma of cynomolgus monkeys after a single oral dose of BIIB042. BIIB042 reduced Aß42 levels and Aß plaque burden in Tg2576 mice, which overexpress human amyloid precursor protein and serve as a model system for Alzheimer's disease. BIIB042 did not inhibit cleavage of other γ-secretase substrates in cell-based and in vivo signaling and cleavage assays. The pharmacodynamic effects of lowering Aß42 in the central nervous system coupled with demonstrated efficacy in reducing plaque pathology suggests modulation of γ-secretase, with molecules like BIIB042, is a compelling therapeutic approach for the treatment of Alzheimer's disease.
Assuntos
Aldeídos/farmacocinética , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/enzimologia , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/metabolismo , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Aldeídos/administração & dosagem , Peptídeos beta-Amiloides/sangue , Animais , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Macaca fascicularis , Masculino , Camundongos , Placa Amiloide/metabolismo , Isoformas de Proteínas/sangue , Ratos , Ratos Endogâmicos F344RESUMO
OBJECTIVE: To determine whether systemic immunologic hyperreactivity exists in horses with chronic laminitis, compared with responses for nonlaminitic horses. ANIMALS: 7 nonlaminitic horses and 7 CL horses. PROCEDURE: In experiment 1, intradermal testing (IDT) was performed on 7 nonlaminitic and 7 CL horses to evaluate the response to a combination of 70 allergens at 15 and 30 minutes and 4 and 24 hours after injection. Three nonlaminitic and 3 CL horses used in experiment 1 were used in experiment 2 to determine whether histologic differences existed between the 2 groups. The H&E-stained tissue sections were evaluated on the basis of 3 criteria. For all analyses, 2-sample t-tests were used to determine significant differences between the groups. RESULTS: In experiment 1, CL horses had significantly higher total responses to IDT than nonlaminitic horses at the first 3 time periods. Also, CL horses had significantly fewer total scores of 0 than nonlaminitic horses at all time periods, except at 24 hours. In experiment 2, we did not detect significant differences between groups for any criterion. CONCLUSIONS AND CLINICAL RELEVANCE: Results support the hypothesis that CL horses develop hyperreactivity to various antigenic stimuli, compared with responses for nonlaminitic horses. Therefore, the possibility that antigenic challenge may result in exacerbation of clinical signs of laminitis should be discussed with horse owners. Chronic laminitis should also be a consideration when a horse becomes lame following antigenic challenges.
Assuntos
Doenças dos Cavalos/imunologia , Hipersensibilidade/imunologia , Hipersensibilidade/veterinária , Testes Intradérmicos/veterinária , Coxeadura Animal/imunologia , Alérgenos/imunologia , Animais , Feminino , Doenças do Pé/complicações , Doenças do Pé/imunologia , Doenças do Pé/patologia , Doenças do Pé/veterinária , Casco e Garras/imunologia , Casco e Garras/patologia , Doenças dos Cavalos/diagnóstico , Doenças dos Cavalos/patologia , Cavalos/imunologia , Hipersensibilidade/complicações , Hipersensibilidade/diagnóstico , Coxeadura Animal/complicações , Coxeadura Animal/patologia , MasculinoRESUMO
A 1-year-old male miniature donkey (Equus asinus) from a herd of eight was presented with a 9-month history of pruritic dermatitis, lethargy and anorexia. Physical examination revealed diffuse lichenification and scales involving the skin of the face, head and dorsum from the neck to the pelvis. The main histological alteration within the superficial and deep dermis was the presence of multiple large, spherical, thick walled, protozoal Besnoitia cysts. In addition, the inflammatory response consisted of a moderate, superficial and deep perivascular, mixed mononuclear cell infiltrate, with epidermal hyperplasia and compact orthokeratosis. Based upon the large size of the protozoal cysts and the ultrastructural features of the bradyzoites contained therein (conoid, polar ring, rhoptries, micronemes and microtubules), a diagnosis of cutaneous besnoitiosis was established. Treatment with trimethoprim-sulphamethoxazole resulted in significant clinical improvement. To our knowledge, this is the first reported case of besnoitiosis in a miniature donkey in North America.
RESUMO
Described herein is a detailed analysis of the impact of three fixatives (10% neutral buffered formalin, modified methacarn and 70% ethanol) on RNA quality and utility using microarray analysis compared to OCT-embedded and flash frozen tissue. From rat livers fixed and stored in paraffin blocks for 1 month or 1 year, RNA was isolated and applied to rat whole genome microarrays. At both time points, RNA isolated from OCT-embedded tissue lost up to 5% of the information contained in snap frozen control liver. Of the fixatives used, modified methacarn was associated with the smallest loss of RNA information content (approximately 10%), while liver fixed in 70% ethanol and 10% neutral buffered formalin lost roughly 25% and 80%, respectively. We conclude that when optimum morphology is required for techniques such as laser microdissection, modified methacarn is the fixative least harmful to nucleic acids of the three tested in this study. In contrast, using traditional isolation techniques, RNA derived from tissue fixed in 10% NBF will not give reliable results on microarray studies, and should be reserved for techniques less affected by the fragmentation and modification of the template RNA, such as quantitative RT-PCR.
Assuntos
Artefatos , Fixadores/química , Análise de Sequência com Séries de Oligonucleotídeos , RNA Complementar/química , Fixação de Tecidos/métodos , Ácido Acético/química , Animais , Clorofórmio/química , Etanol/química , Formaldeído/química , Lasers , Fígado/química , Metanol/química , Microdissecção , Inclusão em Parafina , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
BACKGROUND: Laser scanning cytometry (LSC) is a new technology similar to flow cytometry but generates data from analysis of successive microscopic fields. Unlike its use in other applications, LSC-generated data are not random when used for tissue sections, but are dependent on the microanatomy of the tissue and the distribution and expression of the protein under investigation. For valid LSC analysis, the data generated requires the evaluation of a sufficient tissue area to ensure an accurate representation of expression within the tissue of interest. METHODS: In this report, we describe a simple and common sense method for determining the area of tissue required for sound LSC analysis by tracking the variation in the measure of target expression with increasing number of fields until it approaches zero. RESULTS: This approach was used to evaluate the expression of immunohistochemical markers with differing tissue distributions in liver (PMP70, CYP1A2, and Ki67 positive macrophages) and a colorectal adenocarcinoma (activated caspase-3 positive cells), which exhibited diffuse, regional (centrilobular), random, and irregular distribution patterns respectively. CONCLUSIONS: Analyses of these markers demonstrated that the amount of tissue area required to reach a steady measure of a parameter increased with increasing variability of the tissue distribution.
Assuntos
Citometria de Varredura a Laser/métodos , Coleta de Tecidos e Órgãos , Animais , Biomarcadores/análise , Biomarcadores Tumorais/análise , Humanos , Macaca fascicularis , Masculino , Camundongos , Camundongos SCID , Ratos , Ratos Sprague-Dawley , Estudos Retrospectivos , Distribuição Tecidual , Células Tumorais CultivadasRESUMO
Hepatocellular vacuolation can be a diagnostic challenge since cytoplasmic accumulations of various substances (lipid, water, phospholipids, glycogen, and plasma) can have a similar morphology. Cytoplasmic accumulation of phospholipids following administration of cationic amphiphilic drugs (CAD) can be particularly difficult to differentiate from nonphosphorylated lipid accumulations at the light microscopic level. Histochemical methods (Sudan Black, Oil Red-O, Nile Blue, etc.) can be used to identify both nonphosphorylated and/or phosphorylated lipid accumulations, but these techniques require non-paraffin-embedded tissue and are only moderately sensitive. Thus, electron microscopy is often utilized to achieve a definitive diagnosis based upon the characteristic morphologic features of phospholipid accumulations; however, this is a low throughput and labor intense procedure. In this report, we describe the use of immunohistochemical staining for LAMP-2 (a lysosome-associated protein) and adipophilin (a protein that forms the membrane around non-lysosomal lipid droplets) to differentiate phospholipidosis and lipidosis, respectively in the livers of rats. This staining procedure can be performed on formalin-fixed paraffin embedded tissues, is more sensitive than histochemistry, and easier to perform than ultrastructural evaluation.
Assuntos
Lipidoses/diagnóstico , Fígado/ultraestrutura , Proteína 2 de Membrana Associada ao Lisossomo/análise , Peptídeos/análise , Fosfolipídeos/metabolismo , Vacúolos/efeitos dos fármacos , Animais , Citoplasma/metabolismo , Diagnóstico Diferencial , Feminino , Imuno-Histoquímica , Fígado/efeitos dos fármacos , Fígado/metabolismo , Proteínas de Membrana , Perilipina-2 , Ratos , Ratos Sprague-Dawley , Vacúolos/ultraestruturaRESUMO
Primary inherited disorders of cornification in veterinary medicine are uncommon and rarely reported. Herein described is a unique syndrome associated with keratoderma that occurred in two Bennett's wallaby siblings (Macropus rufogriseus), and was characterized by profound thickening of the pad skin of all feet, generalized scaling of haired skin, and death within 7 weeks of out-of-pouch experience. The male also had depressed serum zinc levels. In addition, the male had, on electron microscopic exam of his skin, the presence of abnormal lipid deposits within the stratum corneum and stratum granulosum. The combination of clinical features and electron microscopic findings strongly suggests a syndrome analogous to harlequin ichthyosis or lamellar ichthyosis in humans.
Assuntos
Eritrodermia Ictiosiforme Congênita/veterinária , Macropodidae , Animais , Diagnóstico Diferencial , Feminino , Eritrodermia Ictiosiforme Congênita/diagnóstico , Eritrodermia Ictiosiforme Congênita/genética , Masculino , LinhagemRESUMO
Molecular characterization of morphologic change requires exquisite tissue morphology and RNA preservation; however, traditional fixatives usually result in fragmented RNA. To optimize molecular analyses on fixed tissues, we assessed morphologic and RNA integrity in rat liver when sections were fixed in 70% neutral-buffered formalin, modified Davidson's II, 70% ethanol, UMFIX, modified Carnoy's, modified methacarn, Bouin's, phosphate-buffered saline, or 30% sucrose. Each sample was subjected to standard or microwave fixation and standard or microwave processing, and sections were evaluated microscopically. RNA was extracted and assessed for preservation of quality and quantity. Modified methacarn, 70% ethanol, and modified Carnoy's solution each resulted in tissue morphology representing a reasonable alternative to formalin. Modified methacarn and UMFIX best preserved RNA quality. Neither microwave fixation nor processing affected RNA integrity relative to standard methods, although morphology was modestly improved. We conclude that modified methacarn, 70% ethanol, and modified Carnoy's solution provided acceptable preservation of tissue morphology and RNA quality using both standard and microwave fixation and processing methods. Of these three fixatives, modified methacarn provided the best results and can be considered a fixative of choice where tissue morphology and RNA integrity are being assessed in the same specimens.
Assuntos
Fixadores/química , Fígado/citologia , Fígado/metabolismo , RNA Ribossômico 18S/química , Fixação de Tecidos/métodos , Animais , Fixadores/farmacologia , Fígado/efeitos dos fármacos , RNA Ribossômico 18S/efeitos dos fármacos , Ratos , Ratos Sprague-DawleyRESUMO
The purpose of this study is to reproduce in vitro a recessive keratinization defect of Norfolk terrier dogs characterized by a lack of keratin 10 (K10) production. Keratinocytes from skin biopsy samples of four normal dogs and two affected dogs were cultured organotypically with growth factor-supplemented media in order to stimulate cornification. The cultured epidermis from the normal dogs closely resembled the normal epidermis in vivo and cornified. The cultured epidermis from the affected dogs displayed many phenotypic alterations identified in skin biopsies from dogs with this heritable defect. Immunohistochemistry and immunoblotting showed a marked decrease in K10 from the cultures of the affected keratinocytes, compared to that in K10 from the cultures of the normal keratinocytes. Real-time reverse transcription polymerase chain reaction quantitation showed a 31-fold decrease in K10, a 1.75-fold increase in K1 and a 136-fold increase in K2e between the affected and the normal epidermis. Organotypic keratinocytes showed a 241-fold decrease in K10, a 31-fold decrease in K1 and a 1467-fold decrease in K2e between the affected and normal cultures. Although in vitro keratin expression did not precisely simulate in vivo, the morphology of the normal and the affected epidermis was largely preserved; thus, this culture system may provide an alternative to in vivo investigations for cutaneous research involving cornification.
Assuntos
Doenças do Cão/patologia , Queratinas/deficiência , Queratinas/genética , Dermatopatias Genéticas/veterinária , Animais , Sequência de Bases , Técnicas de Cultura de Células , Células Cultivadas , DNA/genética , Doenças do Cão/genética , Doenças do Cão/metabolismo , Cães , Queratina-10 , Queratinócitos/metabolismo , Queratinócitos/patologia , Queratinas/metabolismo , Fenótipo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Dermatopatias Genéticas/genética , Dermatopatias Genéticas/metabolismo , Dermatopatias Genéticas/patologiaRESUMO
Hair growth and replacement have been studied extensively in humans, sheep and laboratory rodents, but in dogs and other mammalian species few studies have been published. The objectives of this study were: (1) to determine the time required for the hair to re-grow in dogs after clipping for a surgical procedure; (2) to define whether the season of the year influenced the period of time required for re-growth and; (3) to determine if season might influence the telogen: anagen ratio. Eleven Labrador retrievers were recruited during spring, 10 during summer, six during autumn and 10 during winter. Hairs re-grew to their preclipped length in 14.6 weeks, 14.5 weeks, 13.6 weeks and 15.4 weeks when shaved in the spring, summer, autumn and winter, respectively. The differences in these values were not significant suggesting that season has no effect on the rate of hair re-growth in Labrador retrievers housed indoors (P = 0.12). The mean values for the telogen: anagen ratio in each season were: 5.2 (spring), 6.1 (summer), 9.5 (autumn), and 5.3 (winter). The differences in these values also were not significant (P = 0.89). The percentage of hairs in telogen was over 80% in all four seasons.
Assuntos
Cães/fisiologia , Cabelo/crescimento & desenvolvimento , Animais , Feminino , Cabelo/fisiologia , Abrigo para Animais , Masculino , Linhagem , Estações do AnoRESUMO
Hereditary nasal dermatitis is reported in 14 Labrador Retrievers and 4 Labrador Retriever crosses. This appears to be a newly described inherited disorder for which an autosomal recessive mode of inheritance is suspected. The lesions were first noted between 6 and 12 months of age. Histopathological analysis revealed parakeratotic hyperkeratosis, often with marked multifocal accumulation of proteinaceous fluid between keratinocytes within the stratum corneum and superficial stratum spinosum. There was also a sub-basal lymphoplasmacytic infiltration within the superficial dermis. Immunohistochemistry staining for IgG (n = 4), distemper and papillomaviruses (n = 4) were negative, as were serum antinuclear antibody serology (n = 4) and fungal culture (n = 7). Electron microscopy revealed an altered cornification process: retention of nuclear chromatin, absence of lamellar bodies and marked intercellular oedema. Dogs did not respond to oral administration of zinc methionin (n = 3), cephalexin (n = 4), vitamin A alcohol (n = 1) or topical tretinoin (n = 1). Improvement of the lesions was obtained with topical vitamin E (n = 2), petroleum jelly (n = 2), and propylene glycol (n = 5).