T cell-mediated Immune response and correlates of inflammation and their relationship with COVID-19 clinical severity: not an intuitive guess.

BACKGROUND: Predictors of the outcome of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection remain to be fully determined. We evaluated selected viral characteristics and immunological responses that might predict and/or correlate to the clinical outcome of COVID-19. METHODS: For individuals developing divergent clinical outcomes, the magnitude and breadth of T cell-mediated responses were measured within 36 h of symptom onset. Peripheral Blood Mononuclear Cells (PBMCs) were subjected to in vitro stimulation with SARS-CoV-2-based peptides. In addition, SARS-CoV-2 sequences were generated by metagenome, and HLA typing was performed using Luminex technology. FINDINGS: CD4+ T cell activation was negatively correlated with SARS-CoV-2 basal viral load in patients with severe COVID-19 (p = 0·043). The overall cellular immune response, as inferred by the IFN-γ signal, was higher at baseline for patients who progressed to mild disease compared to patients who progressed to severe disease (p = 0·0044). Subjects with milder disease developed higher T cell responses for MHC class I and II-restricted peptides (p = 0·033). INTERPRETATION: Mounting specific cellular immune responses in the first days after symptom onset, as inferred by IFN-γ magnitude in the ELISPOT assay, may efficiently favor a positive outcome. In contrast, progression to severe COVID-19 was accompanied by stronger cellular immune responses, higher CD4 + T cell activation, and a higher number of in silico predicted high-affinity class I HLA alleles.

Alpha-galactosylceramide as adjuvant induces protective cell-mediated immunity against Leishmania mexicana infection in vaccinated BALB/c mice.

Adjuvants represent a promising strategy to improve vaccine effectiveness against infectious diseases such as leishmaniasis. Vaccination with the invariant natural killer T cell ligand α-galactosylceramide (αGalCer) has been used successfully as adjuvant, generating a Th1-biased immunomodulation. This glycolipid enhances experimental vaccination platforms against intracellular parasites including Plasmodium yoelii and Mycobacterium tuberculosis. In the present study, we assessed the protective immunity induced by a single-dose intraperitoneal injection of αGalCer (2 µg) co-administrated with a lysate antigen of amastigotes (100 µg) against Leishmania mexicana infection in BALB/c mice. The prophylactic vaccination led to 5.0-fold reduction of parasite load at the infection site, compared to non-vaccinated mice. A predominant pro-inflammatory response was observed in challenged vaccinated mice, represented by a 1.9 and 2.8-fold-increase of IL-1ß and IFN-γ producing cells, respectively, in the lesions, and by 23.7-fold-increase of IFN-γ production in supernatants of restimulated splenocytes, all compared to control groups. The co-administration of αGalCer also stimulated the maturation of splenic dendritic cells and modulated a Th1-skewed immune response, with high amounts of IFN-γ production in serum. Furthermore, peritoneal cells of αGalCer-immunized mice exhibited an elevated expression of Ly6G and MHCII. These findings indicate that αGalCer improves protection against cutaneous leishmaniasis, supporting evidence for its potential use as adjuvant in Leishmania-vaccines.

Durable cellular immune response against inactivated ZIKV and envelope proteins in ZIKV-infected women during pregnancy

Introduction: Zika virus (ZIKV) infection has been associated to Guillain-Barré syndrome in adults and congenital malformations during pregnancy, leading to the manifestation of congenital Zika syndrome (CZS). The ZIKV envelope protein (EZIKV), prominently displayed on the virus surface, is a primary target for the humoral immune response. However, limited information exists regarding its capacity to induce cellular immunity, particularly in pregnant women with a history of ZIKV infection. The EZIKV protein comprises three domains: the central domain (EDI), a dimerization domain (EDII), and a domain responsible for binding to the cell surface receptor (EDIII). To examine the regions of EZIKV targeted by cellular immunity, we examined cellular immune responses in a cohort of mothers infected with ZIKV, whose infants exhibited microcephaly. Methods: To assess the ZIKV-specific response, we used inactivated virus and different recombinant viral envelope proteins (EZIKV, EDI/IIZIKV and EDIIIZIKV). All women in the study contracted the infection during pregnancy, with 72% experiencing symptoms such as fever, rash, joint pain, and retro-orbital pain. Peripheral blood mononuclear cells (PMBC) were collected post- ZIKV diagnosis confirmation, with a median time of 18 months (IQR 13.5-19) after parturition. Using the ELISpot assay, we quantified specific interferon-gamma (IFNγ) producing cells by stimulating PBMC with either inactivated ZIKV particles or equimolar amounts of recombinant EZIKV, EDI/IIZIKV and EDIIIZIKV. Results and discussion: Our findings demonstrate the induction of IFN-γ producing cells in PBMC from ZIKV-convalescent mothers, whose infants manifested microcephaly, upon stimulation with both inactivated ZIKV particles and recombinant proteins. The identification of immunodominant regions within ZIKV can contribute for the development of targeted treatments and vaccine candidates tailored for pregnant women.

Association of gestational diabetes mellitus and negative modulation of the specific humoral and cellular immune response against Toxoplasma gondii.

In order to evaluate and compare the specific immune response of pregnant women (PW) chronically infected with Toxoplasma gondii, with and without gestational diabetes mellitus (GDM), and the humoral response of their respective newborns (NB), the study was carried out on 81 PW (34 GDM and 47 controls) from whose medical records the results of the oral glucose tolerance test (OGTT) were obtained, and blood samples were collected at the third trimester of pregnancy; also, on 45 NBs (20 GDM and 25 controls) from whom umbilical cord blood samples were obtained. Humoral immunity was analyzed by measuring anti-T. gondii total IgG, IgG subclasses and IgG avidity. To evaluate cellular immunity, peripheral blood mononuclear cells (PBMC) from 32 PW (16 GDM and 16 controls) were cultured, supernatant cytokines were determined, and flow cytometry was performed to analyze the expression at lymphocytes of surface molecules, cytokines and transcription factors. All PW and NBs were positive for total IgG, and the prevalent subclass was IgG1. There was a negative correlation between the OGTT glycemia of PW and the levels of total IgG, IgG1 and IgG avidity. The IgG avidity of the GDM group was significantly lower than the control group. Patients from the GDM group had a higher number of T lymphocytes expressing markers of cell activation and exhaustion (CD28 and PD-1). In the presence of T. gondii soluble antigen (STAg) the amount of CD4+ T cells producing IFN-γ, IL-10 and IL-17 was significantly lower in the GDM group, while there was no difference between groups in the number of CD4+ CD25HighFOXP3+LAP+ functional Treg cells. Additionally, under STAg stimulus, the secretion of IL-17, IL-4, TNF and IL-2 cytokines at PBMCs culture supernatant was lower in the GDM group. In conclusion, there was a correlation between the increase in blood glucose and the decrease in levels of anti-T. gondii antibodies, associated with the decreased IgG avidity in patients who develop GDM. Also, the GDM group had decreased immune responses in Th1, Th2 and Th17 profiles, suggesting an association between GDM and the negative modulation of the humoral and cellular immune responses against T. gondii.

Comparative efficacy of different Carbopol concentrations as a stabilizer of live attenuated sheep pox virus vaccine against lumpy skin disease

In Egypt, the lyophilized live attenuated sheep pox virus vaccine has been used for the vaccination of cattle against lumpy skin disease virus to control its economic impact on livestock industry. In this endeavor, we validate the efficacy of Carbopol® as a stabilizer and adjuvant to enhance immunogenicity of such a heterologous sheep pox virus vaccine against lumpy skin disease. Lyophilization of sheep pox virus vaccine stabilized with Carbopol® produced better physical and antigenic properties than freeze-drying with lactalbumin/sucrose stabilizer; this was manifested by superior disc uniformity, thermo-stability at 37oC, and less reduction in virus titer. Immunization of calves' groups with variable sheep pox vaccine doses containing different Carbopol® concentrations revealed that 103.5 TCID50 of sheep pox virus vaccine enclosing 0.5 percent Carbopol® is the field dose of choice. Moreover, it induced protective serum neutralizing index of 2.5 and a ELISA S/P ratio of 36, by the 4th week post vaccination. Besides, the inclusion of 0.5 percent Carbopol® in formulation of the sheep pox virus vaccine was safe in bovines and enhanced cellular immune response to lumpy skin disease virus, as evidenced by increased T cell proliferation. Hence, it is recommended to use Carbopol® as 0.5 percent in preparation of live attenuated sheep pox virus vaccine to confer better protection against lumpy skin disease virus infection(AU)

En Egipto, la vacuna atenuada liofilizada contra el virus de la viruela ovina ha sido utilizado para la vacunación del ganado, contra el virus de la dermatosis nodular contagiosa, para controlar su impacto económico en la industria ganadera. En este trabajo, validamos la eficacia del Carbopol®, como estabilizador y adyuvante, para mejorar la inmunogenicidad de dicha vacuna heteróloga contra la dermatosis nodular contagiosa. La liofilización de la vacuna contra el virus de la viruela ovina estabilizada con Carbopol®, resultó en mejores propiedades físicas y antigénicas que la liofilización con el estabilizador de lactoalbúmina/sacarosa; lo anterior se manifestó en la uniformidad superior del disco, la termoestabilidad a 37°C y la menor reducción del título del virus. La inmunización de grupos de terneros con dosis variables de vacuna contra el virus de la viruela ovina, que contenían diferentes concentraciones de Carbopol®, reveló que la dosis de campo de elección fue 103,5 TCID50 de la vacuna contra el virus de la viruela ovina conteniendo 0,5 por ciento de Carbopol®, la que indujo un índice de neutralización sérica protectora de 2,5 y una relación S/P de ELISA de 36 a la cuarta semana después de la vacunación. Además, la inclusión de Carbopol® al 0,5 por ciento en la formulación de la vacuna contra el virus de la viruela ovina fue segura en los bovinos y potenció la respuesta inmunitaria celular contra el virus de la dermatosis nodular contagiosa, como lo demuestra el aumento de la proliferación de células T. Por lo tanto, se recomienda el uso de Carbopol® al 0,5 por ciento en la preparación de la vacuna viva atenuada contra el virus de la viruela ovina para conferir una mejor protección contra la infección por el virus de la dermatosis nodular contagiosa(AU)

Evaluation of systemic immunity in atypical cutaneous leishmaniasis caused by Leishmania (L.) infantum chagasi.

In some central-American countries, Leishmania (L.) infantum chagasi infection can cause non-ulcerated or atypical cutaneous leishmaniasis (NUCL) in addition to the classic clinical form, visceral leishmaniasis (VL). Little is known about the host-parasite relationship that can contribute to the determination of one or another clinical form. The present study had the objective to evaluate the humoral and cellular immunity in the sera of individuals affected by NUCL to improve the comprehension of this atypical host-parasite interaction. Based on clinical and laboratory diagnosis, serum of 80 individuals was collected to evaluate the cytokines and immunoglobulins profile of NUCL (n = 47), VL patients (n = 5), and negative controls (n = 28). Cytokines were detected using Cytokine Bead Array (CBA) Human Th1/Th2/Th17 kit according to the manufacturer's instructions; class (IgG and IgM), and subclass of (IgG1 and IgG2) immunoglobulins was evaluated by ELISA using specific antigens. The concentration of TNF-α, IFN-γ, IL-2 and IL-4 cytokines in NUCL, VL and control was present below the detection threshold of CBA kit. IL-6, IL-10 and IL-17A cytokines was lower in NUCL compared to LV patients. Regarding to immunoglobulins, NUCL patients produced 4.0 times more IgG than the control, while VL patients produced 6.6 times more; and IgM level was 1.6 times higher in NUCL and 2.6 times in VL patients compared to the control. Concerning the immunoglobulins subclass, only VL patients showed positive reaction for IgG1, and IgG2 did not show positive reaction among the groups. The results showed a weak cellular and humoral systemic immune response in NUCL patients.

Comparative Evaluation of Immune Responses and Protection of Chitosan Nanoparticles and Oil-Emulsion Adjuvants in Avian Coronavirus Inactivated Vaccines in Chickens.

Efficient vaccines are the main strategy to control the avian coronavirus (AvCoV), although several drawbacks related to traditional attenuated and inactivated vaccines have been reported. These counterpoints highlight the importance of developing new alternative vaccines against AvCoV, especially those able to induce long-lasting immune responses. This study evaluated and compared two inactivated vaccines formulated with AvCoV BR-I variants, one composed of chitosan nanoparticles (AvCoV-CS) and the second by Montanide oily adjuvant (AvCoV-O). Both developed vaccines were administered in a single dose or associated with the traditional Mass attenuated vaccine. The AvCoV-CS vaccine administered alone or associated with the Mass vaccine was able to induce strong humoral and cell-mediated immune (CMI) responses and complete protection against IBV virulent infection, wherein single administration was characterized by high IgA antibody levels in the mucosa, whereas when associated with the Mass vaccine, the serum IgG antibody was predominantly observed. On the other hand, single administration of the oily vaccine presented poor humoral and CMI responses and consequently incomplete protection against virulent challenge, but when associated with the Mass vaccine, immune responses were developed, and complete protection against infection was observed. Both of our experimental vaccines were able to induce full protection against virulent IBV challenge. A single dose of AvCoV-CS vaccine was sufficient to achieve complete protection, while AvCoV-O required a previous priming by a Mass strain to complete the protection.

Effect of processing methods on the cytotoxicity of methyl methacrylate-based ocular prostheses: An in vitro study.

The study evaluated the influence of cycles and methods of an ocular prosthesis resin on cytotoxicity toward human conjunctival cells. Resins were polymerized by water bath (WB, 74 °C or 100 °C for 30 min to 9 h), microwave (MW, 1200 W, 3 to 14 min and 30 s at 0 to 720 W), or autopolymerization (AP, room temperature for 20 min ± 60 °C for 30 min). Degree of conversion (DC), cytotoxicity, level of inflammatory mediators, gene expression of different markers, and apoptosis were evaluated. Data were submitted to ANOVA and Tukey test (p < 0.05). WB with longer processing time at higher temperature had highest DC (85.6%) and higher TGF ß1-gene expression (1.39); long cycle low power MW showed lowest DC (69.6%), lower cell proliferation (85.4%, MTT), and large IL-2 release (39,297 ng/mL). AP with additional processing time showed lower cell proliferation (75.3%, Alamar Blue), and AP polymerized at room temperature showed higher CASP 9-gene expression (1.21). AP methods showed higher IL-6 release (>277 pg/mL). Short cycle medium power MW had higher IL-23 release (534.2 pg/mL). MW (long and short cycles) and AP polymerizations have triggered a more intense inflammatory response. Among methods recommended by the manufacturer, WB showed high DC and less cytotoxicity.

In situ cellular immune response in non-ulcerated skin lesions due to Leishmania (L.) infantum chagasi infection.

BACKGROUND: Skin lesions of patients affected by non-ulcerated cutaneous leishmaniasis (NUCL) caused by L. (L.) infantum chagasi are characterized by lymphohistiocytic inflammatory infiltrate associated with epithelioid granuloma and scarce parasitism. However, the in situ cellular immune response of these patients is unclear. Therefore, the aim of the present study was to characterize the cellular immune response in the skin lesions of patients affected by NUCL. METHODS: Twenty biopsies were processed by immunohistochemistry using primary antibodies to T lymphocytes (CD4, CD8), NK cells, B lymphocytes, macrophages, nitric oxide synthase and interferon-gamma. RESULTS: Immunohistochemistry revealed higher expression of all cellular types and molecules (IFN-γ, iNOS) in the dermis of diseased skin compared to the skin of healthy individuals (p < 0.05). Morphometric analysis performed in the skin lesions sections showed the predominance of CD8+ T lymphocytes in the mononuclear infiltrate, followed by macrophages, mostly iNOS+, a response that could be mediated by IFN-γ. CONCLUSION: Our study improves knowledge of the cellular immune response in non-ulcerated or atypical cutaneous leishmaniasis caused by L. (L.) infantum chagasi in Central America and pointed to the pivotal participation of CD8+ T lymphocytes in the host defense mechanisms against the parasite in patients with NUCL.

In situ cellular immune response in non-ulcerated skin lesions due to Leishmania (L) infantum chagasi infection

Skin lesions of patients affected by non-ulcerated cutaneous leishmaniasis (NUCL) caused by L. (L.) infantum chagasi are characterized by lymphohistiocytic inflammatory infiltrate associated with epithelioid granuloma and scarce parasitism. However, the in situ cellular immune response of these patients is unclear. Therefore, the aim of the present study was to characterize the cellular immune response in the skin lesions of patients affected by NUCL. Methods Twenty biopsies were processed by immunohistochemistry using primary antibodies to T lymphocytes (CD4, CD8), NK cells, B lymphocytes, macrophages, nitric oxide synthase and interferon-gamma. Results Immunohistochemistry revealed higher expression of all cellular types and molecules (IFN-γ, iNOS) in the dermis of diseased skin compared to the skin of healthy individuals (p < 0.05). Morphometric analysis performed in the skin lesions sections showed the predominance of CD8+ T lymphocytes in the mononuclear infiltrate, followed by macrophages, mostly iNOS+, a response that could be mediated by IFN-γ. Conclusion Our study improves knowledge of the cellular immune response in non-ulcerated or atypical cutaneous leishmaniasis caused by L. (L.) infantum chagasi in Central America and pointed to the pivotal participation of CD8+ T lymphocytes in the host defense mechanisms against the parasite in patients with NUCL.(AU)

In situ cellular immune response in non-ulcerated skin lesions due to Leishmania (L) infantum chagasi infection

Skin lesions of patients affected by non-ulcerated cutaneous leishmaniasis (NUCL) caused by L. (L.) infantum chagasi are characterized by lymphohistiocytic inflammatory infiltrate associated with epithelioid granuloma and scarce parasitism. However, the in situ cellular immune response of these patients is unclear. Therefore, the aim of the present study was to characterize the cellular immune response in the skin lesions of patients affected by NUCL. Methods Twenty biopsies were processed by immunohistochemistry using primary antibodies to T lymphocytes (CD4, CD8), NK cells, B lymphocytes, macrophages, nitric oxide synthase and interferon-gamma. Results Immunohistochemistry revealed higher expression of all cellular types and molecules (IFN-γ, iNOS) in the dermis of diseased skin compared to the skin of healthy individuals (p < 0.05). Morphometric analysis performed in the skin lesions sections showed the predominance of CD8+ T lymphocytes in the mononuclear infiltrate, followed by macrophages, mostly iNOS+, a response that could be mediated by IFN-γ. Conclusion Our study improves knowledge of the cellular immune response in non-ulcerated or atypical cutaneous leishmaniasis caused by L. (L.) infantum chagasi in Central America and pointed to the pivotal participation of CD8+ T lymphocytes in the host defense mechanisms against the parasite in patients with NUCL.(AU)

Th17 lymphocytes in atypical cutaneous leishmaniasis caused by Leishmania (L.) infantum chagasi in Central America.

Skin lesions in nonulcerated cutaneous leishmaniasis (NUCL) caused by Leishmania (L.) infantum chagasi are characterized by a mononuclear inflammatory infiltrate in the dermis, which is composed mainly of lymphocytes, followed by macrophages, few plasma cells and epithelioid granulomas with mild tissue parasitism. Previous studies have shown that the main population of lymphocytes present in the dermal infiltrate is CD8+ T cells, followed by CD4+ T cells, which are correlated with IFN-γ+ cells. To improve the knowledge of cellular immune responses in NUCL, skin biopsies were submitted to immunohistochemistry using anti-ROR-γt, anti-IL-17, anti-IL-6, anti-TGF-ß, and anti-IL-23 antibodies to characterize the involvement of Th17 cells in the skin lesions of patients affected by NUCL. ROR-γt+ , IL-17+ , IL-6+ , TGF-ß+ and IL-23+ cells were observed in the dermal inflammatory infiltrate of NUCL skin lesions. A positive correlation between CD4+ T-lymphocytes and ROR-γt+ and IL-17+ cells suggests that some of the CD4+ T-lymphocytes in NUCL could be Th17 lymphocytes. Moreover, a positive correlation between ROR-γt+ cells and TGF-ß+ , IL-6+ , IL-17+ and IL-23+ cells could indicate the role of these cytokines in the differentiation and maintenance of Th17 lymphocytes. Our findings improve knowledge of the pathogenesis of this rare and atypical clinical form of leishmaniasis.

Mucosal Vaccination with Lactococcus lactis-Secreting Surface Immunological Protein Induces Humoral and Cellular Immune Protection against Group B Streptococcus in a Murine Model.

Group B Streptococcus (GBS) is the primary etiological agent of sepsis and meningitis in newborns and is associated with premature birth and stillbirth. The development of a licensed vaccine is one of the pending challenges for the World Health Organization. Previously, we showed that oral immunization with surface immune protein (SIP) decreases vaginal colonization of GBS and generates functional opsonizing antibodies, which was determined by opsonophagocytic assays (OPA) in vitro. We also showed that the protein has an adjuvant vaccine profile. Therefore, an oral vaccine based on SIP may be an attractive alternative to employ in the development of new vaccines against GBS. Lactococcus lactis is a highlighted oral vaccine probiotic inducer of the mucosal immune response. This bacterium could serve as an antigen-delivering vehicle for the development of an edible vaccine and has been used in clinical trials. In this study, we showed that an oral vaccine with a recombinant L. lactis strain secreting SIP from GBS (rL. lactis-SIP) can induce protective humoral and cellular immunity in an experimental model of GBS vaginal colonization in C57BL/6 mice. Mice immunized with rL. lactis-SIP were protected against clinical symptoms and bacterial colonization after GBS vaginal colonization. Our rL. lactis-SIP vaccine also induces an increase of immunoglobulin G (IgG) and immunoglobulin A (IgA) specifically against SIP. The adoptive transfer of serum from vaccinated mice to naïve mice generated protection against GBS vaginal colonization. Moreover, the rL. lactis-SIP strain induces the activation of SIP-specific T cells, which could decrease GBS vaginal colonization and generate protective antibodies when transferred to other mice. Our experimental observations strongly support the notion that rL. lactis-SIP induces protective humoral and cellular immunity and could be considered as a novel alternative in the development of vaccines for GBS.

IFN-γ Response Is Associated to Time Exposure Among Asymptomatic Immune Responders That Visited American Tegumentary Leishmaniasis Endemic Areas in Peru.

Clinical manifestations of American Tegumentary Leishmaniasis (ATL) include cutaneous (CL) and mucous forms (ML); however, there are asymptomatic individuals who despite being infected do not present any clinical manifestations. This study characterized the cell-mediated immunity of travelers who lived in the Andean highlands of Cusco, free of leishmaniasis transmission, which eventually visited leishmaniasis endemic in the Amazonian basin and returned home without any clinical signs of the disease. Their immune response was compared with CL and ML patients who acquired the disease during their stage in the same region. Fifty-four human subjects from the highlands of Cusco (Peru), who have visited an endemic area, were enrolled: 28 of them did not show any symptoms, 12 showed CL and 14 showed ML. Ten healthy subjects from a non-endemic area (HS) were included as controls. T-cell proliferation was evaluated using peripheral blood mononuclear cells (PBMC) stimulated for 5 days with a total soluble leishmanial antigen (TSLA) of L. (V.) braziliensis. Th1/Th2/Th17 cytokines were also quantified in the supernatants by a flow cytometry multiplex assay. T-cell proliferation was expressed as stimulation index (SI) and the cut off was fixed at SI >2.47. Fifteen out of 28 subjects did not show any signs of disease (54%); subjects with an SI above the cut off. They were defined as asymptomatic immune responders (AIR). CL and ML patients presented a higher SI than HS and AIR. Among the latter group, the exposure time to Leishmania was clearly associated with the IFN-γ response. Increased levels of this cytokine were observed in individuals who remained <90 days in an endemic area of leishmaniasis. Our results evidenced two sub-populations among asymptomatic individuals, one AIR who did not develop clinical disease manifestations when they were exposed to Leishmania in endemic areas. Exposure time to Leishmania in the wild was associated with the IFN-γ response.

Biocompatibilidade da resina acrílica de prótese ocular: análise in vitro e in vivo / Biocompatibility of ocular prosthesis acrylic resin: in vitro and in vivo analysis

O objetivo desse estudo foi avaliar a influência de diferentes ciclos e métodos de polimerização da resina acrílica (RA) branca de próteses oculares sobre a biocompatibilidade de células da conjuntiva humana e resposta inflamatória do tecido subcutâneo de ratos. Para isso, foram confeccionados corpos de prova em RA termopolimerizados em água aquecida (RNAA), por energia de microondas (RNTM) e quimicamente ativados (RNQA). Para a análise in vivo, a resposta inflamatória desses 3 grupos (n=20/grupo) foi avaliada no tecido subcutâneo de 20 ratos Wistar por 7, 15, 30 e 60 dias (d). Células inflamatórias foram contadas no tecido adjacente ao corpo de prova após coloração com hematoxilina e eosina. A análise imunohistoquímica foi realizada para a detecção de IL-1ß, IL-6, TNFα, IL-17 e CCL20. Para a análise in vitro, diferentes ciclos de polimerização para cada método citado foram avaliados, totalizando 11 grupos (n=8/grupo). Foram realizadas análises de grau de conversão (GC), MTT, Alamar Blue, ELISA, RT-PCR em tempo real e dupla marcação de Anexina V e iodeto de propídio. Dados quantitativos foram submetidos à Análise de Variância e ao teste de Tukey com significância de 5%. Os resultados qualitativos foram comparados visualmente. Na análise in vivo, houve infiltrado inflamatório moderado para os grupos RNTM e RNQA e leve para o grupo RNAA após 7 d. O infiltrado inflamatório e a imunomarcação dos alvos testados diminuiu gradativamente ao longo dos 60 d. O grupo RNTM exibiu mais células inflamatórias, com exceção do grupo RNAA, que apresentou mais eosinófilos e linfócitos após 15 d, e do grupo RNQA, onde foi observado mais macrófagos em 15 d e neutrófilos em 60 d. Os grupos RNAA e RNQA apresentaram maior imunomarcação de IL-1ß após 7 d. O grupo RNQA apresentou maior imunomarcação de IL-1ß (15 e 30 d), IL-6 (30 e 60 d), IL-17 (15 e 30 d) e TNF-α (7 d). Os grupos RNAA e RNTM apresentaram maior imunomarcação de TNF-α nos períodos de 15 e 30 d, enquanto o grupo RNTM, aos 60 d. Na análise in vitro, todos os grupos apresentaram proliferação celular maior que 75%. O ciclo longo de polimerização em microondas apresentou menor GC e percentual de proliferação celular no MTT e resultou em grande liberação de IL-2. No ensaio de Alamar Blue, esse grupo apresentou baixo percentual de proliferação celular, assim como o grupo que recebeu ciclo longo de polimerização em água aquecida e grupos submetidos à ativação química. Maior liberação de IL-6 foi observada nos grupos submetidos à ativação química e de IL-23 para o ciclo curto de polimerização em microondas. Maior expressão gênica de TGF ß1 ocorreu para o grupo que recebeu ciclo longo de polimerização em água aquecida seguido de 30 min de armazenamento em água. Maior expressão gênica de CASP9 ocorreu para o grupo ativado quimicamente sobre a bancada. Pode-se concluir que os métodos de polimerização por meio de energia de microondas (ciclos longo e curto) e por ativação química desencadearam uma resposta inflamatória mais intensa. Dentre os métodos de polimerização recomendados pelo fabricante, a polimerização em água aquecida apresentou resultados mais satisfatórios(AU)

The aim of this study was to evaluate the influence of different cycles and methods of white color acrylic resin (AR) for ocular prosthesis on the biocompatibility of human conjunctival cells and on the inflammatory response of rat subcutaneous tissue. For this, AR specimens were prepared in water bath (NRWB), by microwave energy (NRME), and chemically activated (ANR). For in vivo analysis, the inflammatory response of these 3 groups (n=20/group) was assessed in subcutaneous tissue of 20 Wistar rats at 7, 15, 30 and 60 days (d). Inflammatory cells were counted in the tissue adjacent to specimen after staining with hematoxylin and eosin. The immunohistochemical analysis was performed for the detection of IL-1ß, IL-6, TNFα, IL-17, and CCL20. For in vitro analysis, different cycles of polymerization for each method were evaluated, with a total of 11 groups (n=8/group). The degree of conversion (DC), MTT, ELISA, real-time RT-PCR and Annexin V and propidium iodide assays were performed. Quantitative data were submitted to Analysis of Variance and Tukey test with a 5% significance. Qualitative data were submitted to visual comparison. In in vivo analysis, there was a moderate inflammatory infiltrate for groups NRME and ANR, and a light infiltrate for the group NRWB after 7 d. The inflammatory infiltrate and the immunolabeling of tested targets decreased gradually during the 60 d. The group NRME exhibited the highest number of inflammatory cells, except for the group NRWB, which presented a higher number of eosinophils and lymphocytes after 15 d, and for the group ANR, where a higher number of macrophages and neutrophils were observed at 15 d and at 60 d, respectively. Groups NRWB and ANR showed higher IL-1ß immunolabeling after 7 d. The group ANR had the highest immunolabeling of IL-1ß (15 and 30 d), IL-6 (30 and 60 d), IL-17 (15 and 30 d), and TNF-α (7 d). Groups NRWB and NRME showed greater immunolabeling in the periods of 15 and 30 d, while the group NRME had also high results at 60 d. In in vitro analysis, all groups showed cell proliferation higher than 75%. The long cycle of polymerization using microwave energy resulted in lower DC and lower percentage of cell proliferation in the MTT assay and in large release of IL-2. In the Alamar Blue assay, this group had a low percentage of cell proliferation, as well as the group that received a long cycle of polymerization in water bath and groups submitted to chemical activation. A higher release of IL-6 was observed in groups submitted to chemical activation and of IL-23, for the short cycle of polymerization in microwave. Higher TGF ß1 gene expression occurred for the group that received long cycle of polymerization in water bath followed by 30 min of storage in water. Higher CASP 9 gene expression occurred for the chemically activated group on bench. It can be concluded that the polymerization by microwave energy (long and short cycles) and by chemical activation resulted in higher inflammatory response. Among methods recommended by the manufacturer, the water bath polymerization showed more satisfactory results(AU)

Changes in cellular proliferation and plasma products are associated with liver failure.

AIM: To study the differences in immune response and cytokine profile between acute liver failure and self-limited acute hepatitis. METHODS: Forty-six patients with self-limited acute hepatitis (AH), sixteen patients with acute liver failure (ALF), and twenty-two healthy subjects were involved in this study. The inflammatory and anti-inflammatory products in plasma samples were quantified using commercial enzyme-linked immunoassays and quantitative real-time PCR. The cellular immune responses were measured by proliferation assay using flow cytometry. The groups were divided into viral- and non-viral-induced self-limited AH and ALF. Thus, we worked with five groups: Hepatitis A virus (HAV)-induced self-limited acute hepatitis (HAV-AH), HAV-induced ALF (HAV-ALF), non-viral-induced self-limited acute hepatitis (non-viral AH), non-viral-induced acute liver failure (non-viral ALF), and healthy subjects (HC). Comparisons among HAV and non-viral-induced AH and ALF were performed. RESULTS: The levels of mitochondrial DNA (mtDNA) and the cytokines investigated [interleukin (IL)-6, IL-8, IL-10, interferon gamma, and tumor necrosis factor] were significantly increased in ALF patients, independently of etiology (P < 0.05). High plasma mtDNA and IL-10 were the best markers associated with ALF [mtDNA: OR = 320.5 (95%CI: 14.42-7123.33), P < 0.0001; and IL-10: OR = 18.8 (95%CI: 1.38-257.94), P = 0.028] and death [mtDNA: OR = 12.1 (95%CI: 2.57-57.07), P = 0.002; and IL-10: OR = 8.01 (95%CI: 1.26-50.97), P = 0.027]. In the cellular proliferation assay, NKbright, NKT and regulatory T cells (TReg) predominated in virus-specific stimulation in HAV-induced ALF patients with an anergic behavior in the cellular response to mitotic stimulation. Therefore, in non-viral-induced ALF, anergic behavior of activated T cells was not observed after mitotic stimulation, as expected and as described by the literature. CONCLUSION: mtDNA and IL-10 may be predictors of ALF and death. TReg cells are involved in immunological disturbance in HAV-induced ALF.

Immunological findings associated with Argentinean strains of Mycobacterium avium subsp. paratuberculosis in bovine models.

Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of ruminant paratuberculosis. The aim of this study was to evaluate the biological behavior of different Argentinean strains of MAP in two bovine infection models: macrophage (in vitro) and calf (in vivo) through the evaluation of early immune responses at the peripheral and local levels. Two MAP strains (A and C) were selected taking into account the different patterns of TNF-α and IL-10 secretion displayed by infected bovine macrophages in vitro. Two groups of calves were infected with 250mg of total wet weight live MAP: strain A infected group (MA, n=3), strain C infected group (MC, n=2). Another group of animals was mock-infected (MI, n=3). Infection was confirmed by MAP culture of feces and microscopic observation of granulomatous lesions in the gut tissue. All infected calves showed positive results in the DTH skin test. A significant increase in peripheral CD4CD25(+) cells in MC group on day 150 was detected. The specific cellular immune response developed allowed the identification of the infection as early as 30days in the MA group. However, the percentage of CD8CD25(+) cells was significantly increased on day 120 in MC group. Significant differences between groups in proliferation and cellular responses were also detected in ileocecal lymph node samples. In summary, the strains of MAP employed herein induced differential immune responses in peripheral cells, in the proliferative responses and in cell functionality at the local level. Our findings support the hypotheses that the in vitro behavior displayed by macrophages could be a tool to identify differences among MAP strains infecting bovines and that the host-pathogen interactions occurring upon infection are dependent on the strain of MAP involved.

Análise in vitro da proliferação celular e produção de mediadores inflamatórios por células da conjuntiva estimuladas por materiais utilizados na confecção de próteses oculares / In vitro analysis of cell proliferation and the production of inflammatory mediators by conjunctiva cells stimulated by ocular prosthesis materials

A prótese ocular é utilizada para a reabilitação estética e funcional da ausência ocular. O conhecimento da sua biocompatibilidade é importante para a utilização sem reações danosas aos usuários. O objetivo neste estudo foi avaliar a citotoxicidade de materiais utilizados na confecção de próteses oculares, por meio da análise da proliferação celular e da produção de citocinas pró-inflamatórias e de proteínas de matriz extracelular por células da conjuntiva humana. Inicialmente, foi analisada a influência de diferentes períodos de formação e de exposição dos extratos de resina acrílica branca (N1), termopolimerizada em água aquecida, sobre culturas de células da conjuntiva. Foram confeccionados 24 corpos de prova em resina, sendo 12 para cada período de exposição de células da conjuntiva aos extratos da resina testada (24 e 72 horas). Após a formação dos extratos por 24, 48 e 72 horas de imersão em meio de cultura e, 24 horas em água seguido de 24 horas de imersão em meio, os ensaios propostos foram realizados (n=3). Em seguida, foi avaliado o efeito citotóxico de diferentes métodos de polimerização de resina acrílica N1 em células da conjuntiva. Foram confeccionados 9 corpos de prova em resina (n=3), termopolimerizados em água aquecida, por energia de microondas ou ativados quimicamente, utilizados para a formação dos extratos dessas resinas. Os extratos foram obtidos após 72 horas de imersão dos corpos de prova em meio de cultura e, então, expostos às células da conjuntiva por 72 horas para a realização dos ensaios propostos. Adicionalmente, foi analisada a influência da presença do pigmento acrílico na confecção da prótese de resina acrílica N1, termopolimerizada em água aquecida. Foram confeccionados 9 corpos de prova (n=3): resina N1, resina N1 + pigmento e, pigmento, utilizados para a formação dos extratos desses materiais. Os extratos formaram-se por 72 horas de imersão dos corpos de prova em meio de cultura e, então, foram expostos às células da...

Ocular prosthesis is a treatment option for esthetical and functional rehabilitation of ocular absence. The knowledge of ocular prosthesis material’s biocompatibility is important to ensure a safe use in patients. The aim of this study was to evaluate the cytotoxic effect of ocular prosthesis materials, through the analysis of the cell proliferation, and the production of proinflammatory cytokines and extracellular matrix proteins by a human conjunctival cell line. Initially, the influence of different preparation and exposition periods of eluates from heat-polymerized ocular prosthesis N1 color acrylic resin in human conjunctival cell line was evaluated. A total of 24 acrylic resin samples were manufactured and divided into 2 groups, according to the eluate exposition period to conjunctival cell line (24 and 72 hours). Eluates corresponding to 24, 48 and 72 hours of resin sample immersion in medium and, 24 hours of resin sample immersion in water followed by 24 hours of immersion in medium, were prepared (n=3) for the proposed tests. Then, the cytotoxic effect of different polymerization methods of ocular prosthesis N1 color acrylic resin was analyzed. A total of 9 acrylic resin samples were manufactured (n=3), according to the polymerization method: heat-polymerization in water bath, polymerization by microwave energy and auto-polymerization. Eluates corresponding to 72 hours of resin sample immersion in medium were prepared for proposed tests and exposed to conjunctival cell line for 72 hours. Additionally, the influence of pigment incorporation on the cytotoxicity of heat-polymerized ocular prosthesis N1 color acrylic resin was evaluated. A total of 9 samples were manufactured (n=3): N1 color acrylic resin without pigment incorporation, N1 color acrylic resin with pigment incorporation, and acrylic pigment. Eluates corresponding to 72 hours of sample immersion in medium were prepared and exposed to conjunctival cell line for 72 hours. The cytotoxic effect...

Análise in vitro da proliferação celular e produção de mediadores inflamatórios por células da conjuntiva estimuladas por materiais utilizados na confecção de próteses oculares / In vitro analysis of cell proliferation and the production of inflammatory mediators by conjunctiva cells stimulated by ocular prosthesis materials

A prótese ocular é utilizada para a reabilitação estética e funcional da ausência ocular. O conhecimento da sua biocompatibilidade é importante para a utilização sem reações danosas aos usuários. O objetivo neste estudo foi avaliar a citotoxicidade de materiais utilizados na confecção de próteses oculares, por meio da análise da proliferação celular e da produção de citocinas pró-inflamatórias e de proteínas de matriz extracelular por células da conjuntiva humana. Inicialmente, foi analisada a influência de diferentes períodos de formação e de exposição dos extratos de resina acrílica branca (N1), termopolimerizada em água aquecida, sobre culturas de células da conjuntiva. Foram confeccionados 24 corpos de prova em resina, sendo 12 para cada período de exposição de células da conjuntiva aos extratos da resina testada (24 e 72 horas). Após a formação dos extratos por 24, 48 e 72 horas de imersão em meio de cultura e, 24 horas em água seguido de 24 horas de imersão em meio, os ensaios propostos foram realizados (n=3). Em seguida, foi avaliado o efeito citotóxico de diferentes métodos de polimerização de resina acrílica N1 em células da conjuntiva. Foram confeccionados 9 corpos de prova em resina (n=3), termopolimerizados em água aquecida, por energia de microondas ou ativados quimicamente, utilizados para a formação dos extratos dessas resinas. Os extratos foram obtidos após 72 horas de imersão dos corpos de prova em meio de cultura e, então, expostos às células da conjuntiva por 72 horas para a realização dos ensaios propostos. Adicionalmente, foi analisada a influência da presença do pigmento acrílico na confecção da prótese de resina acrílica N1, termopolimerizada em água aquecida. Foram confeccionados 9 corpos de prova (n=3): resina N1, resina N1 + pigmento e, pigmento, utilizados para a formação dos extratos desses materiais. Os extratos formaram-se por 72 horas de imersão dos corpos de prova em meio de cultura e, então, foram expostos às células da...

Ocular prosthesis is a treatment option for esthetical and functional rehabilitation of ocular absence. The knowledge of ocular prosthesis material’s biocompatibility is important to ensure a safe use in patients. The aim of this study was to evaluate the cytotoxic effect of ocular prosthesis materials, through the analysis of the cell proliferation, and the production of proinflammatory cytokines and extracellular matrix proteins by a human conjunctival cell line. Initially, the influence of different preparation and exposition periods of eluates from heat-polymerized ocular prosthesis N1 color acrylic resin in human conjunctival cell line was evaluated. A total of 24 acrylic resin samples were manufactured and divided into 2 groups, according to the eluate exposition period to conjunctival cell line (24 and 72 hours). Eluates corresponding to 24, 48 and 72 hours of resin sample immersion in medium and, 24 hours of resin sample immersion in water followed by 24 hours of immersion in medium, were prepared (n=3) for the proposed tests. Then, the cytotoxic effect of different polymerization methods of ocular prosthesis N1 color acrylic resin was analyzed. A total of 9 acrylic resin samples were manufactured (n=3), according to the polymerization method: heat-polymerization in water bath, polymerization by microwave energy and auto-polymerization. Eluates corresponding to 72 hours of resin sample immersion in medium were prepared for proposed tests and exposed to conjunctival cell line for 72 hours. Additionally, the influence of pigment incorporation on the cytotoxicity of heat-polymerized ocular prosthesis N1 color acrylic resin was evaluated. A total of 9 samples were manufactured (n=3): N1 color acrylic resin without pigment incorporation, N1 color acrylic resin with pigment incorporation, and acrylic pigment. Eluates corresponding to 72 hours of sample immersion in medium were prepared and exposed to conjunctival cell line for 72 hours. The cytotoxic effect...