RESUMEN
As biomarker discovery takes centre-stage, the role of immunohistochemistry within that process is increasing. At the same time, the number of antibodies being produced for "research use" continues to rise and it is important that antibodies to be used as biomarkers are validated for specificity and sensitivity before use. This guideline seeks to provide a stepwise approach for the validation of an antibody for immunohistochemical assays, reflecting the views of a consortium of academic and pharmaceutical based histopathology researchers. We propose that antibodies are placed into a tier system, level 1-3, based on evidence of their usage in immunohistochemistry, and that the degree of validation required is proportionate to their place on that tier.
Asunto(s)
Anticuerpos/química , Biomarcadores/metabolismo , Inmunohistoquímica/métodos , Proteínas/química , Animales , Biomarcadores/química , Biomarcadores de Tumor/metabolismo , Investigación Biomédica/métodos , Línea Celular , Química Farmacéutica/métodos , Epítopos/química , Regulación Neoplásica de la Expresión Génica , Humanos , Neoplasias/inmunología , Neoplasias/metabolismo , Fenotipo , ARN Mensajero/metabolismo , Reproducibilidad de los ResultadosRESUMEN
Despite potential clinical importance, target cells for mother-to-child transmission of HIV-1 have not yet been identified. Cord blood-derived CD4(+) T cells are largely naive and do not express CCR5, the mandatory coreceptor for transmitted HIV-1 R5 strains in infants. In the present study, we demonstrate that in the human fetal and infant gut mucosa, there is already a large subset of mucosal memory CD4(+)CCR5(+) T cells with predominantly a Th1 and Th17 phenotype. Using next-generation sequencing of the TCRß chain, clonally expanded T cells as a hallmark for memory development predominated in the gut mucosa (30%), whereas few were found in the lymph nodes (1%) and none in cord blood (0%). The gut mucosal fetal and infant CD4(+) T cells were highly susceptible to HIV-1 without any prestimulation; pol proviral DNA levels were similar to infected phytohemagglutinin-stimulated adult PBMCs. In conclusion, in the present study, we show that extensive adaptive immunity is present before birth and the gut mucosa is the preferential site for memory CD4(+) T cells. These CD4(+)CCR5(+) T cells in the infant mucosa provide a large pool of susceptible cells for ingested HIV-1 at birth and during breastfeeding, indicating a mucosal route of mother-to-child transmission that can be targeted in prevention strategies.
Asunto(s)
Linfocitos T CD4-Positivos/citología , Infecciones por VIH/transmisión , Memoria Inmunológica , Transmisión Vertical de Enfermedad Infecciosa , Intestinos/inmunología , Receptores CCR5/metabolismo , Adulto , Recuento de Linfocito CD4 , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Células Cultivadas , Femenino , Infecciones por VIH/inmunología , VIH-1/inmunología , Humanos , Memoria Inmunológica/inmunología , Memoria Inmunológica/fisiología , Recién Nacido , Mucosa Intestinal/citología , Mucosa Intestinal/inmunología , Mucosa Intestinal/metabolismo , Intestinos/citología , Intestinos/virología , Masculino , Relaciones Madre-Hijo , Embarazo , Complicaciones Infecciosas del Embarazo/inmunologíaRESUMEN
RATIONALE: Unlike conventional dendritic cells, plasmacytoid DCs (PDC) are poor in antigen presentation and critical for type I interferon response. Though proposed to be present in human atherosclerotic lesions, their role in atherosclerosis remains elusive. OBJECTIVE: To investigate the role of PDC in atherosclerosis. METHODS AND RESULTS: We show that PDC are scarcely present in human atherosclerotic lesions and almost absent in mouse plaques. Surprisingly, PDC depletion by 120G8 mAb administration was seen to promote plaque T-cell accumulation and exacerbate lesion development and progression in LDLrâ»/â» mice. PDC depletion was accompanied by increased CD4⺠T-cell proliferation, interferon-γ expression by splenic T cells, and plasma interferon-γ levels. Lymphoid tissue PDC from atherosclerotic mice showed increased indoleamine 2,3-dioxygenase (IDO) expression and IDO blockage abrogated the PDC suppressive effect on T-cell proliferation. CONCLUSIONS: Our data reveal a protective role for PDC in atherosclerosis, possibly by dampening T-cell proliferation and activity in peripheral lymphoid tissue, rendering PDC an interesting target for future therapeutic interventions.
Asunto(s)
Aterosclerosis/patología , Aterosclerosis/fisiopatología , Linfocitos T CD4-Positivos/patología , Proliferación Celular , Células Dendríticas/patología , Células Dendríticas/fisiología , Animales , Anticuerpos Monoclonales/farmacología , Aterosclerosis/metabolismo , Células Cultivadas , Técnicas de Cocultivo , Células Dendríticas/efectos de los fármacos , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Humanos , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Interferón gamma/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Receptores de LDL/deficiencia , Receptores de LDL/genética , Receptores de LDL/metabolismoRESUMEN
BACKGROUND: Preoperative portal vein embolization (PVE) is used to increase future remnant liver volume through induction of hepatocellular regeneration. This event, however, potentially enhances tumor growth. The aim of our study was to assess tumor growth and liver regeneration after PVE in a rabbit hepatic tumor model. The VX2 carcinoma is derived from a virus-induced papilloma tumor in rabbits. The tumor grows rapidly, and its blood supply is similar to that of human hepatocellular carcinoma. MATERIALS AND METHODS: Two weeks after subcapsular implantation of a VX2 carcinoma in the cranial liver lobe, New Zealand White rabbits were allocated to a control or PVE group (n = 5 per group). In the PVE group, the portal vein branch to the cranial liver lobes (80%) was embolized using particles and coils, leaving the caudal liver lobe (20%) free. In the tumor control group, the liver was mobilized. Computed tomography volumetry was performed on days 3, 7, 10, and 14. Tumor growth rate (TGR), hepatocellular proliferation rate, and liver damage parameters were assessed before PVE and on days 1, 3, 7, 10, and 14. RESULTS: Portography confirmed complete occlusion of the portal vein branch to the cranial liver lobes in all PVE rabbits. The hypertrophy response and proliferation rate in the nonembolized liver lobes were significantly higher in the PVE group, which was confirmed by liver-to-body weight index assessment. TGR was increased in both groups, with a significantly larger increase in the PVE group over time (day 14: mean, 34.4 ± 4.3 mL/d versus control: mean, 24.1 ± 7.2 mL/d; P < 0.05). CONCLUSIONS: TGR was significantly increased after PVE in the rabbit tumor model. This finding supports the notion that PVE potentially enhances tumor growth, along with the regeneration of the nonembolized liver lobe.
Asunto(s)
Embolización Terapéutica/efectos adversos , Neoplasias Hepáticas Experimentales/patología , Vena Porta , Animales , Proliferación Celular , Modelos Animales de Enfermedad , Femenino , Hipertrofia , Regeneración Hepática , Conejos , Tomografía Computarizada por Rayos XRESUMEN
BACKGROUND: Episodes of microvascular proliferation associated with volume expansion have been observed in arteriovenous malformations (AVMs) of skin and soft tissue. OBJECTIVE: We sought to investigate the relationship between a microvascular proliferative response and flow velocity in AVMs. METHODS: Resection specimens of 80 AVMs were clinically categorized as either high- or low-flow lesions, and histopathologically screened for the presence of microvessels, inflammation, thrombosis, or a combination of these. Immunohistochemistry was performed with endoglin (CD105), von Willebrand factor, and fibrinogen antibodies. RESULTS: Clinically, 37 AVMs were classified as high-flow lesions and 43 as low-flow lesions. In 81% of high-flow lesions microvascular proliferations were seen versus in 14% of low-flow lesions (P < .005). In high-flow lesions, which were embolized before surgery (30% of all), 88% showed microvascular proliferation, 88% inflammation, and 33% thrombosis. However, similar vasoproliferative responses were also observed in nonembolized AVM (69% high-flow and 14% low-flow lesions). Endoglin was more frequently expressed in high-flow lesions. Extracellular von Willebrand factor staining was found in most lesions, irrespective of flow type or presence of microvascular proliferations. LIMITATIONS: The study was carried out at a single tertiary referral center. CONCLUSIONS: Microvascular proliferative masses in AVMs appear to be strongly associated with high-flow characteristics. This could be explained to some extent by previous therapeutic embolization and/or inflammation in the lesion. However, occurrence of similar microvascular responses in AVM that were not embolized before surgery suggests that the biomechanical effects of high flow in these lesions may also have an angiogenic effect.
Asunto(s)
Malformaciones Arteriovenosas/patología , Malformaciones Arteriovenosas/fisiopatología , Embolización Terapéutica/efectos adversos , Inflamación/complicaciones , Microvasos/patología , Adolescente , Adulto , Anciano , Velocidad del Flujo Sanguíneo , Niño , Preescolar , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Adulto JovenRESUMEN
OBJECTIVE: Whether and how B lymphocytes contribute to the pathogenesis of spondylarthritis (SpA), a seronegative arthritis associated with gut inflammation, remains unknown. Because innate-like CD5+ B lymphocytes with regulatory functions have been identified in colitis models, we undertook the present study to analyze the presence and function of CD5+ B cells in human SpA. METHODS: Peripheral blood B cells from patients with SpA, patients with rheumatoid arthritis (RA), and healthy controls were analyzed by flow cytometry. Synovial biopsy samples were evaluated by immunohistochemistry analysis. Sorted CD5+ and CD5- B cells were analyzed for somatic hypermutation, expression of costimulatory molecules, and cytokine production. RESULTS: The naive, marginal zone-like, and to a lesser extent memory B cell compartments in patients with SpA exhibited a clear and specific increase of CD5+ B cells, which was not found in patients with RA. This increase was not due to either B cell activation or preferential migration of CD5- B cells to the inflamed synovium. Consistent with their phenotype and the low-affinity polyreactive immunoglobulins produced by their murine counterpart cells, CD5+ B cells from patients with SpA showed low levels of somatic hypermutation. With regard to antigen presentation, CD5+ B cells expressed slightly increased HLA-DR levels but low CD80 and CD86 levels. In vitro activation failed to up-regulate these costimulatory molecules but induced significant production of interleukin-10 and interleukin-6 by CD5+ B cells. CONCLUSION: CD5+ B cells are specifically increased in SpA. Analysis of somatic hypermutation, expression of antigen-presenting and costimulatory molecules, and cytokine production indicates that this B cell subset has regulatory capacities. Further investigation of the potential role of CD5+ cells in SpA is warranted.
Asunto(s)
Linfocitos B Reguladores/inmunología , Antígenos CD5/metabolismo , Espondiloartritis/inmunología , Adulto , Artritis Reumatoide/inmunología , Artritis Reumatoide/metabolismo , Linfocitos B Reguladores/metabolismo , Femenino , Citometría de Flujo , Humanos , Interleucina-10/metabolismo , Interleucina-6/metabolismo , Masculino , Persona de Mediana Edad , Espondiloartritis/metabolismo , Membrana Sinovial/inmunología , Membrana Sinovial/metabolismoRESUMEN
OBJECTIVE: Angiotensin-converting enzyme and its effector peptide angiotensin II have been implicated in the pathogenesis of acute respiratory distress syndrome. Recently, angiotensin-converting enzyme 2 was identified as the counter-regulatory enzyme of angiotensin-converting enzyme that converts angiotensin II into angiotensin-(1-7). The aim of this study was to determine pulmonary angiotensin-converting enzyme and angiotensin-converting enzyme 2 activity in patients with acute respiratory distress syndrome. DESIGN: Prospective observational pilot study. SETTING: A PICU of a university hospital. PATIENTS: Fourteen patients admitted, requiring mechanical ventilation for respiratory syncytial virus lower respiratory tract infection. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Two groups of patients were distinguished at admission: a group fulfilling the criteria for acute respiratory distress syndrome and a non-acute respiratory distress syndrome group. Angiotensin-converting enzyme and angiotensin-converting enzyme 2 activity were measured in bronchoalveolar lavage fluid. Patients with acute respiratory distress syndrome had increased angiotensin-converting enzyme activity and decreased angiotensin-converting enzyme 2 activity (p < 0.001) compared with the control group. CONCLUSION: It is shown for the first time that in acute respiratory distress syndrome, enhanced angiotensin-converting enzyme activity is paralleled by a reduced angiotensin-converting enzyme 2 activity, similar to that found in an experimental rat model of acute respiratory distress syndrome. The reduced angiotensin-converting enzyme 2 activity may be counteracted by restoring angiotensin-(1-7) level, thereby offering a novel treatment modality for this syndrome.
Asunto(s)
Peptidil-Dipeptidasa A/metabolismo , Síndrome de Dificultad Respiratoria del Recién Nacido/enzimología , Enzima Convertidora de Angiotensina 2 , Líquido del Lavado Bronquioalveolar/química , Estudios de Casos y Controles , Femenino , Humanos , Lactante , Recién Nacido , Pulmón/enzimología , Masculino , Peptidil-Dipeptidasa A/análisis , Estudios ProspectivosRESUMEN
Prolactin is best known as the polypeptide anterior pituitary hormone, which regulates the development of the mammary gland. However, it became clear over the last decade that prolactin contributes to a broad range of pathologies, including breast cancer. Prolactin is also involved in angiogenesis via the release of pro-angiogenic factors by leukocytes and epithelial cells. However, whether prolactin also influences endothelial cells, and whether there are functional consequences of prolactin-induced signalling in the perspective of angiogenesis, remains so far elusive. In the present study, we show that prolactin induces phosphorylation of ERK1/2 and STAT5 and induces tube formation of endothelial cells on Matrigel. These effects are blocked by a specific prolactin receptor antagonist, del1-9-G129R-hPRL. Moreover, in an in vivo model of the chorioallantoic membrane of the chicken embryo, prolactin enhances vessel density and the tortuosity of the vasculature and pillar formation, which are hallmarks of intussusceptive angiogenesis. Interestingly, while prolactin has only little effect on endothelial cell proliferation, it markedly stimulates endothelial cell migration. Again, migration was reverted by del1-9-G129R-hPRL, indicating a direct effect of prolactin on its receptor. Immunohistochemistry and spectral imaging revealed that the prolactin receptor is present in the microvasculature of human breast carcinoma tissue. Altogether, these results suggest that prolactin may directly stimulate angiogenesis, which could be one of the mechanisms by which prolactin contributes to breast cancer progression, thereby providing a potential tool for intervention.
Asunto(s)
Células Endoteliales/patología , Neovascularización Patológica/patología , Prolactina/efectos adversos , Transducción de Señal/efectos de los fármacos , Inductores de la Angiogénesis/efectos adversos , Animales , Neoplasias de la Mama/patología , Línea Celular , Embrión de Pollo , Colágeno/metabolismo , Combinación de Medicamentos , Células Endoteliales/metabolismo , Femenino , Inmunohistoquímica , Laminina/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Ratones , Fosforilación , Proteoglicanos/metabolismo , Receptores de Prolactina/antagonistas & inhibidores , Receptores de Prolactina/metabolismo , Factor de Transcripción STAT5/genética , Factor de Transcripción STAT5/metabolismoRESUMEN
OBJECTIVES: Repeated exposure to stress leads to mast cell degranulation, microscopic inflammation, and subsequent visceral hypersensitivity in animal models. To what extent this pathophysiological pathway has a role in patients with the irritable bowel syndrome (IBS) has not been properly investigated. The objective of this study was to assess the relationship between visceral hypersensitivity, microscopic inflammation, and the stress response in IBS. METHODS: Microscopic inflammation of the colonic mucosa was evaluated by immunohistochemistry in 66 IBS patients and 20 healthy volunteers (HV). Rectal sensitivity was assessed by a barostat study using an intermittent pressure-controlled distension protocol. Salivary cortisol to a psychological stress was measured to assess the stress response. RESULTS: Compared with HV, mast cells, T cells, and macrophages were decreased in IBS patients. Similarly, λ-free light chain (FLC)-positive mast cells were decreased but not immunoglobulin E (IgE)- and IgG-positive mast cells. There were no differences between hypersensitive and normosensitive IBS patients. No relation was found between any of the immune cells studied and the thresholds of discomfort, urge, first sensation, or IBS symptoms (e.g., abdominal pain, stool-related complaints, bloating). Finally, stress-related symptoms and the hypothalamic-pituitary-adrenal-axis response to stress were not correlated with the number of mast cells or the presence of visceral hypersensitivity. CONCLUSIONS: Although the number of mast cells, macrophages, T cells, and λFLC-positive mast cells is decreased in IBS compared with HV, this is not associated with the presence of visceral hypersensitivity or abnormal stress response. Our data question the role of microscopic inflammation as an underlying mechanism of visceral hypersensitivity, but rather suggest dysregulation of the mucosal immune system in IBS.
Asunto(s)
Mucosa Intestinal/inmunología , Síndrome del Colon Irritable/inmunología , Síndrome del Colon Irritable/fisiopatología , Recto/fisiopatología , Adulto , Biopsia con Aguja , Recuento de Células , Colon/inmunología , Colon/patología , Colon/fisiopatología , Colonoscopía , Femenino , Humanos , Hidrocortisona/sangre , Inmunohistoquímica , Mucosa Intestinal/patología , Síndrome del Colon Irritable/patología , Síndrome del Colon Irritable/psicología , Macrófagos/inmunología , Macrófagos/patología , Masculino , Mastocitos/inmunología , Mastocitos/patología , Persona de Mediana Edad , Presión , Umbral Sensorial , Estrés Psicológico/fisiopatología , Linfocitos T/inmunología , Linfocitos T/patología , Adulto JovenRESUMEN
AIMS: Vulnerable atherosclerotic plaques are lesions with a high propensity to develop plaque disruption and superimposed thrombosis. No systematic studies have been carried out on tissue markers for plaque vulnerability throughout the entire coronary artery system at the end stages of coronary atherosclerosis. METHODS AND RESULTS: Nine autopsied patients (mean age 77 years) with angiographically severe trivascular coronary atherosclerosis were selected for this study. All visible lesions in postmortem coronary angiograms (n = 125) were histologically and immunohistochemically screened for the presence of intraplaque haemorrhages (activated) microvessels and inflammatory infiltrates. Intraplaque haemorrhages were observed in 76/125 plaques (61%). Chronic inflammation was found superficially in 59/125 plaques (47%) and deeply inside the plaque tissue in 103/125 plaques (83%). Microvessels were found in 100/125 lesions (80%), of which 58% showed endothelial expression of the vascular activation marker CD105. Moreover, microvascular CD105 positivity correlated positively with plaque haemorrhage and deeply seated plaque inflammation. CONCLUSIONS: Plaque inflammation and haemorrhages can be found at a high frequency throughout the coronary artery system of elderly patients with multivessel coronary atherosclerosis. Microvascular expression of endoglin (CD105), which correlates positively with both of these features of plaque vulnerability, can serve as a marker of the risk of developing coronary thrombotic complications.
Asunto(s)
Antígenos CD/metabolismo , Enfermedad de la Arteria Coronaria/metabolismo , Vasos Coronarios/metabolismo , Microvasos/metabolismo , Placa Aterosclerótica/metabolismo , Receptores de Superficie Celular/metabolismo , Anciano , Anciano de 80 o más Años , Autopsia , Biomarcadores/metabolismo , Cadáver , Enfermedad Crónica , Angiografía Coronaria , Enfermedad de la Arteria Coronaria/complicaciones , Enfermedad de la Arteria Coronaria/patología , Vasos Coronarios/patología , Endoglina , Endotelio Vascular/metabolismo , Endotelio Vascular/patología , Femenino , Hemorragia/complicaciones , Hemorragia/patología , Humanos , Inflamación/complicaciones , Inflamación/metabolismo , Inflamación/patología , Masculino , Microvasos/patología , Persona de Mediana Edad , Placa Aterosclerótica/complicaciones , Placa Aterosclerótica/patologíaRESUMEN
Acute respiratory distress syndrome (ARDS) is a devastating clinical syndrome. Angiotensin-converting enzyme (ACE) and its effector peptide angiotensin (Ang) II have been implicated in the pathogenesis of ARDS. A counter-regulatory enzyme of ACE, ie ACE2 that degrades Ang II to Ang-(1-7), offers a promising novel treatment modality for this syndrome. As the involvement of ACE and ACE2 in ARDS is still unclear, this study investigated the role of these two enzymes in an animal model of ARDS. ARDS was induced in rats by intratracheal administration of LPS followed by mechanical ventilation. During ventilation, animals were treated with saline (placebo), losartan (Ang II receptor antagonist), or with a protease-resistant, cyclic form of Ang-(1-7) [cAng-(1-7)]. In bronchoalveolar lavage fluid (BALF) of ventilated LPS-exposed animals, ACE activity was enhanced, whereas ACE2 activity was reduced. This was matched by enhanced BALF levels of Ang II and reduced levels of Ang-(1-7). Therapeutic intervention with cAng-(1-7) attenuated the inflammatory mediator response, markedly decreased lung injury scores, and improved lung function, as evidenced by increased oxygenation. These data indicate that ARDS develops, in part, due to reduced pulmonary levels of Ang-(1-7) and that repletion of this peptide halts the development of ARDS.
Asunto(s)
Angiotensina I/farmacología , Antagonistas de Receptores de Angiotensina/farmacología , Losartán/farmacología , Fragmentos de Péptidos/farmacología , Peptidil-Dipeptidasa A/metabolismo , Síndrome de Dificultad Respiratoria/enzimología , Enzima Convertidora de Angiotensina 2 , Animales , Líquido del Lavado Bronquioalveolar/química , Modelos Animales de Enfermedad , Intubación Intratraqueal , Lipopolisacáridos/análisis , Pulmón/efectos de los fármacos , Pulmón/patología , Macrófagos Alveolares/efectos de los fármacos , Macrófagos Alveolares/enzimología , Macrófagos Alveolares/patología , Masculino , Peptidil-Dipeptidasa A/análisis , Ratas , Ratas Sprague-Dawley , Síndrome de Dificultad Respiratoria/inducido químicamente , Síndrome de Dificultad Respiratoria/patologíaRESUMEN
BACKGROUND: Areas of microvascular proliferation have been observed in a subpopulation of symptomatic congenital vascular malformations later in life. We investigated whether this angiogenic response is followed by a stage of maturation. METHODS: Resections of vascular malformations (n = 15), infantile hemangiomas (IHs) (n = 8) and pyogenic granulomas (PGs) (n = 5) were studied. Histopathologically, all lesions were screened for presence of foci of immature and/or mature microvessels. These areas were further studied immunohistochemically for differential expression of several angiogenic factors, cell cycle-dependent proteins, p53 and active caspase3. Immunostains were scored semiquantitatively. RESULTS: Immature microvessel areas were present in five vascular malformations (all of the arteriovenous type), five IHs and five PGs; these lesions also contained transitions between immature and mature microvessels. Conglomerates of mature microvessels were found in 19 cases (6 vascular malformations, 5 PGs and 8 IHs). Expression of vascular endothelial growth factor-A, angiopoietin-1, Ki-67, p16 and p21/27 ratios were overall significantly lower in mature areas than in immature areas including those in vascular malformations. P53 and caspase3 expression was scarce in all lesions. CONCLUSIONS: Microvascular areas in vascular malformations appear to follow the same pattern of vascular proliferation and maturation as seen in other microvascular lesions of skin and soft tissue.
Asunto(s)
Angiopoyetina 1/biosíntesis , Malformaciones Arteriovenosas , Proteínas de Ciclo Celular/biosíntesis , Ciclo Celular , Microvasos , Neovascularización Patológica , Piel , Factor A de Crecimiento Endotelial Vascular/biosíntesis , Malformaciones Arteriovenosas/metabolismo , Malformaciones Arteriovenosas/patología , Caspasa 3/metabolismo , Femenino , Granuloma Piogénico/metabolismo , Granuloma Piogénico/patología , Hemangioma/metabolismo , Hemangioma/patología , Humanos , Inmunohistoquímica , Masculino , Microvasos/metabolismo , Microvasos/patología , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Piel/irrigación sanguínea , Piel/metabolismo , Piel/patología , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
BACKGROUND: Cerebral vascular malformations were investigated for the presence of the glucose transporter protein GLUT1, which is normally expressed in endothelial cells of the pre-existing microvasculature of the brain and absent in the vasculature of the choroid plexus and extracranial vasculature without a barrier function. Extracranial arteriovenous malformations (AVM) are known to show an absence of GLUT1 expression which distinguishes them from infantile hemangioma of skin and soft tissue. The expression of GLUT1 in cerebrovascular malformations is not systematically investigated. METHODS: Paraffin-embedded sections of cerebral AVM (4), including one choroid plexus AVM, cerebral cavernous malformations (CCM, 3) and extracranial (facial) AVM (3) were immunostained with anti-CD31 and GLUT1 in doublestaining procedure which was further analyzed with the use of spectral analysis software. RESULTS: All 7 cases of cerebral vascular malformations showed colocalization of GLUT1/CD31 of endothelial cells of the vessels within the malformation. Only in the extracranial AVM expression of GLUT1 was completely absent. CONCLUSION: Cerebral AVM differ from extracranial AVM by their endothelial immunoexpression of GLUT1, indicating that the vessels of these malformations retain the endothelial phenotype of the local vascular beds from which they are derived during embryogenesis.
Asunto(s)
Malformaciones Arteriovenosas/metabolismo , Transportador de Glucosa de Tipo 1/biosíntesis , Malformaciones Arteriovenosas Intracraneales/metabolismo , Adolescente , Adulto , Malformaciones Arteriovenosas/patología , Niño , Femenino , Hemangioma Cavernoso del Sistema Nervioso Central/metabolismo , Hemangioma Cavernoso del Sistema Nervioso Central/patología , Humanos , Inmunohistoquímica , Malformaciones Arteriovenosas Intracraneales/patología , MasculinoRESUMEN
Idiopathic pulmonary fibrosis constitutes the most devastating form of fibrotic lung disorders and remains refractory to current therapies. The coagulation cascade is frequently activated during pulmonary fibrosis, but this observation has so far resisted a mechanistic explanation. Recent data suggest that protease-activated receptor (PAR)-2, a receptor activated by (among others) coagulation factor (F)Xa, plays a key role in fibrotic disease; consequently, we assessed the role of PAR-2 in the development of pulmonary fibrosis in this study. We show that PAR-2 is up-regulated in the lungs of patients with idiopathic pulmonary fibrosis and that bronchoalveolar lavage fluid from these patients displays increased procoagulant activity that triggers fibroblast survival. Using a bleomycin model of pulmonary fibrosis, we show that bleomycin induces PAR-2 expression, as well as both myofibroblast differentiation and collagen synthesis. In PAR-2-/- mice, both the extent and severity of fibrotic lesions are reduced, whereas myofibroblast differentiation is diminished and collagen expression is decreased. Moreover, fibrin deposition in the lungs of fibrotic PAR-2-/- mice is reduced compared with wild-type mice due to differential tissue factor expression in response to bleomycin. Taken together, these results suggest an important role for PAR-2 in the development of pulmonary fibrosis, and the inhibition of the PAR-2-coagulation axis may provide a novel therapeutic approach to treat this devastating disease.
Asunto(s)
Diferenciación Celular/genética , Lesión Pulmonar/genética , Miofibroblastos/fisiología , Fibrosis Pulmonar/genética , Receptor PAR-2/fisiología , Tromboplastina/genética , Adulto , Anciano , Animales , Bleomicina , Células Cultivadas , Humanos , Lesión Pulmonar/inducido químicamente , Lesión Pulmonar/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Persona de Mediana Edad , Miofibroblastos/metabolismo , Fibrosis Pulmonar/patología , Receptor PAR-2/genética , Receptor PAR-2/metabolismo , Tromboplastina/metabolismo , Regulación hacia Arriba/genética , Regulación hacia Arriba/fisiologíaRESUMEN
Angiotensin-converting enzyme (ACE) mediates the ventilator-induced inflammatory response in healthy lungs via angiotensin II (Ang II). A rat model was used to examine the role of ACE and Ang II in the inflammatory response during mechanical ventilation of preinjured (ie, lipopolysaccharide [LPS]-exposed) lungs. When indicated, rats were pretreated with the ACE inhibitor captopril and/or intratracheal administration of LPS. The animals were ventilated for 4 hours with moderate pressure amplitudes. Nonventilated animals served as controls. ACE activity and levels of Ang II and inflammatory mediators (interleukin-6, Cytokine-induced Neutrophil Chemoattractant (CINC)-3, interleukin-1beta, and interleukin-10) were determined in bronchoalveolar lavage fluid (BALF). The localization of ACE and Ang II type 1 receptor in lung tissue was determined by immunohistochemistry. The role of the Ang II pathway was assessed by using its receptor antagonist Losartan. Mechanical ventilation of LPS-exposed animals increased ACE activity and levels of inflammatory mediators in BALF compared with ventilated nonexposed and LPS-exposed nonventilated animals. Blocking ACE by captopril attenuated the lung inflammatory response. Furthermore, increased ACE activity in BALF was accompanied by increased levels of Ang II and enhanced expression of its receptor on alveolar cells. Blocking the Ang II receptor attenuated the inflammatory mediator response to a larger extent than by blocking ACE. In conclusion, during mechanical ventilation ACE, via Ang II, mediates the inflammatory response of both healthy and preinjured lungs.
Asunto(s)
Quimiocina CXCL2/metabolismo , Lipopolisacáridos/metabolismo , Pulmón/patología , Peptidil-Dipeptidasa A/metabolismo , Respiración Artificial/efectos adversos , Animales , Líquido del Lavado Bronquioalveolar , Captopril/farmacología , Inflamación , Interleucina-10/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Losartán/farmacología , Pulmón/enzimología , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
In addition to the classical TH1 and TH2 cytokines, members of the recently identified IL-17 cytokine family play an important role in regulating cellular and humoral immune responses. At present nothing is known about the role of these cytokines in atherosclerosis. Expression of IL-17A, -E and -F was investigated in atherosclerotic tissue by rtPCR and immunohistochemistry. IL-17E and its receptor were further studied in cultured smooth muscle cells and endothelial cells, using rtPCR and western blot. rtPCR showed that IL-17A, -E and -F were expressed in the majority of plaques under investigation. IL-17A/F was expressed by mast cells in all stages of plaque development. IL-17A/F(+) neutrophils were always observed in complicated plaques, but hardly in intact lesions. IL-17A/F(+) Tcells ('TH17') were never observed. IL-17E was expressed by smooth muscle cells and endothelial cells in both normal and atherosclerotic arteries, and in advanced plaques also extensively by mature B cells. Cultured smooth muscle cells and endothelial cells were found to express both IL-17E and its functional receptor (IL-17RB). The constitutive expression of IL-17E by resident plaque cells, and the additional presence of IL-17E(+) B cells and IL-17A/F(+) neutrophils in advanced and complicated plaques indicates a complex contribution of IL-17 family cytokines in human atherosclerosis, depending on the stage and activity of the disease.
Asunto(s)
Aterosclerosis/inmunología , Interleucina-17/biosíntesis , Anciano , Anciano de 80 o más Años , Western Blotting , Células Cultivadas , Células Endoteliales/inmunología , Endotelio Vascular/inmunología , Expresión Génica/inmunología , Humanos , Mediadores de Inflamación/metabolismo , Interleucina-17/genética , Persona de Mediana Edad , Músculo Liso Vascular/inmunología , Células Plasmáticas/inmunología , ARN Mensajero/genética , Receptores de Interleucina/metabolismo , Receptores de Interleucina-17 , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodosRESUMEN
Interleukin-17 (IL-17) plays an important role in the regulation of cellular and humoral immune responses. Recent studies suggest a role for IL-17 in transplantation. Our study investigated whether quantifying IL-17(+) cells in renal transplant biopsies during acute rejection could have additional prognostic value for better stratifying patients at risk for nonresponsiveness to anti-rejection therapy and future graft dysfunction. Forty-nine renal biopsies with acute rejection were double immunostained and quantitatively analyzed for IL-17 and CD3 (IL-17(+) T-lymphocytes), tryptase (IL-17(+) mast cells) or CD15 (IL-17(+) neutrophils). Total IL-17(+) cell count correlated with total percentage of inflamed biopsy and estimated GFR during rejection. Most IL-17(+) cells were mast cells and neutrophils. We could hardly find any IL-17(+) T-lymphocytes. IL-17(+) mast cells correlated with interstitial fibrosis/tubular atrophy (IF/TA). None of the IL-17(+) cell counts had an additional prognostic value for response to anti-rejection treatment. Multivariate analysis correcting for C4d positivity and time from transplantation to biopsy showed that total IL-17(+) cell count independently predicts graft dysfunction at the last follow-up, which was validated in an independent cohort of 48 renal biopsies with acute rejection. We conclude that intragraft IL-17(+) cell count during acute allograft rejection could have an additional value for predicting late graft dysfunction.
Asunto(s)
Interleucina-17/biosíntesis , Trasplante de Riñón/métodos , Mastocitos/citología , Insuficiencia Renal/terapia , Adolescente , Adulto , Anciano , Biopsia , Femenino , Tasa de Filtración Glomerular , Rechazo de Injerto , Humanos , Inmunohistoquímica/métodos , Masculino , Mastocitos/metabolismo , Persona de Mediana Edad , Análisis Multivariante , Neutrófilos/metabolismo , Resultado del TratamientoRESUMEN
OBJECTIVE: To investigate the extent of cleaved caspase-3 immunostaining in lung epithelial cells in children with acute respiratory distress syndrome. DESIGN: Observational study in sixteen children who died with acute respiratory distress syndrome and diffuse alveolar damage. SETTING: Pediatric intensive care unit. PATIENTS: Sixteen children with fatal acute respiratory distress syndrome and diffuse alveolar damage. INTERVENTION: None. MEASUREMENTS AND MAIN RESULTS: Double immunohistochemistry for cleaved caspase-3 and (pan)cytokeratin in lung tissues obtained at autopsy. Spectral imaging was used for the quantification of immunohistochemistry colocalization of these markers. We found a wide range in the percentage of alveolar epithelial cell surface area with positive cleaved caspase-3 staining in the lungs of children with acute respiratory distress syndrome (from 1% to almost 20%). The degree of caspase-3 immunostaining in epithelial cells positively correlated with age. CONCLUSION: There is a high variability in the extent of classic apoptosis in lung epithelial cells in pediatric acute respiratory distress syndrome, potentially in part dependent on age.
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Caspasa 3/metabolismo , Pulmón/enzimología , Síndrome de Dificultad Respiratoria/enzimología , Mucosa Respiratoria/patología , Adolescente , Apoptosis , Niño , Preescolar , Femenino , Humanos , Inmunohistoquímica , Unidades de Cuidado Intensivo Pediátrico , Pulmón/patología , Masculino , Síndrome de Dificultad Respiratoria/patologíaRESUMEN
Several staining concepts and color combinations exist to perform successful double immunoenzyme staining on human tissue specimens. Most of these concepts are based on differences between both primary antibodies: animal species, mouse Ig isotype or IgG subclasses, conjugates, or concentrations. Traditionally, double immunoenzyme staining has used chromogens selected to provide maximum color contrast when observed with the unaided eye. Unfortunately, visually good color combinations always include at least one diffuse chromogen, because of the paucity of appropriate chromogen colors. This situation is drastically changed with the use of spectral imaging, where multicolor microscopy can be unmixed in individual images based on their spectral characteristics. Spectral unmixing can be performed even up to quadruple immunoenzyme staining. This work contains practical suggestions for immunoenzyme double staining procedures for some frequently encountered primary antibody combinations: rabbit-mouse, goat-mouse, mouse-mouse, and rabbit-rabbit. The suggested protocols are all suitable for a classical red-brown color combination plus blue nuclear counterstain that is composed of peroxidase activity (diaminobenzidine tetrahydrochloride), alkaline phosphatase activity (Liquid Permanent Red), and hematoxylin, respectively. Although the red and brown chromogens do not contrast very well visually, they both show a crisp localization and can be perfectly unmixed by spectral imaging.
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Técnicas para Inmunoenzimas/métodos , Coloración y Etiquetado/métodos , Animales , Anticuerpos , Reacciones Cruzadas , Cabras , Humanos , Inmunohistoquímica/métodos , Riñón/metabolismo , Ratones , Microscopía Fluorescente , Tonsila Palatina/metabolismo , Conejos , TrasplantesRESUMEN
Regulatory T cells (Treg) are a subset of T lymphocytes that play a central role in immunologic tolerance and in the termination of immune responses. The identification of these cells in normal and inflammatory conditions may contribute to a better understanding of underlying pathology. We investigated the expression of FOXP3 and GITR in normal skin and in a panel of different inflammatory dermatoses. Immunohistochemical double stainings in skin tissue sections revealed that FOXP3 and GITR were almost exclusively present on T cells that express both CD4 and CD25. Further, immunohistochemical double staining, as well as fluorescence-activated cell sorter analysis, on peripheral blood T cells showed that most FOXP3(+) cells expressed GITR and vice versa, whereas a minority were single-positive for these markers. The mean frequency of FOXP3(+) T cells in spongiotic dermatitis, psoriasis, and lichen planus was in the same range (25-29%), but the frequency of these cells in leishmaniasis appeared to be lower (approximately 15%), although this was not statistically significant. The mean frequency of GITR(+) T cells was fairly similar in all conditions studied (14-20%). Normal human skin also contained FOXP3(+) and GITR(+) cells in the same frequency range as in diseased skin, but the absolute numbers were, of course, much lower. In conclusion, frequencies of FOXP3(+) and GITR(+) T cells were similar in all inflammatory skin diseases studied and normal skin, despite the well-known differences among the inflammatory conditions under investigation.