Pan­cancer analysis of transmembrane protease serine 2 and cathepsin L that mediate cellular SARS­CoV­2 infection leading to COVID-19.

Severe acute respiratory syndrome (SARS) coronavirus­2 (SARS­CoV2) is the cause of a new disease (COVID­19) which has evolved into a pandemic during the first half of 2020. Older age, male sex and certain underlying diseases, including cancer, appear to significantly increase the risk for severe COVID­19. SARS­CoV­2 infection of host cells is facilitated by the angiotensin­converting enzyme 2 (ACE­2), and by transmembrane protease serine 2 (TMPRSS2) and other host cell proteases such as cathepsin L (CTSL). With the exception of ACE­2, a systematic analysis of these two other SARS­CoV2 infection mediators in malignancies is lacking. Here, we analysed genetic alteration, RNA expression, and DNA methylation of TMPRSS2 and CTSL across a wide spectrum of tumors and controls. TMPRSS2 was overexpressed in cervical squamous cell carcinoma and endocervical adenocarcinoma, colon adenocarcinoma, prostate adenocarcinoma (PRAD), rectum adenocarcinoma (READ), uterine corpus endometrial carcinoma and uterine carcinosarcoma, with PRAD and READ exhibiting the highest expression of all cancers. CTSL was upregulated in lymphoid neoplasm diffuse large B­cell lymphoma, oesophageal carcinoma, glioblastoma multiforme, head and neck squamous cell carcinoma, lower grade glioma, pancreatic adenocarcinoma, skin cutaneous melanoma, stomach adenocarcinoma, and thymoma. Hypo­methylation of both genes was evident in most cases where they have been highly upregulated. We have expanded on our observations by including data relating to mutations and copy number alterations at pan­cancer level. The novel hypotheses that are stemming out of these data need to be further investigated and validated in large clinical studies.

Intoxication of host cells by the T3SS phospholipase ExoU: PI(4,5)P2-associated, cytoskeletal collapse and late phase membrane blebbing.

Pseudomonas aeruginosa is an opportunistic pathogen that is associated with hospital-acquired infections, ventilator-associated pneumonia, and morbidity of immunocompromised individuals. A subpopulation of P. aeruginosa encodes a protein, ExoU, which exhibits acute cytotoxicity. Toxicity is directly related to the phospholipase A2 activity of the protein after injection into the host cytoplasm via a type III secretion system. ExoU enzymatic activity requires eukaryotic cofactors, ubiquitin or ubiquitin-modified proteins. When administered extracellularly, ExoU is unable to intoxicate epithelial cells in culture, even in the presence of the cofactor. Injection or transfection of ExoU is necessary to observe the acute cytotoxic response. Biochemical approaches indicate that ExoU possesses high affinity to a multifunctional phosphoinositide, phosphatidylinositol 4,5-bisphosphate or PI(4,5)P2 and that it is capable of utilizing this phospholipid as a substrate. In eukaryotic cells, PI(4,5)P2 is mainly located in the cytoplasmic side of the plasma membrane and anchors adaptor proteins that are involved in cytoskeletal structures, focal adhesions, and plasma membranes. Time-lapse fluorescent microscopy analyses of infected live cells demonstrate that ExoU intoxication correlates with intracellular damage in the early phases of infection, such as disruption of focal adhesions, cytoskeletal collapse, actin depolymerization, and cell rounding. At later time points, a membrane blebbing phenotype was prominent prior to the loss of the plasma membrane integrity and barrier function. Membrane blebbing appears to accelerate membrane rupture and the release of intracellular markers. Our data suggest that in eukaryotic host cells, intracellular ExoU targets and hydrolyzes PI(4,5)P2 on the plasma membrane, causing a subsequent disruption of cellular structures and membrane integrity.

A review of molecular modelling studies of dihydrofolate reductase inhibitors against opportunistic microorganisms and comprehensive evaluation of new models.

Dihydrofolate reductase (DHFR) has been used as a target for antimicrobial drug discovery against a variety of pathogenic microorganisms, including opportunistic microorganisms; Pneumocystis carinii (pc), Toxoplasma gondii (tg) and Mycobacterium avium complex (ma). In this regard, several DHFR inhibitors are reported against pc and tg and ma. However, selectivity issue of these inhibitors over human DHFR often preclude their development and clinical use. In the first part of this work, various computational approaches including available crystallographic structures, binding affinity prediction, pharmacophore mapping, QSAR, homology modelling used for design of DHFR inhibitors against opportunistic microorganisms are reviewed, to understand specific interactions required for inhibition of microbial DHFR. Secondly, comprehensive molecular modelling techniques were used, to establish structure-chemical-feature-based pharmacophore models for pcDHFR, tgDHFR and mammalian DHFR. The results show that, the information encoded by ligand based approaches like pharmacophore mapping and 3D-QSAR methods are in well agreement with the information coded in the receptor structure. A combination of ligand and structure based approaches provides understanding of ligand-receptor interactions. The study indicated that the value of small alkyl moieties at position 5 of the bicyclic nitrogen containing nucleus along with a bulky group attached at the C-6 via suitable linker could optimize activity, with regard to both potency and selectivity.

[The etiological pattern of diseases in pregnant women with enhanced blood ALT and AST activities, admitted to the obstetric unit of infectious disease hospital].

AIM: to define a role of hepatotropic (HAV, HBV, HCV, and HDV) and opportunistic hepatotropic (HGV, CMV, EBV, HHV types 1, 2, and 6) viruses in the etiological pattern of diseases accompanied by enhanced blood AlAT and AsA T activities in pregnant women. SUBJECTS AND METHODS: Two hundred and eleven pregnant women, including 123 patients with chronic viral hepatitis, 74 with enhanced blood AlAT activity and no markers of viral hepatitis (EAlA T-NMVH), and 14 with acute viral hepatitis were examined. RESULTS: Most pregnant women with chronic HBV and HCV infections were found to have HBV DNA and HCV RNA in the blood in the presence of normal and enhanced activities of transaminases. In the EAlAT-NMVH group, there was none of the opportunistic hepatotropic viruses in more than 7% of cases. No genetic material of HAV, HBV, HCV, HDV, HGV, CMV, EBV, HHV types 1, 2, and 6 was found in the blood of all 10 patients with hepatitis of unspecified etiology. CONCLUSION: In the absence of serologic data supporting the presence of infectious pathology, blood testing using the polymerase chain reaction is of low informative value in detecting opportunistic hepatotropic viruses in pregnant women with hepatitis of unspecified etiology. However, by keeping in mind that the spectrum of opportunistic hepatotropic viruses is not confined to those included in this study, it is expedient to examine additionally pregnant women with enhanced blood AlAT and AsAT activity in order to identify TTV, B19V, HHV-8, SEN and NV-F in the blood.

Effects of cholecystectomy (laparoscopic versus open) on PMN-elastase.

BACKGROUND/AIMS: Polymorphonuclear leukocytes (PMN) are well recognized as being the principal cells in inflammatory response reaction. During the surgical procedures there is a massive release of elastase (PMN-elastase) from the neutrophils, along with other proteinases. Therefore the measurement of the PMN-elastase might be a useful indicator of the degree of surgical trauma. Laparoscopic cholecystectomy (LC) is a so-called "mini-invasive" surgical procedure and on the basis of this consideration the aim of the present prospective, non-randomized study, is to examine (a) whether the serum levels of PMN-elastase concentration are modified and how, in patients undergoing LC compared to patients undergoing open cholecystectomy (OC), (b) whether these findings are indicative of an increased risk to develop infectious complications and therefore whether they are clinically significant. METHODOLOGY: Plasma granulocyte elastase was determined photometrically, using an immune-activation immunoassay, in 86 patients (42 patients underwent OC and 44 LC). The levels of C reactive protein (CRP), an acute phase protein, were measured using a competitive CRP ELISA kit. Blood samples were collected from all patients a day before operation and at days 1, 3, 6 and 12 after operation. We established a reference range for elastase by measuring the serum elastase concentration in 68 normal control patients without gallbladder cholelithiasis or other diseases. RESULTS: On day, 1, 3 and 6 after surgery, patients that underwent OC showed a significant increase (p < 0.05) in plasma elastase concentration, while it was almost unchanged in LC patients. The mean values of the serum CRP on p.o. days 1, 3 and 6 were also significantly lower in the LC group than those in OC group (p < 0.05). We recorded three cases (7.1%) of postoperative infections in the "open" group. The CRP concentration remained high for 1, 3 and 6 days and normalized 10-12 days after surgery while the PMN-elastase normalized after 13, 14 and 16 days. CONCLUSIONS: The peripheral leukocyte function may be better preserved after LC in comparison to OC. Laparoscopic surgery, associated with a small skin incision and the avoidance of open laparotomy, can thus minimize surgical stress, and provide more favorable postoperative conditions for patients. Indeed excessive and prolonged post-injury elevations of PMN-elastase and CRP are associated with increased morbidity. Moreover, the PMN-elastase is a more sensible marker of inflammation in comparison to the CRP.

Could 2'5'-oligoadenylate synthetase isoforms be biomarkers to differentiate between disease flare and infection in lupus patients? A pilot study.

2'5'-Oligoadenylate synthetase (OAS) was shown to be related to systemic lupus erythematosus (SLE) 20 years ago, and was rediscovered to be involved in type I interferon pathway in SLE by several microarray gene expression studies recently. The goal of this study was to investigate OAS isoform expressions in lupus patients, to evaluate whether they could become biomarkers to differentiate between disease flare and infection. Fifty-four SLE patients presented with fever or systemic inflammatory syndrome, or both, were enrolled. Gene expressions of OAS1, OAS2, and OASL were studied by using real time PCR in active SLE (SLEDAI >or=9, n=29) and in those complicated with infections (n=25). The latter group was composed of 19 patients with invasive bacterial infections, and six patients with viral infections. C reactive protein (CRP) and other clinical parameters were also measured. Twenty-nine healthy individuals made up a normal control group. The mRNA expressions of OAS1, OAS2, and OASL were higher in patients with lupus flares than those with infections (p<0.03), or normal controls (p<0.001). SLE complicated with infections have higher OAS1 expression level (P=0.002), lower OASL (P=0.004), and equivalent OAS2 (P=0.135), when compared with those of normal controls. OASL expression level was negatively correlated with infection in lupus by logistic regression analysis (p=0.008). Area under the receiver operating characteristic curve for the prediction of infection was 0.92 (p<0.0001) for OASL, and 0.77 (p=0.007) for CRP. Therefore, our preliminary data suggest that the pattern of OAS isoform expressions, OASL in particular, may provide useful information in differentiating disease flares from certain infections in SLE.

Identification of superoxide dismutase as a cofactor for the pseudomonas type III toxin, ExoU.

Pseudomonas aeruginosa is an opportunistic pathogen that uses a type III secretion system and four effector proteins to avoid innate immune responses. ExoS, ExoT, ExoY, and ExoU all possess enzymatic activities that disrupt host cellular physiology and prevent bacterial clearance by host defense mechanisms. The specificity of these toxins for eukaryotic cells depends on the presence of substrate targets and eukaryotic cofactors responsible for effector activation. We used a combined biochemical and proteomic approach to identify Cu(2+), Zn(2+)-superoxide dismutase (SOD1) as a cofactor that activates the phospholipase activity of ExoU. Recombinant ExoU (rExoU) was activated in a dose-dependent manner by either bovine liver SOD1 or the yeast ortholog, Sod1p, but not by either Fe or Mn-containing SODs from E. coli or small molecule SOD mimetics. Inhibitor studies indicated that SOD enzymatic activity was not required for the activation of rExoU. The physical interaction between rExoU and SOD was demonstrated by capture techniques using either of the two proteins immobilized onto the solid phase. Identification of SOD as a cofactor allowed us to develop a new assay using a fluorescent substrate to measure the phospholipase activity of rExoU. The ability of SOD to act as a cytoplasmic cofactor stimulating ExoU phospholipase activity has significant implications for the biological activity of the toxin. Further elucidation of the structural mechanism of ExoU activation by this eukaryotic cofactor may provide a rational approach to the design of inhibitors that can diminish tissue damage during infection by ExoU-producing strains of P. aeruginosa.

Posaconazole: SCH 56592.

Posaconazole [SCH 56592, SPRIAFIL, Noxafil] is an orally active triazole derivative that is in phase III trials with the Schering-Plough Research Institute (SPRI) in the US for the treatment of serious opportunistic fungal infections, including aspergillosis, candidiasis, coccidioidomycosis and fusariosis. This profile has been selected from R&D Insight, a pharmaceutical intelligence database produced by Adis International Ltd. Preclinical studies have also been conducted in Italy for the potential treatment of Cryptococcus neoformans infection (cryptococcosis).

Chronic Pneumocystis carinii infection of the liver. A case report and review of the literature.

Pneumocystis carinii infection of the liver is being reported with increasing frequency in patients with acquired immune deficiency syndrome (AIDS). The clinical picture typically resembles hepatitis. We report such an occurrence in a patient with persistent elevation of alkaline phosphatase and gamma-glutamyl transpeptidase with relatively normal transaminases who was found to have P. carinii on antemortem liver biopsy. The differential diagnosis of abnormal alkaline phosphatase and gamma-glutamyl transpeptidase in patients with AIDS should include P. carinii.

[Experimental and clinical studies of drug regulation of the anti-lysozyme activity of microorganisms causing opportunistic infections]. / Eksperimental'no-klinicheskoe izuchenie lekarstvennoi reguliatsii antilizotsimnoi aktivnosti vozbuditelei opportunisticheskikh infektsii.

Experimental materials on choosing antibiotics for etiotropic therapy of opportunistic infections with an account of the regulating effect of the drugs on the ++anti-lysozyme activity of pathogens (the factor of intracellular parasitism) are presented. The in vitro data were applied to the clinical trials in 30 patients with chronic and acute pyelonephritis of the Proteus etiology and to 25 patients with chronic inflammatory diseases of Staphylococcus etiology. It was shown that the use of the antibiotics which lowered the ++anti-lysozyme activity of microorganisms promoted a more rapid disappearance of the disease clinical signs, increased 2- to 3-fold the terms of the remission and resulted in an increase in the number of the persons with complete remission (54.5 to 63.6 per cent) as compared to the use of the drugs which stimulated the pathogen property or were indifferent to it.

[Value of the elastase-alpha 1-proteinase inhibitor complex in pediatric oncologic patients with fever]. / Wertigkeit des Elastase-alpha1-Proteinaseinhibitor-Komplexes bei pädiatrisch-onkologischen Patienten mit Fieber.

The elastase-alpha1-proteinase inhibitor complex (E-alpha 1-PI) is a well known, sensitive indicator of infection. The significance of this parameter was investigated in 12 febrile paediatric-oncological patients. With neutrophil counts greater than 500/microliters infections caused a definitive increase in E-alpha 1-PI serving as an additional diagnostic parameter. E-alpha 1-PI provided no additional information in situations of sepsis with leukopenia and simultaneous agranulocytosis. In patients with metastatic tumours, the assay of E-alpha 1-PI may differentiate between infection-related and tumour-related serological disturbances.

Pathogenic potentials of bacterial proteases.

Six separate molecular mechanisms for pathogenesis attributed to bacterial proteases are described. (I). Enhancements of vascular permeability and edema formation which result from the activation of kinin generating cascade such as Hageman factor by the proteases. (II). Degradation of defense oriented proteins including IgG and IgA as well as destruction of structural matrices such as fibronectin, proteoglycan and collagen. (III). Inactivation of complement system and generated chemotactic factor from C3 and C5. (IV). Degradation of regulatory plasma protease inhibitors (serpins) including alpha 1-protease inhibitor, alpha 2-macroglobulin (alpha 2M), C1-esterase inhibitor, alpha 2-antiplasmin and antithrombin-III. (V). The protease forms a transitory stable enzyme/inhibitor(alpha 2M) complex. It binds to and internalizes into the cells which possess alpha 2M-receptor such as fibroblasts via the alpha 2M-receptor, and the protease activity is regenerated in cells, and subsequently intracellular integrity is destroyed resulting in cell killing. (VI). The serratial 56 kDa (56K) protease is found to potential viral yield 100 fold more when influenza virus infected mice were subjected to administrations of this protease intranasally. This results in rapid and much elevated lethality.