RESUMO
The COVID-19 pandemic, promoted by the SARS-CoV-2 respiratory virus, has resulted in widespread global morbidity and mortality. The immune response against this pathogen has shown a thin line between protective effects and pathological reactions resulting from the massive release of cytokines and poor viral clearance. The latter is possibly caused by exhaustion, senescence, or both of TCD8+ cells and reduced activity of natural killer (NK) cells. The imbalance between innate and adaptive responses during the early stages of infection caused by SARS-CoV-2 contributes to the ineffective control of viral spread. The present study evaluated the tissue immunoexpression of the tissue biomarkers (Arginase-1, CCR4, CD3, CD4, CD8, CD20, CD57, CD68, CD138, IL-4, INF-α, INF-γ, iNOS, PD-1, Perforin and Sphingosine-1) to understand the cellular immune response triggered in patients who died of COVID-19. We evaluated twenty-four paraffin-embedded lung tissue samples from patients who died of COVID-19 (COVID-19 group) and compared them with ten lung tissue samples from patients who died of H1N1pdm09 (H1N1 group) with the immunohistochemical markers mentioned above. In addition, polymorphisms in the Perforin gene were genotyped through Real-Time PCR. Significantly increased tissue immunoexpression of Arginase, CD4, CD68, CD138, Perforin, Sphingosine-1, and IL-4 markers were observed in the COVID-19 group. A significantly lower immunoexpression of CD8 and CD57 was also found in this group. It is suggested that patients who died from COVID-19 had a poor cellular response concerning viral clearance and adaptive response going through tissue repair.
Assuntos
COVID-19 , Vírus da Influenza A Subtipo H1N1 , Humanos , Arginase , Perforina , Esfingosina , Interleucina-4 , Pandemias , SARS-CoV-2 , Imunidade CelularRESUMO
COVID-19 is a viral disease associated with an intense inflammatory response. Macrophage Activation Syndrome (MAS), the complication present in secondary hemophagocytic lymphohistiocytosis (sHLH), shares many clinical aspects observed in COVID-19 patients, and investigating the cytolytic function of the responsible cells for the first line of the immune response is important. Formalin-fixed paraffin-embedded lung tissue samples obtained by post mortem necropsy were accessed for three groups (COVID-19, H1N1, and CONTROL). Polymorphisms in MAS cytolytic pathway (PRF1; STX11; STXBP2; UNC13D and GZMB) were selected and genotyping by TaqMan® assays (Thermo Fisher Scientific, MA, USA) using Real-Time PCR (Applied Biosystems, MA USA). Moreover, immunohistochemistry staining was performed with a monoclonal antibody against perforin, CD8+ and CD57+ proteins. Histopathological analysis showed high perforin tissue expression in the COVID-19 group; CD8+ was high in the H1N1 group and CD57+ in the CONTROL group. An association could be observed in two genes related to the cytolytic pathway (PRF1 rs885822 G/A and STXBP2 rs2303115 G/A). Furthermore, PRF1 rs350947132 was associated with increased immune tissue expression for perforin in the COVID-19 group. The genotype approach could help identify patients that are more susceptible, and for this reason, our results showed that perforin and SNPs in the PRF1 gene can be involved in this critical pathway in the context of COVID-19.
Assuntos
COVID-19 , Vírus da Influenza A Subtipo H1N1 , Síndrome de Ativação Macrofágica , Biópsia , COVID-19/genética , Humanos , Vírus da Influenza A Subtipo H1N1/metabolismo , Proteínas de Membrana/genética , Perforina/genética , Perforina/metabolismo , Polimorfismo de Nucleotídeo ÚnicoRESUMO
Mast cells (MCs) have relevant participation in inflammatory and vascular hyperpermeability events, responsible for the action of the kallikrein-kinin system (KKS), that affect patients inflicted by the severe form of COVID-19. Given a higher number of activated MCs present in COVID-19 patients and their association with vascular hyperpermeability events, we investigated the factors that lead to the activation and degranulation of these cells and their harmful effects on the alveolar septum environment provided by the action of its mediators. Therefore, the pyroptotic processes throughout caspase-1 (CASP-1) and alarmin interleukin-33 (IL-33) secretion were investigated, along with the immunoexpression of angiotensin-converting enzyme 2 (ACE2), bradykinin receptor B1 (B1R) and bradykinin receptor B2 (B2R) on post-mortem lung samples from 24 patients affected by COVID-19. The results were compared to 10 patients affected by H1N1pdm09 and 11 control patients. As a result of the inflammatory processes induced by SARS-CoV-2, the activation by immunoglobulin E (IgE) and degranulation of tryptase, as well as Toluidine Blue metachromatic (TB)-stained MCs of the interstitial and perivascular regions of the same groups were also counted. An increased immunoexpression of the tissue biomarkers CASP-1, IL-33, ACE2, B1R and B2R was observed in the alveolar septum of the COVID-19 patients, associated with a higher density of IgE+ MCs, tryptase+ MCs and TB-stained MCs, in addition to the presence of intra-alveolar edema. These findings suggest the direct correlation of MCs with vascular hyperpermeability, edema and diffuse alveolar damage (DAD) events that affect patients with a severe form of this disease. The role of KKS activation in events involving the exacerbated increase in vascular permeability and its direct link with the conditions that precede intra-alveolar edema, and the consequent DAD, is evidenced. Therapy with drugs that inhibit the activation/degranulation of MCs can prevent the worsening of the prognosis and provide a better outcome for the patient.
Assuntos
Enzima de Conversão de Angiotensina 2/metabolismo , COVID-19/patologia , Permeabilidade Capilar , Sistema Calicreína-Cinina/fisiologia , Pulmão/patologia , Mastócitos/imunologia , SARS-CoV-2/imunologia , Adulto , Idoso , Autopsia , COVID-19/imunologia , COVID-19/virologia , Caspase 1/metabolismo , Feminino , Humanos , Interleucina-33/metabolismo , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/virologia , Masculino , Mastócitos/metabolismo , Mastócitos/virologia , Pessoa de Meia-Idade , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/patogenicidadeRESUMO
The COVID-19 fatality rate is high when compared to the H1N1pdm09 (pandemic Influenza A virus H1N1 subtype) rate, and although both cause an aggravated inflammatory response, the differences in the mechanisms of both pandemic pneumonias need clarification. Thus, our goal was to analyze tissue expression of interleukins 4, 13, (IL-4, IL-13), transforming growth factor-beta (TGF-ß), and the number of M2 macrophages (Sphingosine-1) in patients who died by COVID-19, comparing with cases of severe pneumopathy caused by H1N1pdm09, and a control group without lung injury. Six lung biopsy samples of patients who died of SARS-CoV-2 (COVID-19 group) were used and compared with ten lung samples of adults who died from a severe infection of H1N1pdm09 (H1N1 group) and eleven samples of patients who died from different causes without lung injury (CONTROL group). The expression of IL-4, IL-13, TGF-ß, and M2 macrophages score (Sphingosine-1) were identified through immunohistochemistry (IHC). Significantly higher IL-4 tissue expression and Sphingosine-1 in M2 macrophages were observed in the COVID-19 group compared to both the H1N1 and the CONTROL groups. A different mechanism of diffuse alveolar damage (DAD) in SARS-CoV-2 compared to H1N1pdm09 infections were observed. IL-4 expression and lung remodeling are phenomena observed in both SARS-CoV-2 and H1N1pdm09. However, SARS-CoV-2 seems to promote lung damage through different mechanisms, such as the scarce participation Th1/Th17 response and the higher participation of the Th2. Understanding and managing the aggravated and ineffective immune response elicited by SARS-CoV-2 merits further clarification to improve treatments propose.
Assuntos
Infecções por Coronavirus/metabolismo , Interleucina-13/metabolismo , Interleucina-4/metabolismo , Pulmão/metabolismo , Pneumonia Viral/metabolismo , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/metabolismo , COVID-19 , Infecções por Coronavirus/patologia , Feminino , Humanos , Interleucina-13/genética , Interleucina-4/genética , Pulmão/patologia , Macrófagos/metabolismo , Masculino , Pessoa de Meia-Idade , Pandemias , Pneumonia Viral/patologia , Esfingosina/metabolismo , Fator de Crescimento Transformador beta/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
SUMMARY: The aim of this study was to evaluate the effects of stretching and therapeutic ultrasound (TUS) on desmin and laminin contents of rat muscle after contusion. Male Wistar rats (n = 35, 8-9 weeks of age, 271 ± 14g body weight) were divided into five groups: Control group (CG) (n= 03); Injured group (IG) (n= 8); Injured + ultrasound group (IUSG) (n= 8); Injured+stretching group (ISG) (n= 8); Injured +ultrasound + stretching group (IUSSG) (n= 8). The application of ultrasound started 72 hours after the contusion, using the 50 % pulsed mode, 0.5 W/cm2, 5 min, once a day, for five consecutive days. Passive manual stretching was started on the tenth day after injury, with four repetitions of 30 s each and 30 s rest between repetitions, once a day, five times per week, for a total of ten applications. After 22 days, the rats were euthanazied and the gastrocnemius of both limbs removed for desmin and laminin immunohistochemistry morphometric measurement. Analysis was conducted using ANOVA one way post-hoc Tukey to parametric data and Kruskall-Wallis for non-parametric data. The IUSSG animals showed a larger area of desmin than ISG (p<0.05). It was found a decrease in laminin comparing IUSG to IG. However, laminin area was higher in ISG than all groups (p<0.05). UST isolated or in combination with stretching influenced gastrocnemius regeneration in different manners. While stretching applied isolated enhanced gastrocnemius regeneration noticed by the increase in laminin area, in combination with TUS strengthened the muscle healing rising desmin area.
RESUMEN: El objetivo de este estudio fue evaluar los efectos del estiramiento y la ecografía en los contenidos de desmina y laminina del músculo de rata después de la lesión. Ratas Wistar macho (n = 35, 8-9 semanas de edad, 271 ± 14 g de peso corporal) se dividieron en cinco grupos: grupo de control (CG) (n = 03); Grupo lesionado (GL) (n = 8); Lesionado + grupo de ultrasonido (LGU) (n= 8); Lesionado + grupo de estiramiento (LGE) (n = 8); Lesionado + ultrasonido + grupo de estiramiento (LUGE) (n = 8). La aplicación de ultrasonido comenzó 72 horas después de la lesión, usando el modo pulsado al 50 %, 0,5W / cm2, 5 min, una vez al día, durante cinco días consecutivos. El estiramiento manual pasivo se inició el décimo día después de la lesión, con cuatro repeticiones de 30 seg cada una y 30 seg de descanso entre repeticiones, una vez al día, cinco veces por semana, para un total de diez aplicaciones. Las ratas fueron sacrificadas después de 22 días, y se extrajo el músculo gastrocnemio de ambos miembros para la medición morfométrica de desmina y laminina a través de inmunohistoquímica. El análisis se realizó utilizando ANOVA unidireccional Tukey post-hoc para datos paramétricos y Kruskall-Wallis para datos no paramétricos. Los animales LUGE mostraron un área mayor de desmina que LGE (p <0,05). Se encontró una disminución en la laminina comparando LGU con GL. Sin embargo, el área de laminina fue mayor en LGE que en todos los grupos (p <0,05). El tratamiento con ultrasonido aislado o en combinación con estiramiento influyó en la regeneración del músculo gastrocnemio de diferentes maneras. Si bien el estiramiento aplicado, en combinación con tratamiento de ultrasonido, fortaleció el área de desmina, la regeneración del músculo gastrocnemio mejoró por el aumento en el área de laminina aumentando la curación muscular.
Assuntos
Animais , Masculino , Ratos , Terapia por Ultrassom/métodos , Músculo Esquelético/patologia , Contusões/terapia , Exercícios de Alongamento Muscular/métodos , Imuno-Histoquímica , Análise de Variância , Laminina/análise , Ratos Wistar , Músculo Esquelético/lesões , Desmina/análiseRESUMO
BACKGROUND: Cardiovascular disease in chronic kidney disease (CKD) has peculiar characteristics. The aim of this study was to analyze atherosclerosis, vascular calcification and nitration in arteries from CKD patients. METHODS: External iliac and renal artery segments from 27 stage 5 CKD patients and 25 donor controls, respectively, were collected during the transplantation procedure. RESULTS: CKD patients presented a significantly higher degree of lesion. In a large proportion (72%) of CKD patients, we observed vascular calcifications. Immunohistochemistry for nitrotyrosine revealed a significant increase in nitrotyrosine production in arteries from CKD patients compared with control donors. In addition, within CKD patients, nitrotyrosine staining was significantly stronger in arteries with media calcification when compared with arteries without media calcification. CONCLUSION: The arteriopathy in the CKD patients appears in an early age and seems to be distinct from the arteriopathy of the general population, especially due to intense calcification and vascular oxidative stress.
Assuntos
Falência Renal Crônica/metabolismo , Falência Renal Crônica/patologia , Proteínas/metabolismo , Calcificação Vascular/patologia , Adulto , Aterosclerose/patologia , Estudos de Casos e Controles , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Artéria Renal/metabolismo , Artéria Renal/patologia , Fatores de Risco , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMO
Objetivos: Descrever e validar uma técnica alternativa econômica e eficiente para a confecção de amostras teciduais com arranjo matricial (tissue microarrays, TMA). Materiais e métodos: Utilizou-se um motor, um micromotor, um contra-ângulo redutor 16:1 e brocas trefina de aço inoxidável para osso. Análise histomorfométrica do volume das células acinares de glândulas parótidas foi realizada. Para testar marcadores imunoistoquímicos para células mioepiteliais, acinares e ductais das parótidas foram utilizados calponina e PCNA. Resultados: Na análise macroscópica e microscópica das lâminas, não foi encontrada perda total do tissue e nem mesmo deslocamento (parcial e/ou total) deste, sendo as perdas teciduais observadas apenas parciais. Das 90 lâminas analisadas, 59 (65%) obtiveram de 50% a 100% do tissue com ausência de artefato, deslocamento ou perda de tecido. Conclusão: O equipamento proposto pelos autores para a confecção deamostras teciduais com arranjo matricial representa uma alternativa econômica e eficiente.
Objectives: To describe and validate an inexpensive and efficient alternative for the production of tissue microarrays (TMA). Materials and methods: An electric-motor, a hand-piece, a reducing contra-angle hand-piece 16:1 and trephine stainless steel drills for bones were used in this study. A histomorphometric assessment of the volume of the acinar cells of parotid glands was performed. Calponin and PCNA were used to test the immunohistochemical markers for myoepithelial, acinar and ductal cells of parotid glands. Results: During the macroscopic and microscopic analysis, total loss of sections was not observed in any slide as well as artifactual ungluing (total and/or partial) of the sections. The loss of sections was partial. Fifty nine (65%) out of 90 slides showed 50%-100% of the tissue without technical artifact, artifactual ungluing or loss of the section. Conclusion: The equipment proposed by the authors for the production of arrays represents an inexpensive and efficient alternative.
Assuntos
Humanos , Análise Serial de Tecidos/instrumentação , Glândula Parótida/patologia , Análise Serial de Tecidos/métodos , Desenho de Equipamento , Imuno-Histoquímica , Reprodutibilidade dos TestesRESUMO
OBJECTIVE: To study the effect of bone marrow derived-mononuclear stem cells transplantation in the growth, VEGF-R and TNF-alpha expression of surgically induced endometriosis in an experimental model. STUDY DESIGN: This is an experimental study conducted in the Center for Health and Biological Sciences at the Pontifical Catholic University of Parana, Brazil. Endometriotic implants were surgically induced in 120 female Wistar rats. The animals with viable endometrial implant (larger than 25 mm(2)) were randomically divided into 3 groups to receive an intraperitoneal injection of 0.2 cc of saline solution (C group; n=30), a subcutaneous injection of 1mg/kg of leuprolide (L group; n=34), or an intraperitoneal injection of 5×10(6) bone marrow derived-mononuclear stem cells (SC group; n=36). They were sacrificed after 21 days to assess the implants' size and the tissue expression of vascular endothelial growth factor receptor (VEGF-R) and tumor necrosis factor-alpha (TNF-alpha). RESULTS: Treatment with leuprolide decreased the surface area of the endometriotic implant compared to the SC group and the C group. The absolute reduction in the surface area of the implant was 16.5mm, 0mm, and 0mm (p=0.007), respectively, and the percent reduction was 40.2%, 0%, and 0% (p=0.001). VEGF-R expression in the endometriotic implant decreased after treatment in the L and SC groups compared to the C group (409.6 µm(2) vs. 465 µm(2) vs. 920.9 µm(2), respectively; p=0.021). TNF-alpha expression also reduced in the L and SC groups compared to the C group (585.7 µm(2) vs. 549.3 µm(2) vs. 2402.1 µm(2), respectively; p<0.001). CONCLUSION: Bone marrow derived-mononuclear stem cells transplantation decreased the expression of VEGF-R and TNF-alpha in the endometriotic implant but did not reduce the surface area of the lesion.
Assuntos
Endometriose/metabolismo , Endometriose/terapia , Receptores de Fatores de Crescimento do Endotélio Vascular/biossíntese , Transplante de Células-Tronco , Fator de Necrose Tumoral alfa/biossíntese , Animais , Medula Óssea , Modelos Animais de Doenças , Endometriose/patologia , Feminino , Fármacos para a Fertilidade Feminina/administração & dosagem , Injeções Intraperitoneais , Leuprolida/administração & dosagem , Ratos , Ratos WistarRESUMO
Objetivos: Comparar a expressão imuno-histoquímica da E-caderina e da Beta-catenina de lesões escamosaspré-neoplásicas e neoplásicas de mucosa oral de amostras emblocadas em parafina. Materiais e métodos:Foram selecionadas 15 amostras de mucosa oral de pacientes apresentando hiperplasia com ou sem displasialeve (grupo 1); 5 amostras apresentando displasia moderada, acentuada ou carcinoma in situ (grupo 2); e12 amostras apresentando carcinoma de células escamosas invasor (grupo 3). Essas amostras foram submetidasà técnica de imuno-histoquímica com anticorpos primários monoclonais anti-E-caderina e anti-Betacatenina.A leitura em microscopia óptica compreendeu a expressão tecidual desses marcadores no epitélioescamoso das amostras de mucosa oral lesadas ou não. A expressão imuno-histoquímica dessas moléculasde adesão foi classificada, segundo a sua intensidade de marcação tecidual, em negativa, positiva fraca epositiva forte. Resultados: A expressão de E-caderina foi forte em 93,3% dos casos do grupo 1 (hiperplasia/displasia leve), e 100% dos casos demonstraram forte expressão para a Beta-catenina nesse mesmo grupo. Contudo, no grupo 3 (carcinoma de célula escamosa), somente 42% dos casos foram fortemente positivospara E-caderina e 25% deles para Beta-catenina. Conclusões: A E-caderina e a Beta-catenina diminuíram asua expressão segundo a progressão tumoral do carcinoma de mucosa oral, reforçando um dos mecanismosrelacionados com a sua carcinogênese.
Objectives: To compare the immunohistochemical expression of E-cadherin and Beta-catenin in squamous pre-malignant and malignant lesions of formalin fixed paraffin embedded buccal mucosa samples. Materials e methods: Selected 15 samples of buccal mucosa of patients with hyperplasia with or without mild dysplasia (group 1), 5 samples showing moderate dysplasia, severe or carcinoma in situ (group 2) and 12 samples presenting invasive squamous cell carcinoma (group 3). These samples were subjected to immunohistochemistry with anti-E-cadherin and anti-Beta-catenin monoclonal antibodies. The expression of these markers in tissue samples injured or not were analyzed in accordance of positivity that was observed in epithelium stratum. The immunohistochemical expression of these adhesion molecules was classified according to their intensity in negative, weak positive and strong positive. Results: The expression of E-cadherin was strong at 93.3% of patients in group 1, and 100% of the cases showed strong expression of Beta-catenin in the same group. However, in group 3, only 42% of cases were strongly positive for E-cadherin and 25% of them to Beta-catenin. Conclusions: The E-cadherin and Beta-catenin decreased their expression according to tumor progression, from hiperplasia/mild dysplasia lesion to buccal invasive carcinoma and this fact may be related of the carcinogenesis mechanisms.
Assuntos
Humanos , Masculino , Feminino , Adulto , Pessoa de Meia-Idade , Idoso de 80 Anos ou mais , Caderinas/química , Carcinoma de Células Escamosas/patologia , Mucosa Bucal/patologia , Neoplasias Bucais/patologia , beta Catenina/química , Fatores Etários , Distribuição de Qui-Quadrado , Estudos Prospectivos , Fatores SexuaisRESUMO
INTRODUCTION AND OBJECTIVE: The aim of the present study is the immunohistochemical assessment of tumor progression markers (E-cadherin, β-catenin, CEACAM-1 and PTEN) in primary cutaneous melanomas and their correlation with prognostic factors. METHOD: Primary lesions in patients with cutaneous melanoma were recorded as to clinical data (age, gender, location and metastases) and anatomopathologic data (Breslow, Clark level, margins, histological type, mitosis, ulceration, regression, satellitosis and TIL type). It was made a comparison between immunohistochemical expression of the markers and prognostic and anatomopathologic factors. RESULTS: Breslow thickness was > 1 mm (thick) in 21 patients (48.83 percent) and < 1 mm (thin) in 22 (51.16 percent). There was a higher CEACAM-1 positive expression in thick melanomas than in thin ones (p = 0.002). There was a more frequent E-cadherin (p = 0.008), b-catenin (p = 0.001) and PTEN (p = 0.005) positive expression in thin melanomas than in thick ones. There was a more frequent CEACAM-1 positive expression in superficial (p = 0.003) and deep (p = 0.002) samples of thick melanomas than in thin ones. No statistically significant differences between clinical and histopathological data were found when comparing patients with (n = 6) and without metastasis (n = 15). DISCUSSION AND CONCLUSION: There was a higher positivity for E-cadherin, b-catenin and PTEN in thin melanomas, whereas there was a higher positivity for CEACAM-1 in thick melanomas.
INTRODUÇÃO E OBJETIVO: O objetivo do presente estudo está na avaliação imuno-histoquímica de marcadores de progressão tumoral (E-caderina, β-catenina, CEACAM-1 e PTEN) em melanomas cutâneos primários e sua correlação com fatores prognósticos. MÉTODO: Lesões primárias de pacientes portadores de melanoma cutâneo foram tabuladas quanto a dados clínicos (idade, sexo, localização e metástases) e anatomopatológicos (Breslow, nível de Clark, margens, tipo histológico, mitoses, ulceração, regressão, satelitose e tipo de TIL). Foi realizada comparação da expressão imuno-histoquímica dos marcadores em estudo com fatores prognósticos clínicos e anatomopatológicos. RESULTADOS: Breslow foi > 1 mm (espesso) em 21 pacientes (48,83 por cento) e ≤ 1 mm (fino) em 22 (51,16 por cento). CEACAM-1 foi mais positivo em melanomas grossos que em finos (p = 0,002). E-caderina (p = 0,008), β-catenina (p = 0,001) e PTEN (p = 0,005) foram mais frequentemente positivos em melanomas finos que em grossos. CEACAM-1 foi mais frequentemente positivo nas porções superficiais (p = 0,003) e profundas (p = 0,002) dos melanomas grossos que nas dos finos. Não houve diferenças estatisticamente significativas entre dados clínicos e histopatológicos quando comparamos os pacientes com (n = 6) e sem (n = 15) metástases. DISCUSSÃO E CONCLUSÃO: Observou-se maior tendência de positividade para E-caderina, b-catenina e PTEN em melanomas finos. Por sua vez, CEACAM-1 demonstrou maior frequência de positividade nos melanomas grossos.