[Impact of days elapsed from the onset of symptoms to hospitalization in COVID-19 in-hospital mortality: Time matters]. / Impacto de los días transcurridos desde el inicio de los síntomas hasta la hospitalización en la mortalidad hospitalaria por COVID-19: el tiempo importa.

Background: COVID-19 shows different clinical and pathophysiological stages over time. Theeffect of days elapsed from the onset of symptoms (DEOS) to hospitalization on COVID-19prognostic factors remains uncertain. We analyzed the impact on mortality of DEOS to hospital-ization and how other independent prognostic factors perform when taking this time elapsedinto account. Methods: This retrospective, nationwide cohort study, included patients with confirmed COVID-19 from February 20th and May 6th, 2020. The data was collected in a standardized online datacapture registry. Univariate and multivariate COX-regression were performed in the generalcohort and the final multivariate model was subjected to a sensitivity analysis in an earlypresenting (EP; < 5 DEOS) and late presenting (LP; ≥5 DEOS) group. Results: 7915 COVID-19 patients were included in the analysis, 2324 in the EP and 5591 in theLP group. DEOS to hospitalization was an independent prognostic factor of in-hospital mortalityin the multivariate Cox regression model along with other 9 variables. Each DEOS incrementaccounted for a 4.3% mortality risk reduction (HR 0.957; 95% CI 0.93---0.98). Regarding variationsin other mortality predictors in the sensitivity analysis, the Charlson Comorbidity Index onlyremained significant in the EP group while D-dimer only remained significant in the LP group. Conclusion: When caring for COVID-19 patients, DEOS to hospitalization should be consideredas their need for early hospitalization confers a higher risk of mortality. Different prognosticfactors vary over time and should be studied within a fixed timeframe of the disease.

Impact of days elapsed from the onset of symptoms to hospitalization in COVID-19 in-hospital mortality: time matters.

BACKGROUND: COVID-19 shows different clinical and pathophysiological stages over time. The effect of days elapsed from the onset of symptoms (DEOS) to hospitalization on COVID-19 prognostic factors remains uncertain. We analyzed the impact on mortality of DEOS to hospitalization and how other independent prognostic factors perform when taking this time elapsed into account. METHODS: This retrospective, nationwide cohort study, included patients with confirmed COVID-19 from February 20th and May 6th, 2020. The data was collected in a standardized online data capture registry. Univariate and multivariate COX-regression were performed in the general cohort and the final multivariate model was subjected to a sensitivity analysis in an early presenting (EP; <5 DEOS) and late presenting (LP; ≥5 DEOS) group. RESULTS: 7915 COVID-19 patients were included in the analysis, 2324 in the EP and 5591 in the LP group. DEOS to hospitalization was an independent prognostic factor of in-hospital mortality in the multivariate Cox regression model along with other 9 variables. Each DEOS increment accounted for a 4.3% mortality risk reduction (HR 0.957; 95% CI 0.93-0.98). Regarding variations in other mortality predictors in the sensitivity analysis, the Charlson Comorbidity Index only remained significant in the EP group while D-dimer only remained significant in the LP group. CONCLUSION: When caring for COVID-19 patients, DEOS to hospitalization should be considered as their need for early hospitalization confers a higher risk of mortality. Different prognostic factors vary over time and should be studied within a fixed timeframe of the disease.

The Mycobacterium tuberculosis sigma factor sigmaB is required for full response to cell envelope stress and hypoxia in vitro, but it is dispensable for in vivo growth.

The numerous sigma (sigma) factors present in Mycobacterium tuberculosis are indicative of the adaptability of this pathogen to different environmental conditions. In this report, we describe the M. tuberculosis sigma(B) regulon and the phenotypes of an M. tuberculosis sigB mutant strain exposed to cell envelope stress, oxidative stress, and hypoxia. The sigB mutant was especially defective in survival under hypoxic conditions in vitro, but it was not attenuated for growth in THP-1 cells or during mouse and guinea pig infection.

Antibodies to streptococcal surface enolase react with human alpha-enolase: implications in poststreptococcal sequelae.

The pathogenic mechanisms for developing acute rheumatic fever after group A streptococcal pharyngitis are still poorly understood. The glycolytic enzyme enolase is one of the major proteins on the surface of group A streptococci. Herein, significant cross-reactivity was shown between streptococcal enolase and human enolase. Fluorocytometric analysis revealed that antistreptococcal enolase antibodies react with the enolase expressed on the surface of hematopoietic cells. Furthermore, the enolase on the leukocyte surface was found to be up-regulated by inflammatory stimuli. Evaluation of antibody titers indicated that serum samples from patients with acute rheumatic fever have higher levels of antibodies that react with the human and bacterial enolases than do serum samples from patients with streptococcal pharyngitis or healthy control subjects. These results show that streptococcal enolase is a novel cross-reactive antigen that may play an important role in the initiation of the autoimmune diseases related to streptococcal infection.

Contribution of novel choline-binding proteins to adherence, colonization and immunogenicity of Streptococcus pneumoniae.

The surface of Streptococcus pneumoniae is decorated with a family of choline-binding proteins (CBPs) that are non-covalently bound to the phosphorylcholine of the teichoic acid. Two examples (PspA, a protective antigen, and LytA, the major autolysin) have been well characterized. We identified additional CPBs and characterized a new CBP, CbpA, as an adhesin and a determinant of virulence. Using choline immobilized on a solid matrix, a mixture of proteins from a pspA-deficient strain of pneumococcus was eluted in a choline-dependent fashion. Antisera to these proteins passively protected mice challenged in the peritoneum with a lethal dose of pneumococci. The predominant component of this mixture, CbpA, is a 75-kDa surface-exposed protein that reacts with human convalescent antisera. The deduced sequence from the corresponding gene showed a chimeric architecture with a unique N-terminal region and a C-terminal domain consisting of 10 repeated choline-binding domains nearly identical to PspA. A cbpA-deficient mutant showed a >50% reduction in adherence to cytokine-activated human cells and failed to bind to immobilized sialic acid or lacto-N-neotetraose, known pneumococcal ligands on eukaryotic cells. Carriage of this mutant in an animal model of nasopharyngeal colonization was reduced 100-fold. There was no difference between the parent strain and this mutant in an intraperitoneal model of sepsis. These data for CbpA extend the important functions of the CBP family to bacterial adherence and identify a pneumococcal vaccine candidate.

Neutrophil apoptosis induced by proteolytic enzymes.

In this study, we show that three proteolytic enzymes of different specificity-pronase, chymotrypsin, and trypsin-induced a dramatic stimulation of neutrophil apoptosis as shown by morphologic characteristics, analysis of cell DNA content, and presence of a characteristic "ladder" pattern of DNA fragmentation. The action of either chymotrypsin or trypsin was completely prevented by the serine protease inhibitor aprotinin, indicating that the proteolytic activity of the enzymes accounts for apoptosis induction. Stimulation of neutrophil apoptosis by proteases was observed in culture medium supplemented with either inactivated fetal calf serum (0.1-50%), autologous serum (0.1-50%), bovine serum albumin (0.1%), or in protein-free medium. Other cell types such as human peripheral blood monocytes and lymphocytes, human leukemic cells from THP-1, HL-60 and K562 lines, murine L929 fibroblasts, and unstimulated murine macrophages harvested from the peritoneal cavity were not induced to undergo apoptosis after the treatment with proteases. In an attempt to determine whether neutrophil serine proteases could induce apoptosis as chymotrypsin and trypsin do, the effect of elastase was assessed. A significant increase in the percentage of apoptotic cells was observed in elastase-treated neutrophils. We propose that the selective stimulation of neutrophil apoptosis by proteolytic enzymes may play an important role in the normal resolution of inflammation by limiting the autotoxic potential of the neutrophil.

Haemophilus influenzae type b exoproducts induce chemotaxis and macrolide antibiotic release by human polymorphonuclear leukocytes.

The capacity of phagocytes to concentrate macrolide antibiotics was suggested by previous reports. In this study, we evaluated the capacity of Haemophilus influenzae type b culture supernatants (HICS) to induce polymorphonuclear leukocyte (PMNL) migration and macrolide antibiotic delivery. Using a Boyden multiwell chamber and a chemotaxis assay under agarose combined with a bioassay to measure antibiotic levels in agar, we demonstrated the chemotactic activity of HICS. Preincubation of PMNL with either erythromycin or azithromycin did not affect PMNL chemotaxis. By the agar diffusion test, we established that HICS increased the release of antibiotic from PMNL when compared with spontaneous release. Furthermore, we determined that the antibiotics remain bioactive after release. These results suggest that HICS may have a modulatory effect on transport and delivery of macrolide antibiotics by PMNL at the infection site.

Tumor necrosis factor alpha plus interleukin 1 beta treatment protects granulocytopenic mice from Pseudomonas aeruginosa lung infection: role of an unusual inflammatory response.

We have recently demonstrated that treatment with interleukin 1 beta (IL-1 beta) plus tumor necrosis factor alpha (TNF alpha) protects granulocytopenic hosts from Pseudomonas aeruginosa aerosol challenge. In this study we characterized the inflammatory response induced by P. aerugionsa in granulocytopenic mice treated with 2,000 U IL-1 beta plus 2,000 U TNF alpha. Treatment with the nonsteroidal anti-inflammatory agent piroxicam abolished both the protective effect of cytokine treatment and the increase in myeloperoxidase (MPO) pulmonary activity. Histopathological studies revealed that, after aerosol challenge with P. aeruginosa, treatment with these cytokines induced migration and extravasation of mononuclear cells of immature appearance into the lung parenchyma. These cells contained MPO in their cytoplasm and displayed phagocytic capacity. Resident alveolar macrophages exhibited signs of activation and appeared in reduced numbers in bronchoalveolar lavage fluid. We suggest that the inflammatory response promoted by low TNF alpha plus IL-1 beta doses may be one mechanism responsible for protection of granulocytopenic hosts from P. aeruginosa aerosol challenge.

Neutrophil chemotaxis induced by immune complexes.

In the current work we have analyzed the ability of different soluble immune complexes (IC) prepared with IgG antibodies to induce neutrophil chemotactic responses in vitro. While, in all cases, IC were able to induce neutrophil migration in a concentration-dependent fashion, IgG antibodies alone were completely unable to induce locomotor responses. Checkerboard analysis indicated the chemotactic nature of motility. On the other hand, chemotaxis induced by IC was markedly inhibited by IV. 3, a monoclonal antibody (mAb) to Fc gamma RII, slightly reduced by 3G8 F(ab')2, a mAb to Fc gamma RIII, and nearly abrogated by both mAbs. The impact of IC on neutrophil migration induced by FMLP was also studied. We found that when a suboptimal concentration of FMLP was employed, the simultaneous addition of IC increased the migration acting in additive form. The significance of these results is discussed.

Modulation of human polymorphonuclear leukocyte chemotaxis and superoxide anion production by Pseudomonas aeruginosa exoproducts, IL-1 beta and piroxicam.

Whereas addition of 200 ng ml-1 exotoxin A (exoA) did not modify PMNL chemotaxis, 20 U ml-1 human recombinant interleukin-1 beta (hrIL-1 beta) primed polymorphonuclear leukocytes (PMNL) for migration towards Pseudomonas aeruginosa peptide chemotactins (PAPCs). Piroxicam (100 micrograms ml-1), a non-steroidal anti-inflammatory agent (NSAIA), inhibited PMNL chemotaxis and abolished the priming effect of hrIL-1 beta. Both PAPCs and exoA induced PMNL superoxide anion production, but neither hrIL-1 beta nor piroxicam modified significantly PMNL superoxide anion production induced by PAPCs. The fact that hrIL-1 beta can prime PMNL for chemotaxis towards PAPCs and that piroxicam can abolish activation by primed PMNL are findings relevant to the pharmacological control of lung tissue damage during P. aeruginosa pneumonia.

The effect of treatment with interleukin-1 and tumor necrosis factor on Pseudomonas aeruginosa lung infection in a granulocytopenic mouse model.

The efficacy of treatment with interleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF alpha) on Pseudomonas aeruginosa pneumonia was evaluated in a granulocytopenic mouse model. Combined intravenous administration of 2000 U IL-1 beta plus 2000 U TNF alpha significantly diminished mortality from aerosol challenge with P. aeruginosa. Mice treated with IL-1 beta, TNF alpha, or both also exhibited a significant enhancement in pulmonary clearance of P. aeruginosa. Combined cytokine administration induced an increase in the pulmonary content of myeloperoxidase activity. Mature leukocytes were not detected in either circulation or bronchoalveolar lavage fluid from granulocytopenic, cytokine-treated mice. In conclusion, IL-1 beta and TNF alpha treatment exhibited a synergistic protective effect from pulmonary P. aeruginosa challenge in granulocytopenic hosts, probably due to enhancement of nonspecific antibacterial mechanisms.

The effect of a cyclooxygenase inhibitor on the in vivo polymorphonuclear leukocyte migration to Pseudomonas aeruginosa peptide chemotactins.

Pseudomonas aeruginosa chemotactic peptides (PAPCs) induced migration of polymorphonuclear leukocytes (PMNL) into the lungs when administered by the aerosol route. Migration of PMNL into the lungs and total protein content of lung lavage fluids in response to PAPCs aerosol challenge, and mortality from lethal challenge with P. aeruginosa were decreased by piroxicam treatment.

Piroxicam-copper complexes: inhibition of polymorphonuclear leukocyte migration to Pseudomonas aeruginosa chemotactins in vivo and superoxide dismutase-like activity in vitro.

Piroxicam-copper (Cu2+) complexes, formed spontaneously by mixing solutions of piroxicam and CuSO4 (1:1 Cu2+:piroxicam), inhibited the superoxide anion-catalyzed reduction of ferricytochrome C in a dose-related fashion. Addition of ethylenediaminetetraacetate to the mixture decreased in a dose-related manner the superoxide dismutase (SOD)-like activity of piroxicam-Cu2+. Piroxicam alone (10(-5) M, final concentration) did not display SOD-like activity but 10(-5) M Cu2+ exhibited significant activity, similar to that of piroxicam-Cu2+. Intraperitoneal treatment of mice with either 0.64 mg/kg piroxicam or its Cu2+ complexes (0.64 mg/kg piroxicam + 0.12 mg/kg Cu2+) was equally effective in diminishing both the migration of polymorphonuclear leukocytes (PMNL) to the airways and the content of myeloperoxidase activity in the lungs, induced by aerosol challenge with Pseudomonas aeruginosa peptide chemotactins. Therefore, piroxicam-Cu2+ complexes may provide both the anti-inflammatory activity of piroxicam plus the SOD-like activity of Cu2+.

Treatment with a non-steroidal anti-inflammatory agent delays the growth of spontaneous pulmonary metastases of a mammary adenocarcinoma of non-detected immunogenicity.

Previous reports showed that treatment with non-steroidal anti-inflammatory agents (NSAIA) can alter the growth profile of a variety of tumours. In this study, the effect of NSAIA treatment on the growth of the primary tumour and the appearance of spontaneous pulmonary metastases, was investigated. A mammary adenocarcinoma of non-detected immunogenicity, C7HI, was grafted subcutaneously in the lateral flank of Balb/c mice. Oral treatment with approximately 1 mg kg-1 day-1 piroxicam delayed both tumour growth and the growth of pulmonary metastases. Survival of mice bearing the primary tumour was significantly lengthened by anti-inflammatory treatment. Similarly, in separate experiments, after surgical removal of the primary tumour by day 34 after grafting, the group of mice treated orally with piroxicam also exhibited a higher survival rate than the control group. Upon surgical removal of the primary tumour 34 days after grafting, piroxicam treatment significantly decreased both the number and size of pulmonary metastases. The results of this study lends support to the hypothesis that inhibition or modulation of inflammation may delay tumour organisation and growth. It is suggested that piroxicam treatment may be an appropriate adjunct therapy to delay the appearance of pulmonary metastases and to increase life-expectancy in a host whose primary tumour has to be surgically removed.

Preliminary characterization of Pseudomonas aeruginosa peptide chemotactins for polymorphonuclear leukocytes.

In a previous report, we showed that supernatants of Pseudomonas aeruginosa cultures exhibit chemotactic activity for polymorphonuclear leukocytes (PMNL). In this study, P. aeruginosa chemotactins were isolated, purified, and partially characterized. The organisms were cultured in Vogel-Bonner defined medium, and cultures were stopped in late log phase. Chemotactins withstood heating, remained unaltered after acid or alkali treatment in a pH range from 4 to 10, and resisted digestion by trypsin or carboxypeptidase, but chemotactic activity was decreased by 73% after incubation with pronase. Only 2% of the total chemotactic activity of culture supernatants could be extracted with chloroform. Chemotactins with molecular sizes less than 3 kDa constituted the largest contribution to the chemotactic activity of culture supernatants. Pretreatment of PMNL with 10(-5) M formylmethionyl-leucyl-phenylalanine (FMLP) inhibited chemotaxis towards FMLP and P. aeruginosa culture supernatants but not towards complement component C5a. In conclusion, the total chemotactic activity for PMNL of P. aeruginosa culture supernatants was due, almost exclusively, to chemotactins that have properties similar, if not identical, to those exhibited by formylmethionyl peptides.

Age-dependent pulmonary clearance of Pseudomonas aeruginosa in a mouse model: diminished migration of polymorphonuclear leukocytes to N-formyl-methionyl-leucyl-phenylalanine.

Ten-, 20-, and 35-day-old mice were subjected to an aerosol containing Pseudomonas aeruginosa. The lung clearance of the organism was decreased in mice under 20 days of age. This deficiency was accompanied by decreased migration of polymorphonuclear leukocytes (PMN) to the airways in response to the P. aeruginosa challenge. Similar results were obtained in both outbred, C5-sufficient Swiss mice and inbred, C5-deficient DBA/2 mice. The diminished clearance of P. aeruginosa was related to a transient, age-related decrease in PMN chemotaxis to formyl-methionyl oligopeptides. PMN chemotaxis levels similar to those seen in adults were regained by day 35 after birth.

Neutrophil erythrotoxicity induced by phorbol myristate acetate: mechanisms involved in neutrophil activation.

The capacity of phorbol myristate acetate (PMA) to prime neutrophil cytotoxic responses induced by a second stimulus was investigated. Treatment of neutrophils with low concentrations of PMA (0.2-0.5 ng/ml) for 18 hr at 37 degrees C markedly enhanced cytotoxicity triggered by Ca2+ ionophore A23187, N-formyl-methionyl-leucyl-phenylalanine (FMLP) and PMA. Pretreatment with PMA also enabled neutrophils to mediate significant cytotoxicity when triggered by platelet-activating factor (PAF), a stimulus unable to induce untreated cells to display cytotoxicity. Conversely, neutrophil cytotoxicity triggered by immune complexes (IC) was not modified by PMA treatment, whereas cytolytic activity of neutrophils against antibody-sensitized target cells was significantly increased. Treatment with PMA concentrations higher than 1 ng/ml directly triggered neutrophil cytotoxicity. Interestingly, we found that PMA-triggered neutrophils were able to sustain maximal levels of cytotoxicity for at least 8 hr after stimulation. With regard to the mechanisms involved in neutrophil activation by PMA, we found that catalase but not superoxide dismutase (SOD) prevented neutrophil activation measured as 1) induction of cytotoxic responses, 2) increase of neutrophil adhesiveness to cell-free surfaces, and 3) inhibition of chemotactic responses to FMLP. These findings suggest that H2O2 may play a major role in neutrophil activation induced by PMA.

Anti-inflammation induced by counter-irritation or by treatment with non-steroidal agents inhibits the growth of a tumour of non-detected immunogenicity.

Counter-irritation (CI) triggered by different non-specific irritant stimuli delayed the growth of a murine tumour of non-detected immunogenicity. The syngeneic LB tumour transplant by itself also induced CI and decreased the number of leukocytes migrating to a secondary s.c. irritant stimulus, e.g. sponge or carrageenan. On the other hand, partial inhibition of cell migration by treatment with either 0.5 mg kg-1 indomethacin or 0.3 mg kg-1 piroxicam retarded LB tumour growth, presumably by a mechanism unrelated to inhibition of immune responses by PGE2. It is suggested that CI may play a role in the early stages of concomitant resistance.