Identification of Potential Antiviral Hops Compounds against Chikungunya Virus.

Chikungunya virus (CHIKV) is an arthropod-borne virus that belongs to the genus Alphavirus (family Togaviridae). CHIKV causes chikungunya fever, which is mostly characterized by fever, arthralgia and, sometimes, a maculopapular rash. The bioactive constituents of hops (Humulus lupulus, Cannabaceae), mainly acylphloroglucinols, known as well as α- and ß-acids, exerted distinct activity against CHIKV, without showing cytotoxicity. For fast and efficient isolation and identification of such bioactive constituents, a silica-free countercurrent separation method was applied. The antiviral activity was determined by plaque reduction test and was visually confirmed by a cell-based immunofluorescence assay. All hops compounds demonstrated a promising post-treatment viral inhibition, except the fraction of acylphloroglucinols, in mixture. ß-acids fraction of 125 µg/mL expressed the strongest virucidal activity (EC50 = 15.21 µg/mL), in a drug-addition experiment on Vero cells. Hypothesis for mechanism of action were proposed for acylphloroglucinols based on their lipophilicity and chemical structure. Therefore, inhibition of some steps of the protein kinase C (PKC) transduction cascades was also discussed.

Accessibility and adherence to positive airway pressure treatment in patients with obstructive sleep apnea: a multicenter study in Latin America.

PURPOSE: Information on access and adherence to positive airway pressure (PAP) treatment is lacking at the regional level in Latin America. This study characterized access and adherence to PAP in patients with moderate-severe obstructive sleep apnea (OSA) in Latin America. METHODS: Cross-sectional study, conducted at 9 sleep centers across Argentina, Brazil, Chile, Colombia, Mexico, and Peru. Adults diagnosed with moderate-severe OSA (apnea-hypopnea index [AHI] ≥ 15/h) in the previous 12-18 months were eligible. Anthropometrics, health coverage, and OSA severity data were collected. Data on access to therapy, barriers to access, adherence, and factors related to non-compliance were obtained via standardized telephone survey. RESULTS: Eight hundred eighty patients (70% male, 54 ± 13 years, AHI 49 ± 28/h, body mass index 32 ± 7 kg/m2) were included. Four hundred ninety patients (56%) initiated PAP, 70 (14%) discontinued therapy during the first year (mainly due to intolerance), and 420 (48%) were still using PAP when surveyed. Health insurance was private in 36.9% of patients, via the social security system in 31.1%, and via the state in 13.3%, and 18.7% did not have any coverage; 49.5% of patients had to pay all equipment costs. Reasons for not starting PAP were unclear or absent indication (42%), coverage problems (36%), and lack of awareness of OSA burden (14%). Patients with better adherence were older (55.3 ± 13 vs 52 ± 13; p = 0.002) and had more severe OSA (AHI 51.8 ± 27 vs 45.6 ± 27; p = 0.001). CONCLUSIONS: Less than half moderate-severe OSA patients started and continue to use PAP. Unclear or absent medical indication and financial limitations were the most relevant factors limiting access to therapy.

Herpes simplex virus 1 and cytomegalovirus are associated with pemphigus vulgaris but not with pemphigus foliaceus disease.

Pemphigus vulgaris (PV) and pemphigus foliaceus (PF) are blistering autoimmune diseases that depend on interaction between genetic and environmental factors. Viral infections, like herpes simplex viruses 1 and 2 (HSV1/2), cytomegalovirus (CMV), Epstein-Barr virus and dengue virus, could trigger or exacerbate pemphigus. IgM and IgG antibodies against these viruses in serum from PV and PF, their relatives and controls were determined. HSV1/2 expression was evaluated by direct immunofluorescence (DIF) and qPCR in affected or not oral mucosa from PV patients compared with uninjured PF mucosa. IgG anti-HSV1 was higher in the PV group compared with all groups. IgG anti-CMV resulted higher in PV group compared with PF patients and PV relatives. HSV1 was confirmed by DIF and qPCR on oral samples from patients with PV. Lack of HSV1 expression in the oral mucosa of patients with PF corroborate that immunosuppressive therapy cannot be the main cause for HSV1 replication in PV disease.

Measurement of pesticides in hair samples from pemphigus foliaceus and pemphigus vulgaris patients in Southeastern Brazil.

BACKGROUND: Pesticides, mainly organophosphates (OP), have been related to increased risk of pemphigus vulgaris (PV) and pemphigus foliaceus (PF), nevertheless, their measurement has not been determined in pemphigus patients. OBJECTIVE: To evaluate pesticide exposure and pesticide measurement, comparing PV, PF and control groups in Southeastern Brazil. METHODS: Information about urban or rural residency and exposure to pesticides at the onset of pemphigus was assessed by questionnaire interview; hair samples from the scalp of PV, PF, and controls were tested for OP and organochlorines (OC) by gas-phase chromatography coupled to mass spectrometry. RESULTS: The minority of PV (2 [7.1%] of 28) and PF (7 [18%] of 39), but none of the 48 controls, informed living in rural areas at the onset of pemphigus (p = 0.2853). PV (33.3%), PF (38.5%), and controls (20%) informed exposure to pesticides (p = 0.186). Twenty-one (14.8%) of 142 individuals tested positive for OP and/or OC: PV (2 [6.3%] of 32) and PF (11 [25.6%] of 43) had similar pesticides contamination as controls (8 [11.9%] of 67) (p = 0.4928; p = 0.0753, respectively), but PF presented higher contamination than PV (p = 0.034). PV did not present any positivity for OP. Three (7%) PF tested positive for both OP and OC. Some PF tested positive for three or four OP, mainly diazinon and dichlorvos. STUDY LIMITATION: Lack of data for some controls. CONCLUSION: Although the frequency of PV and PF patients exposed to pesticides was similar, pesticides were more frequently detected in hair samples from PF compared to PV. The cause-effect relationship still needs to be determined.

Mitochondrial DNA and TLR9 activation contribute to SARS-CoV-2-induced endothelial cell damage.

BACKGROUND AND PURPOSE: Mitochondria play a central role in the host response to viral infection and immunity, being key to antiviral signaling and exacerbating inflammatory processes. Mitochondria and Toll-like receptor (TLR) have been suggested as potential targets in SARS-CoV-2 infection. However, the involvement of TLR9 in SARS-Cov-2-induced endothelial dysfunction and potential contribution to cardiovascular complications in COVID-19 have not been demonstrated. This study determined whether infection of endothelial cells by SARS-CoV-2 affects mitochondrial function and induces mitochondrial DNA (mtDNA) release. We also questioned whether TLR9 signaling mediates the inflammatory responses induced by SARS-CoV-2 in endothelial cells. EXPERIMENTAL APPROACH: Human umbilical vein endothelial cells (HUVECs) were infected by SARS-CoV-2 and immunofluorescence was used to confirm the infection. Mitochondrial function was analyzed by specific probes and mtDNA levels by real-time polymerase chain reaction (RT-PCR). Inflammatory markers were measured by ELISA, protein expression by western blot, intracellular calcium (Ca2+) by FLUOR-4, and vascular reactivity with a myography. KEY RESULTS: SARS-CoV-2 infected HUVECs, which express ACE2 and TMPRSS2 proteins, and promoted mitochondrial dysfunction, i.e. it increased mitochondria-derived superoxide anion, mitochondrial membrane potential, and mtDNA release, leading to activation of TLR9 and NF-kB, and release of cytokines. SARS-CoV-2 also decreased nitric oxide synthase (eNOS) expression and inhibited Ca2+ responses in endothelial cells. TLR9 blockade reduced SARS-CoV-2-induced IL-6 release and prevented decreased eNOS expression. mtDNA increased vascular reactivity to endothelin-1 (ET-1) in arteries from wild type, but not TLR9 knockout mice. These events were recapitulated in serum samples from COVID-19 patients, that exhibited increased levels of mtDNA compared to sex- and age-matched healthy subjects and patients with comorbidities. CONCLUSION AND APPLICATIONS: SARS-CoV-2 infection impairs mitochondrial function and activates TLR9 signaling in endothelial cells. TLR9 triggers inflammatory responses that lead to endothelial cell dysfunction, potentially contributing to the severity of symptoms in COVID-19. Targeting mitochondrial metabolic pathways may help to define novel therapeutic strategies for COVID-19.

SARS-CoV-2 productively infects primary human immune system cells in vitro and in COVID-19 patients.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection is associated with a hyperinflammatory state and lymphocytopenia, a hallmark that appears as both signature and prognosis of disease severity outcome. Although cytokine storm and a sustained inflammatory state are commonly associated with immune cell depletion, it is still unclear whether direct SARS-CoV-2 infection of immune cells could also play a role in this scenario by harboring viral replication. We found that monocytes, as well as both B and T lymphocytes, were susceptible to SARS-CoV-2 infection in vitro, accumulating double-stranded RNA consistent with viral RNA replication and ultimately leading to expressive T cell apoptosis. In addition, flow cytometry and immunofluorescence analysis revealed that SARS-CoV-2 was frequently detected in monocytes and B lymphocytes from coronavirus disease 2019 (COVID-19) patients. The rates of SARS-CoV-2-infected monocytes in peripheral blood mononuclear cells from COVID-19 patients increased over time from symptom onset, with SARS-CoV-2-positive monocytes, B cells, and CD4+ T lymphocytes also detected in postmortem lung tissue. These results indicated that SARS-CoV-2 infection of blood-circulating leukocytes in COVID-19 patients might have important implications for disease pathogenesis and progression, immune dysfunction, and virus spread within the host.

Infection of human lymphomononuclear cells by SARS-CoV-2.

Although SARS-CoV-2 severe infection is associated with a hyperinflammatory state, lymphopenia is an immunological hallmark, and correlates with poor prognosis in COVID-19. However, it remains unknown if circulating human lymphocytes and monocytes are susceptible to SARS-CoV-2 infection. In this study, SARS-CoV-2 infection of human peripheral blood mononuclear cells (PBMCs) was investigated both in vitro and in vivo . We found that in vitro infection of whole PBMCs from healthy donors was productive of virus progeny. Results revealed that monocytes, as well as B and T lymphocytes, are susceptible to SARS-CoV-2 active infection and viral replication was indicated by detection of double-stranded RNA. Moreover, flow cytometry and immunofluorescence analysis revealed that SARS-CoV-2 was frequently detected in monocytes and B lymphocytes from COVID-19 patients, and less frequently in CD4 + T lymphocytes. The rates of SARS-CoV-2-infected monocytes in PBMCs from COVID-19 patients increased over time from symptom onset. Additionally, SARS-CoV-2-positive monocytes and B and CD4+T lymphocytes were detected by immunohistochemistry in post mortem lung tissue. SARS-CoV-2 infection of blood circulating leukocytes in COVID-19 patients may have important implications for disease pathogenesis, immune dysfunction, and virus spread within the host.

Trace element profile in pemphigus foliaceus and in pemphigus vulgaris patients from Southeastern Brazil.

Pemphigus foliaceus (PF) and pemphigus vulgaris (PV) are autoimmune bullous diseases; they are endemic in the northeastern region of the state of São Paulo, Southeastern Brazil. Patients' copper (Cu), zinc (Zn), and selenium (Se) metabolic deficiencies have already been associated with PV pathogenesis in the Middle East but not in Brazil. Lead (Pb), Cu, Zn, and Se concentrations were determined in whole blood or serum samples obtained from 56 PV patients, 62 PF patients, and 135 healthy controls from the endemic region and compared. The PV patients had higher (above the reference values) Pb and lower Zn levels as compared to controls. Both the PV and the PF patients presented decreased Se levels as compared to controls. The PV, PF, and control groups did not differ in terms of Cu concentration. These results indicate that high Pb blood levels are related with environmental contamination and consequently with PV pathogenesis. Moreover, Zn and Se depletion, observed in the PV patients and in the PV and the PF patients, respectively, may result from metabolic consumption verified in chronic diseases.

Nocturnal aspects of narcolepsy with cataplexy.

Even though the most impressive manifestation of narcolepsy is excessive sleepiness, paradoxically a significant number of patients have trouble sleeping at night. A wide array of alterations can affect the night-time sleep of a narcoleptic patient, and the aim of this review is to increase awareness on this issue, thereby enhancing the care of narcoleptic patients by more specific approaches to their disturbed night sleep. This review covers a broad variety of nocturnal sleep features in narcolepsy. Starting from animal models and the clinical features of patients, the paper then discusses the many comorbid conditions found in narcolepsy at night, the most advanced methods of analysis and the few recent advances in the specific treatment of night sleep in narcoleptic patients.

Cataplexy features in childhood narcolepsy.

Cataplexy, the hallmark of narcolepsy, has been well characterized in adults but not in children. This study systematically used structured clinical assessments and video-recordings (49 episodes in eight cases) to evaluate cataplexy in 23 patients diagnosed before the age of 18 years. Forty-three percent of patients had falls as part of their attacks. During cataplexy knees, head, and jaw were the most frequently compromised body segments; eyelids, arms, and trunk being less commonly involved. More rarely, blurred vision, slurred speech, irregular breathing, or a sudden loss of smiling mimics were reported. One-third of the sample presented with a previously unrecognized description of cataplexy that we coined "cataplectic facies," consisting of a state of semipermanent eyelid and jaw weakness, on which partial or complete cataplectic attacks were superimposed. The usual triggering emotions, such as laughter, joking, or anger, were not always present, especially when close to an abrupt onset, hampering diagnosis. Video-recordings of cataplectic attacks may be useful to document the attack, allowing a comparison with archived presentations.

Measurement of pesticides in hair samples from pemphigus foliaceus and pemphigus vulgaris patients in Southeastern Brazil

Abstract Background Pesticides, mainly organophosphates (OP), have been related to increased risk of pemphigus vulgaris (PV) and pemphigus foliaceus (PF), nevertheless, their measurement has not been determined in pemphigus patients. Objective To evaluate pesticide exposure and pesticide measurement, comparing PV, PF and control groups in Southeastern Brazil. Methods Information about urban or rural residency and exposure to pesticides at the onset of pemphigus was assessed by questionnaire interview; hair samples from the scalp of PV, PF, and controls were tested for OP and organochlorines (OC) by gas-phase chromatography coupled to mass spectrometry. Results The minority of PV (2 [7.1%] of 28) and PF (7 [18%] of 39), but none of the 48 controls, informed living in rural areas at the onset of pemphigus (p = 0.2853). PV (33.3%), PF (38.5%), and controls (20%) informed exposure to pesticides (p = 0.186). Twenty-one (14.8%) of 142 individuals tested positive for OP and/or OC: PV (2 [6.3%] of 32) and PF (11 [25.6%] of 43) had similar pesticides contamination as controls (8 [11.9%] of 67) (p = 0.4928; p = 0.0753, respectively), but PF presented higher contamination than PV (p = 0.034). PV did not present any positivity for OP. Three (7%) PF tested positive for both OP and OC. Some PF tested positive for three or four OP, mainly diazinon and dichlorvos. Study limitation Lack of data for some controls. Conclusion Although the frequency of PV and PF patients exposed to pesticides was similar, pesticides were more frequently detected in hair samples from PF compared to PV. The cause-effect relationship still needs to be determined.

Evaluating the use of fluorescence-based flow cytometry assay for dengue diagnosis using peripheral blood mononuclear cells.

INTRODUCTION: Dengue virus (DENV) is the most important arthropod-borne viral disease worldwide with an estimated 50 million infections occurring each year. METHODS: In this study, we present a flow cytometry assay (FACS) for diagnosing DENV, and compare its results with those of the non-structural protein 1 (NS1) immunochromatographic assay and reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: All three assays identified 29.1% (39/134) of the patients as dengue-positive. The FACS approach and real-time RT-PCR detected the DENV in 39 and 44 samples, respectively. On the other hand, the immunochromatographic assay detected the NS1 protein in 40.1% (56/134) of the patients. The Cohen's kappa coefficient analysis revealed a substantial agreement among the three methods. CONCLUSIONS: The FACS approach may be a useful alternative for dengue diagnosis and can be implemented in public and private laboratories.

Spatial Distribution of Pemphigus Occurrence over Five Decades in Southeastern Brazil.

Well-defined locations of pemphigus cases support the hypothesis of environmental factors' involvement in its etiopathogenesis; however, these foci have never been described using specialized geographical tools. This is the first report to geo-reference pemphigus cases in a high-prevalence Brazilian region using geographic information systems. We aimed to report the spatio-temporal behavior of pemphigus foliaceus (PF) and vulgaris (PV) in southeastern Brazil, over the last five decades to describe geographical clusters, as well as to characterize the land use in the city with the highest number of cases. Patients were identified from 1965 to 2014. Maps were developed using ArcGIS software and organized into decades from 1965 to 2014. Ribeirão Preto was identified as the city with the greatest number of cases. Land use was analyzed within a 2 km-buffer surrounding the residence of each patient. A total of 426 cases of pemphigus were identified. PF was the predominant form (285 cases); notwithstanding, the number of new cases of PV rose, overtaking the number of new cases of PF in the last decade studied. Agricultural area (42%) and exposed soil (33.2%) are the most predominant land uses in Ribeirão Preto surrounding patients' residences. This study shows high-confidence geographical foci of PF and PV, as well as provides evidence of an increase of both clinical forms over the last five decades. All cases of PV and PF are in proximity to rivers and agricultural areas which reinforce the hypothesis that environmental factors play a role in pemphigus etiopathogenesis.

Evaluating the use of fluorescence-based flow cytometry assay for dengue diagnosis using peripheral blood mononuclear cells

Abstract INTRODUCTION: Dengue virus (DENV) is the most important arthropod-borne viral disease worldwide with an estimated 50 million infections occurring each year. METHODS: In this study, we present a flow cytometry assay (FACS) for diagnosing DENV, and compare its results with those of the non-structural protein 1 (NS1) immunochromatographic assay and reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: All three assays identified 29.1% (39/134) of the patients as dengue-positive. The FACS approach and real-time RT-PCR detected the DENV in 39 and 44 samples, respectively. On the other hand, the immunochromatographic assay detected the NS1 protein in 40.1% (56/134) of the patients. The Cohen's kappa coefficient analysis revealed a substantial agreement among the three methods. CONCLUSIONS: The FACS approach may be a useful alternative for dengue diagnosis and can be implemented in public and private laboratories.

Olfactory dysfunction in narcolepsy with cataplexy.

BACKGROUND: Narcolepsy-cataplexy (NC) is caused by the loss of hypocretin neurons. Several lines of evidence suggest a role for hypocretin in olfactory function. Recent data have documented that NC is associated with olfactory dysfunction but smell testing has been performed only in small studies. METHODS: One hundred thirty NC patients were recruited from two sleep disorders centers in France and Italy. They were compared to 129 age- and gender-matched healthy controls on two well standardized olfactory tests: the Sniffin' Sticks (France) and Brief Smell Identification (Italy) tests. Olfactory dysfunction was defined as a score below the tenth centile on each smell test. RESULTS: Altogether, olfactory deficit was higher in NC compared to controls (23.8% vs. 13.9%, p=0.042, OR: 1.93 CI 95%, 1.01-3.66); olfactory identification deficit was found in 35.8% of NC compared to 13.9% of controls in Italy (p=0.03), and in 11.2% vs. 8.2% in France (NS). Using the Sniffin' Sticks (France) we noted significant lower discrimination and global olfactory performance in NC compared to controls. Based on standardized criteria for Sniffin' Sticks, none of the NC patients or controls presented anosmia or severe hyposmia, but 6.4% of the NC patients had moderate hyposmia and 22.2% mild, in contrast to 6.4% of controls with only mild hyposmia. Finally except tobacco smoking, clinical potential factors including age at onset and severity of the condition did not modify olfactory performances in the whole population. CONCLUSION: Our study is the largest investigation of olfactory performance in NC showing that the disease perse is associated with mild/moderate dysfunction in a quarter of patients.