Requirements for Brazilian Outpatient Centers of Excellence in Hidradenitis Suppurativa: Consensus Co-Creative Study.

Background: Hidradenitis suppurativa (HS) is a chronic skin condition. Its complexity and impact on patients highlight the need for multidisciplinary care that can address the physical, psychological, and social aspects. Centers of excellence can ideally provide the necessary infrastructure, resources, and expertise to effectively treat HS. However, there are still no consolidated models of centers of excellence in HS, and establishing their foundations is an intricate research challenge. Purposely, design and co-creation as innovation techniques are helpful approaches to this type of research. Methods: In this study, we conducted a co-creation with consensus among HS specialists to propose the criteria and requirements to establish outpatient centers of excellence of HS in Brazil. We followed a linear process with mixed methods in 6 stages. Results: The process resulted in 10 categories for establishing outpatient centers, including their respective requirements, rationale, and classification. The categories include onboarding and welcoming; infrastructure and procedures; infusion therapy; flows and referrals; staffing; disease management; metrics during diagnosis; metrics during treatment; awareness and advocacy; research and education. Discussion: The idealized outpatient centers can play a role in the complete multidisciplinary treatment for HS and advancing the science of healthcare services by providing a focus for research, training, and translation of findings into practice.

Asthma control in Brazil: a systematic review.

OBJECTIVES: To explore asthma control in patients undergoing pharmacotherapy on studies in the last 20 years in Brazil. Asthma is a chronic airway inflammation disease with a high prevalence worldwide. Even with a variety of drug treatment improvements, attaining asthma control is challenging, since it should have a personalized approach. In Brazil, studies on the prevalence of asthma control are scarce and usually from a small sample size. DATA SOURCES: A systematic review was performed to assess asthma control in Brazilian population. Terms related to "asthma", "asthma control" and "Brazil" were used in the search strategies in PubMed, BVSalud, Embase and Cochrane Library, including Brazilian Journal of Allergy and Immunology as data sources. A narrative synthesis was performed to report key outcome. STUDY SELECTIONS: In total, 23 studies were included. Most of them were conducted in the Southeastern and Northeast regions, in a short duration. RESULTS: Pediatric and non-pediatric population were assessed, with a higher proportion of female. In pediatric population, those with poorly controlled asthma usually had severe or persistent disease. In elderly, an increased asthma severity was found, although proper treatment might be effective. Most studies (70%) also described exacerbations, hospitalizations (48%), quality of life (39%), and emergency visits (30%). Despite heterogeneity of outcomes and population, studies show an important prevalence of uncontrolled asthma even in patients being treated, with better disease control with treatment improvements. CONCLUSIONS: Studies in Brazil have shown that asthma control remains a challenge and there is still a need for improvement on disease management.

Development of an enzyme-linked immunosorbent assay for diagnosis of human herpesvirus-7 infection.

INTRODUCTION: Human herpesvirus (HHV)-7 establishes a latent infection during the lifetime of the host and can reactivate after the primary infection, leading to lytic replication in immunosuppressed patients. METHODS: This study aimed to develop an enzyme-linked immunosorbent assay (ELISA) to identify HHV-7 serum antibodies and compare its performance with that of an indirect immunofluorescence assay (IFA). RESULTS: Serum samples (n=102) were tested by IgG-IFA and by ELISA. IFA and ELISA showed IgG-positive results in 77 and 73 samples, respectively. Qualitative concordance of 96% was demonstrated between the two techniques. CONCLUSIONS: ELISA may be useful to diagnose HHV-7 infection.

Development of an enzyme-linked immunosorbent assay for diagnosis of human herpesvirus-7 infection

INTRODUCTION: Human herpesvirus (HHV)-7 establishes a latent infection during the lifetime of the host and can reactivate after the primary infection, leading to lytic replication in immunosuppressed patients. METHODS :This study aimed to develop an enzyme-linked immunosorbent assay (ELISA) to identify HHV-7 serum antibodies and compare its performance with that of an indirect immunofluorescence assay (IFA). RESULTS: Serum samples (n=102) were tested by IgG-IFA and by ELISA. IFA and ELISA showed IgG-positive results in 77 and 73 samples, respectively. Qualitative concordance of 96% was demonstrated between the two techniques. CONCLUSIONS: ELISA may be useful to diagnose HHV-7 infection.

Development of an enzyme-linked immunosorbent assay for diagnosis of human herpesvirus-7 infection

INTRODUCTION: Human herpesvirus (HHV)-7 establishes a latent infection during the lifetime of the host and can reactivate after the primary infection, leading to lytic replication in immunosuppressed patients. METHODS :This study aimed to develop an enzyme-linked immunosorbent assay (ELISA) to identify HHV-7 serum antibodies and compare its performance with that of an indirect immunofluorescence assay (IFA). RESULTS: Serum samples (n=102) were tested by IgG-IFA and by ELISA. IFA and ELISA showed IgG-positive results in 77 and 73 samples, respectively. Qualitative concordance of 96% was demonstrated between the two techniques. CONCLUSIONS: ELISA may be useful to diagnose HHV-7 infection.

Development of an enzyme-linked immunosorbent assay for diagnosis of human herpesvirus-7 infection

Abstract INTRODUCTION: Human herpesvirus (HHV)-7 establishes a latent infection during the lifetime of the host and can reactivate after the primary infection, leading to lytic replication in immunosuppressed patients. METHODS: This study aimed to develop an enzyme-linked immunosorbent assay (ELISA) to identify HHV-7 serum antibodies and compare its performance with that of an indirect immunofluorescence assay (IFA). RESULTS: Serum samples (n=102) were tested by IgG-IFA and by ELISA. IFA and ELISA showed IgG-positive results in 77 and 73 samples, respectively. Qualitative concordance of 96% was demonstrated between the two techniques. CONCLUSIONS: ELISA may be useful to diagnose HHV-7 infection.

An optimization study for expression of the rabies virus glycoprotein (RVGP) in mammalian cell lines using the Semliki Forest virus (SFV).

The Semliki Forest virus (SFV) viral vector has been widely used for transient protein expression. This study aimed to analyze comprehensively the capacity of SFV vector to express rabies lyssavirus glycoprotein (RVGP) in mammalian cells. The assessed parameters were transfection strategy, multiplicity of infection (MOI), harvest time and mammalian cell host. Two transfection approaches, electroporation and lipofection were evaluated to obtain the recombinant SFV, and the electroporation was found to be the most effective. Viral quantification by RT-qPCR was performed to elucidate the relation between the amount of recombinant virus utilized in the infection process and the production levels of the heterologous protein. Four different multiplicities of infection (MOIs = 1; 10; 15; 50) were evaluated using five mammalian cell lines: BHK-21, HuH-7, Vero, L929, and HEK-293T. Protein expression was assessed at two harvest times after infection (24 and 48 h). The recombinant protein generated was characterized by western blot, dot blot, and indirect immunofluorescence (IIF), while its concentration was determined by enzyme-linked immunosorbent assay (ELISA). Similar expression patterns were observed in cell lines BHK-21, HEK-293T, L929, and Vero, with higher RVGP production in the first 24 h. The BHK-21 cells showed yields of up to 4.3 µg per 106 cells when lower MOIs (1 and 10) were used. The HEK-293 T cells also showed similar production (4.3 µg per 106 cells) with MOI of 1, while the L929 and Vero cell lines showed lower expression rates of 2.82 and 1.26 µg per 106 cells, respectively. These cell lines showed lower expression levels at 48 h after infection compared to 24 h. Controversially, in the case of the HuH-7 cell line, RVGP production was higher at 48 h after infection (4.0 µg per 106 cells) and using MOIs of 15 and 50. This work may contribute to optimize the RVGP production using SFV system in mammalian cells. This study can also substantiate for example, the development of approaches that use of SFV for applications for other protein expressions and suggests values for relevant parameters and cell lines of this biotechnique.

An optimization study for expression of the rabies virus glycoprotein (RVGP) in mammalian cell lines using the Semliki Forest virus (SFV)

The Semliki Forest virus (SFV) viral vector has been widely used for transient protein expression. This study aimed to analyze comprehensively the capacity of SFV vector to express rabies lyssavirus glycoprotein (RVGP) in mammalian cells. The assessed parameters were transfection strategy, multiplicity of infection (MOI), harvest time and mammalian cell host. Two transfection approaches, electroporation and lipofection were evaluated to obtain the recombinant SFV, and the electroporation was found to be the most effective. Viral quantification by RT-qPCR was performed to elucidate the relation between the amount of recombinant virus utilized in the infection process and the production levels of the heterologous protein. Four different multiplicities of infection (MOIs = 1; 10; 15; 50) were evaluated using five mammalian cell lines: BHK-21, HuH-7, Vero, L929, and HEK-293T. Protein expression was assessed at two harvest times after infection (24 and 48 h). The recombinant protein generated was characterized by western blot, dot blot, and indirect immunofluorescence (IIF), while its concentration was determined by enzyme-linked immunosorbent assay (ELISA). Similar expression patterns were observed in cell lines BHK-21, HEK-293T, L929, and Vero, with higher RVGP production in the first 24 h. The BHK-21 cells showed yields of up to 4.3 µg per 106 cells when lower MOIs (1 and 10) were used. The HEK-293 T cells also showed similar production (4.3 µg per 106 cells) with MOI of 1, while the L929 and Vero cell lines showed lower expression rates of 2.82 and 1.26 µg per 106 cells, respectively. These cell lines showed lower expression levels at 48 h after infection compared to 24 h. Controversially, in the case of the HuH-7 cell line, RVGP production was higher at 48 h after infection (4.0 µg per 106 cells) and using MOIs of 15 and 50. This work may contribute to optimize the RVGP production using SFV system in mammalian cells. This study can also substantiate for example, the development of approaches that use of SFV for applications for other protein expressions and suggests values for relevant parameters and cell lines of this biotechnique.

An optimization study for expression of the rabies virus glycoprotein (RVGP) in mammalian cell lines using the Semliki Forest virus (SFV)

The Semliki Forest virus (SFV) viral vector has been widely used for transient protein expression. This study aimed to analyze comprehensively the capacity of SFV vector to express rabies lyssavirus glycoprotein (RVGP) in mammalian cells. The assessed parameters were transfection strategy, multiplicity of infection (MOI), harvest time and mammalian cell host. Two transfection approaches, electroporation and lipofection were evaluated to obtain the recombinant SFV, and the electroporation was found to be the most effective. Viral quantification by RT-qPCR was performed to elucidate the relation between the amount of recombinant virus utilized in the infection process and the production levels of the heterologous protein. Four different multiplicities of infection (MOIs = 1; 10; 15; 50) were evaluated using five mammalian cell lines: BHK-21, HuH-7, Vero, L929, and HEK-293T. Protein expression was assessed at two harvest times after infection (24 and 48 h). The recombinant protein generated was characterized by western blot, dot blot, and indirect immunofluorescence (IIF), while its concentration was determined by enzyme-linked immunosorbent assay (ELISA). Similar expression patterns were observed in cell lines BHK-21, HEK-293T, L929, and Vero, with higher RVGP production in the first 24 h. The BHK-21 cells showed yields of up to 4.3 µg per 106 cells when lower MOIs (1 and 10) were used. The HEK-293 T cells also showed similar production (4.3 µg per 106 cells) with MOI of 1, while the L929 and Vero cell lines showed lower expression rates of 2.82 and 1.26 µg per 106 cells, respectively. These cell lines showed lower expression levels at 48 h after infection compared to 24 h. Controversially, in the case of the HuH-7 cell line, RVGP production was higher at 48 h after infection (4.0 µg per 106 cells) and using MOIs of 15 and 50. This work may contribute to optimize the RVGP production using SFV system in mammalian cells. This study can also substantiate for example, the development of approaches that use of SFV for applications for other protein expressions and suggests values for relevant parameters and cell lines of this biotechnique.

Zika virus selectively kills aggressive human embryonal CNS tumor cells in vitro and in vivo

Zika virus (ZIKV) is largely known for causing brain abnormalities due to its ability to infect neural progenitor stem cells during early development. Here, we show that ZIKV is also capable of infecting and destroying stem-like cancer cells from aggressive human embryonal tumors of the central nervous system (CNS). When evaluating the oncolytic properties of Brazilian Zika virus strain (ZIKV(BR)) against human breast, prostate, colorectal, and embryonal CNS tumor cell lines, we verified a selective infection of CNS tumor cells followed by massive tumor cell death. ZIKV(BR) was more efficient in destroying embryonal CNS tumorspheres than normal stem cell neurospheres. A single intracerebroventricular injection of ZIKV(BR) in BALB/c nude mice bearing orthotopic human embryonal CNS tumor xenografts resulted in a significantly longer survival, decreased tumor burden, fewer metastasis, and complete remission in some animals. Tumor cells closely resembling neural stem cells at the molecular level with activated Wnt signaling were more susceptible to the oncolytic effects of ZIKV(BR). furthermore, modulation of Wnt signaling pathway significantly affected ZIKV(BR)-induced tumor cell death and viral shedding. Altogether, these preclinical findings indicate that ZIKV(BR) could be an efficient agent to treat aggressive forms of embryonal CNS tumors and could provide mechanistic insights regarding its oncolytic effects.

Zika virus selectively kills aggressive human embryonal CNS tumor cells in vitro and in vivo

Zika virus (ZIKV) is largely known for causing brain abnormalities due to its ability to infect neural progenitor stem cells during early development. Here, we show that ZIKV is also capable of infecting and destroying stem-like cancer cells from aggressive human embryonal tumors of the central nervous system (CNS). When evaluating the oncolytic properties of Brazilian Zika virus strain (ZIKV(BR)) against human breast, prostate, colorectal, and embryonal CNS tumor cell lines, we verified a selective infection of CNS tumor cells followed by massive tumor cell death. ZIKV(BR) was more efficient in destroying embryonal CNS tumorspheres than normal stem cell neurospheres. A single intracerebroventricular injection of ZIKV(BR) in BALB/c nude mice bearing orthotopic human embryonal CNS tumor xenografts resulted in a significantly longer survival, decreased tumor burden, fewer metastasis, and complete remission in some animals. Tumor cells closely resembling neural stem cells at the molecular level with activated Wnt signaling were more susceptible to the oncolytic effects of ZIKV(BR). furthermore, modulation of Wnt signaling pathway significantly affected ZIKV(BR)-induced tumor cell death and viral shedding. Altogether, these preclinical findings indicate that ZIKV(BR) could be an efficient agent to treat aggressive forms of embryonal CNS tumors and could provide mechanistic insights regarding its oncolytic effects.

Impact of recombinant Drosophila S2 cell population enrichment on expression of rabies virus glycoprotein

Recombinant Drosophila S2 cells have been used for the expression of many proteins of medical interest. However, membrane-attached glycoproteins, which commonly exhibit lower expression levels compared to soluble proteins, may require special procedures in order to attain high levels of expression. In this study, two S2 cell population enrichment methods (antibiotic and immunomagnetic selection) were evaluated for their ability to enhance expression of the membrane-anchored rabies virus glycoprotein (RVGP). Quantification of RVGP production and determination of its cDNA copy number in transformed cells showed that both enrichment methods increased RVGP expression without significantly affecting its gene copy number. More interestingly, RVGP mRNA levels measured after cycloheximide treatment were poorly correlated with glycoprotein levels. Both enrichment methods enhanced expression of RVGP by recombinant S2 cells, with the highest level of expression achieved using immunomagnetic selection

Transient expression of rabies virus G-glycoprotein using BHK-21 cells cultured in suspension.

OBJECTIVE: To assess the expression of rabies virus G-glycoprotein (RVGP) expression using Semliki Forest virus as a vector in combination with BHK-21 cells cultured in suspension. RESULTS: A multilevel factorial design was used to quantify effects of temperature (33-37 °C), fresh medium addition after the viral adsorption step (100-200 % with respect to the initial cell suspension volume before infection) and harvest time (8-40 h) on RVGP production. Experimental runs were performed in 24-well cell culture plates at a multiplicity of infection (MOI) of 16. An additional experiment in spinner-flask was performed at MOI of 9, using the optimal conditions determined in cell culture plates. Values for temperature, fresh medium addition and harvest time of 33 °C, 100 % and 16 h, respectively, ensured the optimal RVGP production in culture plates. The volumetric yield (239 ng ml(-1)) in these conditions was higher than that reported previously for adherent cell culture. In spinner-flasks, the volumetric yield was improved (559 ng ml(-1)). CONCLUSION: These results establish the basis for designing bioprocess to produce RVGP.