Anti-TNF-α agents Modulate SARS-CoV-2 Receptors and Increase the Risk of Infection Through Notch-1 Signaling.

Although millions of patients with underlining conditions are treated primarily with anti-TNF-α agents, little is known about the safety of this standard therapy during the coronavirus disease-2019 (COVID-19) pandemic. In this study, we investigated the effect of anti-TNF-α monoclonal antibodies on the cellular entry mechanism of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and increasing the risk of COVID-19 development. We focused on the expression of angiotensin-converting enzyme II (ACE2), type II transmembrane serine proteases (TMPRSS2)/TNF-α converting enzyme (TACE) ratio. We also investigated the involvement of Notch-1 signaling and its downstream influence on IL-6, myeloid cell leukemia sequence-1(MCL-1) in the anti-TNF-α mode of action and increased the susceptibility to Mycobacterium avium subspecies paratuberculosis (MAP) infection. Surprisingly, anti-TNF-α downregulated ACE2 expression by 0.46-fold and increased TMPRSS2/TACE ratio by 44% in THP-1 macrophages. Treatment of macrophages with rIL-6 also downregulated ACE2 and increased TMPRSS2/TACE ratio by 54%. Interestingly, anti-TNF-α treatment upregulated Notch-1, IL-6, and MCL-1 by 1.3, 1.2, and 1.9-fold, respectively, and increased viability and burden of MAP infection in macrophages. Blocking Notch signaling doubled ACE2 expression, decreased TMPRSS2/TACE ratio by 38%, and reduced MAP viability by 56%. In a small group of patients, ACE2 level was significantly lower in the plasma from rheumatoid arthritis (RA) patients on anti-TNF-α treatment compared to healthy control. The data in this critical study demonstrated that through Notch-1/IL-6 signaling, anti-TNF-α agents decreased ACE2 expression and shedding through TMPRSS2/TACE modulation and increased the susceptibility to infection. Overall, this study warns against anti-TNF-α therapy in some patients with underlining inflammatory conditions during the COVID-19 pandemic. The findings should impact current guidelines regarding treatment decisions of patients on anti-TNF-α during the COVID-19 pandemic.

Identification of proteins from tuberculin purified protein derivative (PPD) by LC-MS/MS.

The tuberculin purified protein derivative (PPD) is a widely used diagnostic antigen for tuberculosis, however it is poorly defined. Most mycobacterial proteins are extensively denatured by the procedure employed in its preparation, which explains previous difficulties in identifying constituents from PPD to characterize their behaviour in B- and T-cell reactions. We here described a proteomics-based characterization of PPD from several different sources by LC-MS/MS, which combines the solute separation power of HPLC, with the detection power of a mass spectrometer. The technique is able to identify proteins from complex mixtures of peptide fragments. A total of 171 different proteins were identified among the four PPD samples (two bovine PPD and two avium PPD) from Brazil and UK. The majority of the proteins were cytoplasmic (77.9%) and involved in intermediary metabolism and respiration (24.25%) but there was a preponderance of proteins involved in lipid metabolism. We identified a group of 21 proteins that are present in both bovine PPD but were not detected in avium PPD preparation. In addition, four proteins found in bovine PPD are absent in Mycobacterium bovis BCG vaccine strain. This study provides a better understanding of the tuberculin PPD components leading to the identification of additional antigens useful as reagents for specific diagnosis of tuberculosis.

Humoral response to Mycobacterium avium subsp. avium in naturally infected ring-neck doves (Streptopelia risoria).

Creation of a reliable and easy to use serologic test would greatly improve ante mortem diagnosis of Mycobacterium avium subsp. avium and aid in the control of avian mycobacteriosis, particularly in captive birds. In order to determine whether serodiagnostics could be of value in testing ring-neck doves (Streptopelia risoria) for M. a. avium infection, Western blot analysis was used to assess the humoral response of ring-neck doves exposed to M. a. avium, and to evaluate whether an association could be made between the humoral response and necropsy findings, histopathology, culture, and PCR testing. Western blot results were examined for reactivity patterns associating humoral response with infection status, severity and type of lesions (diffuse vs. multifocal granulomatous inflammation) and phenotype (white vs. non-white). A sensitivity of 88.24% and a specificity of 100% were achieved utilizing Western blot analysis to detect M. a. avium infection in ring-neck doves, offering a negative predictive value of 93% and a positive predictive value of 100%. While Western blot analysis results did not reflect lesion severity, lesion type did partially correspond with the humoral response. The findings of the present study indicate that serologic testing can be used as a valuable ante mortem screening tool for identifying ring-neck doves infected with M. a. avium.

Effect of different cell fractions of Mycobacterium avium and vaccination regimens on Mycobacterium avium infection.

Because of the availability of uniform genetic stocks and the ability to modulate stress levels, chickens were investigated as a host for the development of an antimycobacterial vaccine. The imposition and the timing of stress significantly influenced the outcome of Mycobacterium avium infection in chickens. Simple, whole cell or lysate vaccines and combinations of vaccine preparations were identified that led to high levels of protection. In addition, short-term stress at the time of vaccination significantly increased the protective efficacy of M. avium vaccine preparations. Post-infection vaccination of M. avium-infected chickens was also shown to significantly reduce the number of lesions and colony counts.

T-cell responses to Mycobacterium avium PPD antigens in gastro-intestinal helminth co-infected chickens in Central Ethiopia.

A cross-sectional study was conducted on extensively reared chickens of three selected agro-climatic zones in Central Ethiopia to examine the predisposing effect of gastro-intestinal helminthes to intestinal Mycobacterium avium when it occurs as co-infection. This was done through a Lymphocyte Stimulation Test (LST) using avian PPD on peripheral blood mononuclear cells obtained from the blood of chickens and gross examination of digestive tract for the presence of helminth parasites. Data were analyzed using the statistical softwares SAS (1994) and Intercooled STATA version 6. Fourteen (14.7%) out of the 95 examined chickens were positive in in vitro LST showing stimulation index (SI) > or = 2. There was a significant (chi2 = 9.93, P < 0.01) difference in prevalence of M. avium by altitude: highest in chickens from lowland (27.8%) areas, followed by 13.3% in chickens from mid altitude and none was reacted to LST from highland region. A significant relationship (chi2 = 9.58, P < 0.01) in cestode co-infection with M. avium was found. There was no significant (chi2 = 1.66, P > 0.05) relationship in nematode co-infection with M. avium.

Avian immune responses to Mycobacterium avium: the wildfowl example.

Immunological responses of wildfowl (Order Anseriformes: ducks, geese, swans and screamers) to mycobacteria have been investigated as part of studies to develop a vaccine and diagnostic assay for avian tuberculosis. 10(9) killed Mycobacterium vaccae protected the Cairinini (perching ducks) from avian tuberculosis (p<0.02) but did not achieve statistically significant protection in the other taxonomic tribes. The Cairinini includes the threatened, yet highly susceptible, white-winged duck (Cairina scutulata).Together, loss of cell-mediated responses to common mycobacterial antigens, increased responsiveness to the species specific antigens of M. avium, and increased antibody production are reminiscent of the T(H1) to T(H2) shift seen in mammalian mycobacterial infections. It is speculated that excessive exposure to environmental mycobacteria prior to vaccination is detrimental and common antigens play an important role in wildfowl immunity to mycobacteria. A new vaccination trial using killed M. vaccae is being undertaken. Antibody responses are a useful ante mortem diagnostic indicator in most taxonomic tribes with the exception of the primitive Dendrocygnini (whistling ducks).

[Comparison of the pathogenesis and epizootiologic importance of avian mycobacteriosis in various types of domestic and free-living syntropic birds]. / Srovnání patogeneze a epizootologického významu aviární mykobakteriózy ruzných druhu domestikovaných a volne zijících synantropních ptáku.

The sensitivity of domestic and different sorts of synanthropic free living fowl to M. avium and pathogenesis of tuberculous process were observed by experimental infections. The partridge, domestic fowl, pheasant and sparrow were the most sensitive to intramuscular infection by suspension of M. avium. The sparrow, domestic fowl, laughing gull were the most sensitive to peroral infection by food contaminated by tbc poultry liver. After free contact between the poultry, the domestic fowl was the most sensitive to infection and then the sparrow, turkey, guinea fowl, pheasant and partridge were sensitive, too. The tuberculous lesions were observed in majority of infected fowls in cases of guinea fowl, turkey, laughing gull, domestic fowl, sparrow, partridge and pheasant after all types of infections. The pigeon, rook and turtle-dove were very resistant to M. avium infection. The largest quantities of mycobacteria in tissues of infected fowl were found in sparrow and guinea fowl and then in domestic fowl, pheasant and partridge. From the results of patho-morphological and culture examinations, we can distribute the experimentally infected fowl in four groups: very sensitive-domestic fowl, sparrow, pheasant, partridge, laughing gull; little sensitive-guinea fowl, turkey; resistant-goose, duck; very resistant-pigeon, turtle-dove, rook. It is necessary to know that the sensitivity of these sorts of fowl can be affectable by conditions of experimental infection. If we want to criticize the epizootological importance of individual sorts of fowl as a source of M. avium, we must take into account their sensitivity to M. avium, pathogenesis of tuberculous process and their breeding or zoological specificities of individual sorts.

Susceptibility of captive wildfowl to avian tuberculosis: the importance of genetic and environmental factors.

This study reports the findings of an epidemiological survey of death due to avian tuberculosis in the captive collection of wildfowl at The Wildfowl and Wetlands Trust Centre, Slimbridge, Gloucestershire. Both genetic and environmental factors have been shown to affect the incidence of, and the birds' susceptibility to, the disease. Seasonal body condition was related to the occurrence of death due to the disease in both males and females. Birds from either hot or cold climates appeared to have a higher incidence than those from temperate climates. What the birds ate did not affect incidence but the method they used for obtaining their food did. Higher susceptibility was found in those species evolved for marine or arboreal habitats. Anomalies in susceptibility which suggest a higher level of genetic immunity in some groups have also been found. Reasons are put forward to explain these findings.

Effect of environmental-genetic interactions on Mycobacterium avium challenge infection.

Chickens from lines selectively bred for either a high-antibody (HA) or low-antibody (LA) response to sheep erythrocytes were injected intravenously with Mycobacterium avium while being held in low, medium, or high levels of social stress for 5 days (first environment). During the remaining 6 weeks, they were held under either low or medium levels of social stress (second environment). Infection led to lesions consisting of granulomas, some of which had necrotic centers. There was a positive correlation between numbers of lesions with necrotic centers and M. avium cells recovered from livers. The numbers and nature of lesions were influenced by both genetic and environmental factors. Numbers of necrotizing lesions increased with stressfulness of the first environment. Total numbers of lesions were reduced by the medium-stress second environment, and the total number of necrotizing lesions was reduced among LA chickens in the low-stress second environment.

[Biological and allergenic properties of various strains of Mycobacterium avium and atypical Mycobacterium isolated from swine]. / Badania nad wlasciwosciami biologicznymi i alergennymi niektórych pratków ptasich i atypowych wydzielonych ze swin.

Thirty two strains of Myc. avium showing typical or atypical cultural and biochemical properties, and 8 strains of atypical bacilli, belonging to II, III and IV Runyon's groups, were inoculated into guinea-pigs, rabbits and chickens. All the Myc. avium strains were not pathogenic for guinea-pigs but were very virulent to rabbits and chickens. The atypical bacilli were not pathogenic for all the animals to be used, but some of them induced tuberculin allergy. Myc. avium, Myc. fortuitum, strain of scotochromogenic bacilli--all isolated from pigs and Myc. avium D4, were used for study of the sensitizing properties. The experiments were performed on albinotic guinea-pigs sensitized intramuscularly and young pigs sensitized per os. The results showed that avian bacilli possessed much stronger sensitizing properties than Myc. fortuitum or scotochromogenic bacilli. Tuberculin allergy caused by atypical bacilli usually lasts for a short time. However, infection of pigs with atypical bacilli can sometimes allergical diagnosis of swine tuberculosis difficult.

Effect of repeated injections of iron dextran on the haematological, serological and pathological changes in experimental avian tuberculosis.

Eight intramuscular injections of 200 mg/kg of iron (DFe), given as iron dextran twice weekly in the week before and the three weeks after intravenous infection with about 10(7.5) colony-forming units of Mycobacterium avium, significantly prolonged (by about 11 days) the mean 'time-to-death' of immature male fowl (Gallus domesticus) compared with corresponding regimes using dextran (Dx) only or saline, When a proportion of the birds were examined 21 days after infection many of the abnormalities associated with the disease, including a marked hypochromic anaemia, were less severe in DFe-treated than in the Dx- or saline-treated chicks and there were about 10- to 85-fold fewer viable tubercle bacilli in the liver and spleen of the DFe-treated birds.

Specific detection of mycobacteria by the immunofluorescent technique.

Using an indirect immunofluorescent technique, M. bovis var. BCG can be differentiated from M. avium in pure culture. The test involves using an unlabeled antiserum prepared against B-24, a type specific antigen of M. bovis var. BCG. In the same system M. bovis could be differentiated from other pathogenic my cobacteria in tissue sections of naturally and experimentally infected animals.

Experimentally induced Mycobacterium avium serotype 8 infection in swine.

Five of 6 swine experimentally inoculated with Mycobacterium avium serotype 8 had microgranulomas in the cervical or mesenteric lymph nodes at necropsy 92 days later. In vivo tuberculin skin reactivity and in vitro lymphocyte immunostimulation responses were evaluated at 10 and 12 weeks after the pigs were inoculated. Positive responses were obtained on both tests in inoculated pigs, whereas test results in noninoculated pigs and pigs given killed bacterial cells were negative. Mycobacterium avium serotype 8 was isolated at necropsy from the cervical or mesenteric lymph nodes of each of the pigs inoculated with viable microorganisms and from the 2 pigs kept in the pen with inoculated swine. Mycobacteria were not isolated from tissues of the noninoculated swine or those given killed cells.

The specificity of the fluorescent antibody test using the sera of rabbits inoculated with strains of myocobacteria.

Sera from experimentally infected rabbits were used to test the specificity of the fluorescent antibody test. It was possible using mono-specific sera to differentiate antigenically Mycobacterium phlei, M fortuitum, M smegmatis, M avium, M intracellulare, M bovis (BCG) and M johnei. The cross-reactivity within the M avium and M intracellulare group was such that one antigen from these groups would detect infection within that group and exclude M johnei infection. The M phlei growth factor independent strain M johnei 316F was shown to be antigenically distinct from a M phlei dependent strain 9N96. There was loss of specificity when M avium infection was superimposed on a previous M johnei infection and when M johnei infection was superimposed on M avium infection.

The specificity and sensitivity of the fluorescent antibody test in cattle experimentally infected with Mycobacterium avium and Mycobacterium johnei.

In five calves experimentally infected with Mycobacterium avium and in 10 with M johnei it was shown that the serological response by the fluorescent antibody (FA) test was specific. The serological response in the M avium infected calves was transitory and lasted up to five months. The first evidence of a serological response with the FA test in the M johnei dosed calves occurred at four months, and all calves had reacted by nine months after dosing. At the 23 tests carried out between four and 32 months after dosing, when the experiment was terminated, an average of 5-8 animals was positive at any one test. Johne's disease was confirmed bacteriologically and histologically in six of the 10 calves though none had shown clinical signs.