Detection of the antiviral activity of epicatechin isolated from Salacia crassifolia (Celastraceae) against Mayaro virus based on protein C homology modelling and virtual screening.

Mayaro fever, caused by Mayaro virus (MAYV) is a sub-lethal disease with symptoms that are easily confused with those of dengue fever, except for polyarthralgia, which may culminate in physical incapacitation. Recently, outbreaks of MAYV have been documented in metropolitan areas, and to date, there is no therapy or vaccine available. Moreover, there is no information regarding the three-dimensional structure of the viral proteins of MAYV, which is important in the search for antivirals. In this work, we constructed a three-dimensional model of protein C of MAYV by homology modelling, and this was employed in a manner similar to that of receptors in virtual screening studies to evaluate 590 molecules as prospective antiviral agents. In vitro bioassays were utilized to confirm the potential antiviral activity of the flavonoid epicatechin isolated from Salacia crassifolia (Celastraceae). The virtual screening showed that six flavonoids were promising ligands for protein C. The bioassays showed potent antiviral action of epicatechin, which protected the cells from almost all of the effects of viral infection. An effective concentration (EC50) of 0.247 µmol/mL was observed with a selectivity index (SI) of 7. The cytotoxicity assay showed that epicatechin has low toxicity, with a 50% cytotoxic concentration (CC50) greater than 1.723 µmol/mL. Epicatechin was found to be twice as potent as the reference antiviral ribavirin. Furthermore, a replication kinetics assay showed a strong inhibitory effect of epicatechin on MAYV growth, with a reduction of at least four logs in virus production. Our results indicate that epicatechin is a promising candidate for further testing as an antiviral agent against Mayaro virus and other alphaviruses.

Saturated salt solution: a further step to a formaldehyde-free embalming method for veterinary gross anatomy.

In the field of veterinary anatomy, most of the specimens used in practical sessions are perfused with fixatives. Thus, they can be used for a longer time, reducing the number of animals for educational purposes. Formalin is the most commonly used fixative, consisting of a 37% formaldehyde solution. However, formaldehyde is a powerful irritant of the eyes and airways and is considered carcinogenic, causing nasopharyngeal cancer in exposed workers and professionals. In the present study, we explored an alternative method to avoid the use of formaldehyde in specimens used for gross anatomy practical sessions. We propose an inexpensive, non-toxic fixative that is available worldwide, such as sea salt. This method consists of a continuous perfusion of saturated salt solution for a period of 6-8 h, enabling drainage of the solution to avoid a weight increase of the specimen, and allowing salt to be retained in the tissue. The method is based on recirculation of the saturated salt solution instead of maceration. Perfused specimens retained their natural consistency and joint mobility, with no blood, resembling a piece of meat from the slaughterhouse. They could be used immediately without a maceration period, or stored in the fridge until use and then kept in a bath of saturated salt solution for future conservation. In the case of the former, no refrigeration was needed. The specimens did not have an irritating or offensive smell, and could be used for long sessions (several hours per day) and stored for long periods. However, the blood vessels used for perfusion determine the results: a less invasive approach (through common carotid arteries) gave good preservation of the musculoskeletal system, whereas more invasive access to cannulate the abdominal aorta and vena cava caudalis was required to achieve better preservation of the viscera. In conclusion, we propose that perfusion followed by immersion in a saturated salt solution is a good alternative method for the preservation of specimens used in the practical teaching of gross veterinary anatomy. It is a very simple and inexpensive technique, and is much healthier for users than traditional formalin. Moreover, specimens can be preserved for prolonged periods, and maintain a similar appearance and consistency to fresh material.

Strong Constraints on Cosmological Gravity from GW170817 and GRB 170817A.

The detection of an electromagnetic counterpart (GRB 170817A) to the gravitational-wave signal (GW170817) from the merger of two neutron stars opens a completely new arena for testing theories of gravity. We show that this measurement allows us to place stringent constraints on general scalar-tensor and vector-tensor theories, while allowing us to place an independent bound on the graviton mass in bimetric theories of gravity. These constraints severely reduce the viable range of cosmological models that have been proposed as alternatives to general relativistic cosmology.

Reliability and validity of the Chester step test in patients with interstitial lung disease.

BACKGROUND: The Chester Step Test (CST) is a simple and inexpensive field test, which requires minimal physical space to assess exercise capacity. Such characteristics make the CST suitable to be used in different settings, however, its measurement properties in patients with interstitial lung diseases (ILD) are unknown. METHODS: A cross-sectional study was conducted in patients with ILD. First, a CST-1 and a 6-minute walk test (6MWT) were performed. After 48-72 hours, a CST-2 was repeated. A 2nd rater was present in one of the sessions. Relative reliability was measured using intraclass correlation coefficient (ICC1,1 and ICC2,1). Absolute reliability was determined using standard error of measurement (SEM), minimal detectable change at 95% confidence interval (MDC95) and the Bland-Altman method. The values of SEM and MDC95 were also expressed as a percentage of the mean. Construct validity was explored using Spearman correlation coefficient (rs) between the number of steps taken in the best CST and the distance performed in the 6MWT. RESULTS: Sixty-six patients with ILD (65.5±12.9 years; 48.5%men; FVC 79.4±18.8pp; DLCO 49.0±18.3pp) participated in the study. Relative (ICC 0.95-1.0) and absolute reliability were excellent without evidence of systematic bias. The SEM and MDC95 were 11.8 (14.7%) and 32.6 steps (40.7%), respectively. The correlation between CST and 6MWT was significant, positive, and high (rs=0.85, p=0.001). CONCLUSION: The CST is a reliable and valid test and might be especially useful to assess exercise capacity in patients with ILD in limited space environments.

Criteria for progressive fibrotic hypersensitivity pneumonitis in a Portuguese patient cohort.

Background: Hypersensitivity pneumonitis (HP) is a syndrome caused by sensitisation to inhaled antigens that leads to an abnormal immune response in the airways and lung parenchyma. Some patients previously diagnosed with certain types of fibrotic interstitial lung diseases (f-ILDs), including fibrotic HP (f-HP), are susceptible to develop a progressive fibrosing phenotype (PF-ILD), despite initial state-of-the-art management. Objectives: To characterise a cohort of patients with a multidisciplinary diagnosis (MTD) of chronic f-HP, who were followed up in an ILD outpatient clinic of a hospital in Portugal, and to assess the prevalence of PF-ILD criteria in these patients. Methods: Data were collected from all patients with a definite or provisional diagnosis of f-HP after a multidisciplinary team discussion. Patients were followed up between December 2014 and July 2019. Data included clinical characteristics, high-resolution chest tomography (HRCT) disease patterns, lung function tests, bronchoalveolar lavage and further immunological work-up, biopsy reports (conventional transbronchial lung biopsy, transbronchial lung cryobiopsy or surgical video-assisted thoracoscopic lung biopsy), all ILD multidisciplinary team records and diagnostic confidence levels. Patients were assessed according to PF-ILD criteria as defined in the INBUILD trial. Results: We identified 83 patients with an MTD of HP, who had been followed up for at least 12 months. Of these, 63 (75.9%) were diagnosed with f-HP. Of the 63 f-HP patients, 33.3% (n=21) fulfilled the predefined criteria for PF-HP: 66.7% had a relative decline of ≥10% forced vital capacity (FVC); 5% a relative decline of 5 - 9% FVC, with worsening symptoms or increased fibrosis on HRCT; and 23.8% had worsening respiratory symptoms with radiological progression. Conclusion: This single-centre cohort study demonstrated that a third of f-HP patients presented with PF-ILD, as determined by progression during initial standard-of-care treatment. A usual interstitial pneumonia (UIP)/UIP-like pattern was present in >70% of patients with f-HP, and two-thirds of these patients had an FVC decline of ≥10%. PF-HP patients were also more exacerbation prone. According to recent trial data, this segment of patients can be considered possible candidates for antifibrotic treatment, with a reasonable prospect of effectiveness. Further efforts should focus on refining knowledge of longitudinal behaviour of large multicentric cohorts of f-HP patients, establishing a consensual and uniform definition of progression for use in clinical practice, as well as developing prognostic prediction tools to better (and early) inform the disease course.

A baseline evaluation of PAH body burden in sardines from the southern Brazilian shelf.

The concentrations of 37 polycyclic aromatic hydrocarbons (PAHs) and their potential risk to human health were determined in fifty sardine muscle (Sardinella brasiliensis) samples collected along the southern Brazilian shelf. Parental and alkylated PAHs were identified and quantified using a pressurized liquid extraction with in-cell purification method and gas chromatography-mass spectrometry identification and quantification. The concentrations of Σ37 PAHs in muscle ranged between 6.02 and 4074 µg kg-1 wet weight, which are comparable to levels reported for commercially important fish worldwide. The most abundant compounds were pyrene and fluoranthene, which originate from both petrogenic and pyrolytic hydrocarbon inputs. In only 4% of the samples the benzo[a] pyrene equivalent concentration was above the threshold of 6 µg kg-1 suggested for safe fish consumption in Brazil. These findings will serve as baseline data for monitoring the quality of sardines consumed in the country and for studying fish populations.

Adult rabbits acquire resistance to lethal calicivirus infection by adoptive transfer of sera from infected young rabbits.

Calicivirus infection of adult rabbits induces the so-called rabbit haemorrhagic disease (RHD) that kills 90% or more of the infected animals; in contrast, young rabbits (up to 8-week-old animals) are resistant to the same infectious agent. We report that calicivirus inoculation of young rabbits induced moderate titres of antiviral antibodies. When these rabbits reached adulthood, a second calicivirus inoculation resulted in resistance to RHD and boosting of antibody titres in half of the rabbits. Adoptive transfer of sera from calicivirus-infected young rabbits to naïve adult rabbits conferred resistance to RHD. We conclude that calicivirus infection of young rabbits induces specific anti-calicivirus antibodies that will protect them from RHD when they reach adulthood.

Liver disease in young rabbits infected by calicivirus through nasal and oral routes.

Calicivirus infection causes rabbit haemorrhagic disease (RHD) that kills more than 90% of adult animals, whereas young rabbits are naturally resistant to this viral disease. It has been proposed that the different response of adult and young rabbits to calicivirus infection is due to absence of viral receptors in respiratory and digestive systems of young animals. We have searched for liver disease in 4-week-old rabbits inoculated with a calicivirus suspension by intranasal and oral routes. These young rabbits showed cell damage and mononuclear infiltration of the liver. The hepatic lesions were associated with mild to moderate increase in circulating transaminases. We conclude that the previously reported reduction of viral receptors in the epithelium of respiratory and digestive systems of young rabbits does not inhibit calicivirus from inducing liver disease in these hosts.

Severe leukopenia and liver biochemistry changes in adult rabbits after calicivirus infection.

Calicivirus infection is the major cause of the severe decrease in the stocks of wild and farm rabbits that has occurred worldwide during the last two decades. Adult rabbits (10-weeks-old) were experimentally infected with a calicivirus inoculum that killed all animals by causing rabbit haemorrhagic disease (RHD) within 24-62 h of infection. The rabbits were used to evaluate blood cell numbers and serum biochemistry every 6h, starting 12h after the inoculation of the caliciviruses. No significant changes in blood parameters were observed in most of the rabbits up to 18 h of infection. Severe leukopenia was seen 6h before death of the infected rabbits; both heterophils and lymphocytes contributed to the decrease in circulating white blood cells. Platelets were also severely decreased in number. Marked enhancement in liver enzymes was seen 6-12 h before death of the infected rabbits. There was also evidence both for cholestasis, as expressed by the elevated levels of direct (conjugated) bilirubin, and for hypoglycemia, an alteration that it is likely to contribute for the seizures that rabbits show during the late stages of RHD. Liver ultrastructure of rabbits that died from RHD revealed extensive hepatocyte vacuolization, severe changes in mitochondrial structure, and depletion of glycogen granules. We conclude that: (i) severe leukopenia characterizes the final hours of calicivirus-induced RHD; (ii) hypoglycemia and cholestasis precede death of rabbits from RHD; (iii) the kinetics of liver enzymes allows an accurate prediction of the time of death of rabbits from calicivirus-induced RHD.

Liver enzymes and ultrastructure in rabbit haemorrhagic disease (RHD).

Rabbit haemorrhagic disease (RHD) is caused by a calicivirus infection that kills most adult rabbits 24-72 h after viral inoculation. Two liver enzymes (AST, aspartate aminotransferase, and ALT, alanine aminotransferase) were monitored in blood samples of calicivirus-infected rabbits during the short course of RHD. Values of AST were used to differentiate three stages of hepatocellular degeneration in RHD: mild (up to 20-fold increase in AST), moderate (150-200-fold elevation of AST) and severe (more than 1000-fold elevation in AST). Liver samples of rabbits from these three biochemical stages of hepatocellular degeneration of RHD were studied by transmission electron microscopy to define the fine structure of the hepatocytes. In the mild hepatocellular degeneration there was proliferation (microvesiculation) of the smooth endoplasmic reticulum and swelling of mitochondria into spheroid bodies with loss of cristae. In moderate hepatocellular degeneration, vacuolization of cytoplasm and mitochondrial damage continued to be present, and there was also formation of autophagic vesicles. In the severe hepatocellular degeneration of RHD, the altered mitochondria also showed loss of density of their matrix; rupture of cytoplasmic vacuoles led to the formation of large vesicles. Marked depletion of liver glycogen was also found in this late stage of RHD. These data offer a correlation between biochemical and cytological features of the liver during the hepatocellular degeneration of RHD.

Leukocyte-hepatocyte interaction in calicivirus infection: differences between rabbits that are resistant or susceptible to rabbit haemorrhagic disease (RHD).

Calicivirus infection is lethal for adult rabbits, whereas young rabbits (less than 8-weeks-old) are resistant to the same infectious agent. The virus replicates in the liver and causes a fulminant hepatitis in adult rabbits leading to rabbit haemorrhagic disease (RHD); this is in contrast with the mild and transient hepatitis observed in infected young rabbits. We have used electron microscopy to compare liver leukocyte infiltrates between young (resistant) and adult (susceptible) rabbits, 36-48 h after inoculation of the animals with caliciviruses. In adult rabbits, liver infiltrates were made up mostly of heterophils, and they were located near hepatocytes showing severe cellular damage. In contrast, liver leukocyte infiltrates of RHD-resistant young rabbits were dominated by lymphocytes that depicted membrane contacts with the cell surface of undamaged hepatocytes. We conclude that: (i) the cellular inflammatory response of the liver to calicivirus infection is different in rabbits that are susceptible (adult) or resistant (young) to RHD; (ii) leukocyte infiltration of the adult liver by heterophils is probably directed at the removal of dead hepatocytes, whereas the liver lymphocytic infiltration of young rabbits suggests the expression of viral antigens on the surface of liver cells of the RHD-resistant animals.

Transient decrease in blood heterophils and sustained liver damage caused by calicivirus infection of young rabbits that are naturally resistant to rabbit haemorrhagic disease.

Young rabbits are naturally resistant to rabbit haemorrhagic disease (RHD) caused by the same calicivirus that kills, within 3 days, nearly all adult animals. We have investigated changes in blood leukocytes, and in the morphology and biochemistry of the liver (the organ where caliciviruses replicate) of young rabbits undergoing benign infection by the RHD virus. Four-week-old rabbits were infected with a calicivirus inoculum having a titre of 2(12) haemagglutination units either sacrificed 18, 24, 48 and 72 h later, or kept for follow-up studies up to 21 days after inoculation. The infection caused an acute and transient decrease in blood heterophils, and sustained enhancement in hepatic transaminases. Inflammatory infiltrates of the liver were seen in all animals after 24 h of infection; they had a predominant midlobular location. Hepatocytes could present different degrees of cell damage, including cell death; these lesions were limited to the liver cells located around the inflammatory infiltrates. Liver transaminases peaked 24-48 h after calicivirus infection; this was the same timing when liver infiltration and hepatocyte damage were more evident. No alterations of other parameters of liver biochemistry were observed. We conclude that calicivirus infection of young rabbits causes a subclinical disorder characterised by an acute and transient decrease in circulating heterophils, and focal liver damage that is expressed by intralobular infiltration by heterophils, initially, and, later on, by mononuclear cells. Our finding of persistence of increased values of liver transaminases suggests chronicity of the infection in young rabbits. We propose that, although resistant to RHD, young rabbits infected by calicivirus may be long-term carriers of the infectious agent and, thus, become a major source of transmission of the virus.

Bulging mesothelial cells of the visceral pleura of the rat mimic the network of subpleural lymphatics.

The mesothelial surface of the visceral pleura of the Wistar rat was viewed at high resolution by scanning electron microscopy (SEM). The pleural surface showed exquisite linear arrangements made up of bulging mesothelial cells. They were organized in irregular circles that often presented anastomotic junctures. This arrangement of pleural mesothelial cells mimics the organization of subpleural lymphatics of the lung. A low density of microvilli was seen inside the irregular circles, contrasting with the microvilli-rich mesothelial cells seen on or outside these arrangements. These SEM features of the mesothelium may be related with the formation of microdomains for fluid absorption across the visceral pleura into subpleural lymphatics.

Urinothorax as an unusual type of pleural effusion - clinical report and review.

BACKGROUND: Despite the fact that there are a great number of established etiologies for pleural effusion, there are grounds for believing that there are also causes from unusual pathophysiological mechanisms, seen in certain clinical contexts and from potential iatrogenic interventions. Urinothorax is such a rare type of pleural effusion as there are fewer than 70 cases reported worldwide. CLINICAL CASE: A patient with a persistent left pleural effusion was admitted to the Urology ward for a lithiasic obstructive uropathy with hydronephrosis. A left percutaneous nephrostomy was performed. The effusion was unclassified at the initial workup and recurred after first drainage. A second approach confirmed a citrine fluid with borderline criteria for exudate, ammoniacal odour and an elusive pleural fluid-to-serum creatinine ratio. A retroperitoneal urinoma was recognized on CT, and the patient underwent a left nephrectomy with resolution of the pleural effusion. CONCLUSIONS: Urinothorax most frequently develops in patients with excretory uropathy or blunt abdominal trauma, although other mechanisms have been reported. Traditionally, a pleural fluid to serum creatinine ratio higher than one is a hallmark of this condition. In certain settings, taking this diagnosis into account at an early stage might be crucial for a good outcome.

Early inflammatory response of young rabbits attending natural resistance to calicivirus (RHDV) infection.

Young rabbits (i.e. up to 4 weeks of age) are naturally resistant to infection by rabbit haemorrhagic disease virus (RHDV), the same calicivirus that kills more than 90% of adult rabbits in 3 days or less. To characterize this fascinating model of age-related natural resistance to viral infection, we have studied the kinetics (from 6h up to 7 days) of cytokines and of leukocyte subpopulations in the liver (the target organ for calicivirus replication) and spleen (host systemic response) of RHDV infected young rabbits. Infection was associated with early (6h) elevation of proinflammatory cytokines (TNF-α, IL-1, IFN-α, IFN-γ, IL-6, IL-8). We found that all three major leukocyte subpopulations (macrophages, B and T lymphocytes) were increased in the liver 48h after the RHDV inoculation. At 7 days of infection, B and T lymphocytes were still elevated in the liver of the rabbits. In the spleen, both macrophages and B lymphocytes (but not T cells) were also enhanced. At 7 days, anti-RHDV specific antibodies were present in sera of all young rabbits infected by the virus. We conclude that natural resistance of young rabbits to RHDV infection is associated with a rapid and effective inflammatory response by the liver, with few hepatocytes being infected, and also with a sustained elevation in local and systemic B and T cells.

Partial sequencing of recent Portuguese myxoma virus field isolates exhibits a high degree of genetic stability.

To study genetic changes underlying myxoma virus evolution in its new host, the European rabbit (Oryctolagus cuniculus), we sequenced selected genomic regions of nine recent virulent field strains and a live attenuated vaccine strain ("MAV", Germany). DNA was extracted from cell culture passaged myxoma virus. A total of 4863 bp (approximately 3% of the genome) of 10 regions spanning 12 genes of the myxoma viruses was sequenced and compared to the original virulent strain "Lausanne" and its attenuated field derivative strain "6918". The field strains displayed a maximum of three (strains C43, C95) and a minimum of one (strains CD01, CD05) nucleotide substitutions. These were distributed through all analysed coding regions, except gene M022L (major envelope protein), where all strains were identical to "Lausanne" and "6918". Two new single nucleotide insertions were observed in some of the field strains: within the intergenic region M014L/M015L and within gene M009L, where it leads to a frameshift. These insertions were located after homopolymeric regions. The vaccine strain displayed 37 nucleotide substitutions, predominantly (95%) located in genes M022L and M036L. Interestingly, regions M009L and M014L/M015L of the vaccine were not amplified successfully, suggesting major genomic changes that could account for its attenuated phenotype. Our results support a high degree of genetic stability of myxoma virus over the past five decades. None of the analysed genome regions by its own seems sufficient for the genetic characterisation of field strains.