Elevated Plasma Level of Interferon-λ1 in Chronic Spontaneous Urticaria: Upregulated Expression in CD8(+) and Epithelial Cells and Induction of Inflammatory Cell Accumulation.

Interferon- (IFN-) λ1 is regarded as a potent bio-active molecule in innate immunity. However, little is known about its role in chronic spontaneous urticaria (CSU). We therefore investigated expression of IFN-λ1 in CSU, its cellular location, and its influence on inflammatory cell accumulation by using flow cytometry analysis, skin tissue dispersion, immunohistochemical stain, and a mouse peritoneal inflammation model. The results showed that level of IFN-λ1 was 2.0-fold higher in plasma of the patients with CSU than the level in healthy control (HC) subjects. Among leukocytes examined, only CD8(+) T cells expressed more IFN-λ1 in CSU blood. Double labeling immunohistochemical staining revealed that IFN-λ1(+) inflammatory cells such as mast cells, eosinophils, B cells, neutrophils, and macrophages were mainly located in dermis, whereas epidermis tissue highly expressed IFN-λ1. IFN-λ1 induced a dose-dependent increase in number of eosinophils, lymphocytes, mast cells, macrophages, and neutrophils in the peritoneum of mice at 6 h following injection, which was inhibited by pretreatment of the animals with anti-intercellular adhesion molecule- (ICAM-) 1 and/or anti-L-selectin antibodies. In conclusion, IFN-λ1 is likely to play a role in the pathogenesis of CSU. Blocking IFN-λ1 production may help to reduce the accumulation of inflammatory cells in the involved CSU skin.

[Epidemiological analysis of pathogens causing bloodstream infections in department of hematology in Guangdong Province].

Objective: To evaluate the epidemiology of bacterial bloodstream infections in patients submitted to hematologic wards in southern China. Methods: A total of 50 teaching hospitals were involved based on the China Antimicrobial Resistance Surveillance System. The data of clinical isolates from blood samples were collected from January 1, 2019, to December 31, 2019. Antimicrobial susceptibility testing was conducted by the Kirby-Bauer automated systems, and the results were interpreted using the CLSI criteria. Results: The data of 1,618 strains isolated from hematologic wards in 2019 were analyzed, of which gram-negative bacilli and gram-positive cocci accounted for 71.8% and 28.2%, respectively. Of those, the five major species were most often isolated, including Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, coagulase-negative staphylococcus, and Streptococcus viridans. The prevalence rates of methicillin-resistant strains in Staphylococcus aureus and coagulase-negative staphylococcus were 19.7% and 80.6%, respectively. No gram-positive cocci were resistant to vancomycin, linezolid, and teicoplanin, and none of the enterococci were resistant to linezolid. The resistance rate of S. viridans to penicillin G was 6.9%, and those to ceftriaxone and cefotaxime were more than 25%. The resistance rate of E. coli and K. pneumoniae in Enterobacteriaceae was higher in children than that in adults. The resistance rate of K. pneumoniae to meropenem was 14.1%. The resistant rate of Enterobacter cloacae to carbapenem was more than 25%. P. aeruginosa was more sensitive to more antibiotics than 80%, but the resistance rate to meropenem in children was higher than that in adults (11.8% vs. 6.5%). The proportion of gram-positive cocci in the ICU and respiratory departments was higher than that in the hematology department. The detection rates of carbapenem-resistant E. coli and K. pneumoniae in the respiratory department were the lowest with 0.3% and 3.7%, respectively, while those of CRPA and CRAB in the hematology department were the lowest with 8.3% and 25.8%, respectively. The detection rate of all carbapenem-resistant organisms in the ICU was the highest among the three departments. Conclusion: The etiology and drug resistance of bacteria from blood samples in the hematology department are different from those in the ICU and respiratory departments. The proportions of K. pneumoniae, P. aeruginosa, E. cloacae, and S. viridans dominating in the department of Hematology were significantly higher than those in the ICU and respiratory departments in Guangdong region.

Polymorphisms in P450 CYP1B1 affect the conversion of estradiol to the potentially carcinogenic metabolite 4-hydroxyestradiol.

Most drug metabolizing cytochrome P450s (P450) are predominantly expressed in the liver. In contrast, human CYP1B1 is an extrahepatic P450 which is overexpressed in many tumours and has been strongly implicated in the activation of carcinogens. Rare allelic variants of the CYP1B1 gene which encode an inactive protein have been identified. However, four polymorphisms which most likely do not abolish functionality have been described. In this report, we have characterized the functional consequences of these. A CYP1B1 cDNA, identical to a cDNA published previously, served as a template to introduce allelic changes either separately or in combination. The resulting effects on CYP1B1 activity were determined in membranes isolated from Escherichia coli which coexpressed CYP1B1 together with P450 reductase. None of the allelic changes affected the CYP1B1 expression level. The allelic changes Arg48 to Gly, Ala19 to Ser and Asn453 to Ser had little influence on the Vmax and the Km of the CYP1B1 mediated 2- and 4-hydroxylation of estradiol. In contrast, the Km of these metabolic pathways was increased at least three-fold by the allelic change Va432 to Leu or by simultaneously changing Val432 to Leu and Asn453 to Ser. However, these alterations had little effect on the kinetic parameters of other CYP1B1 mediated reactions such as the epoxidation of (-)-trans-(7R,8R)-benzo[a]pyrene 7,8-dihydrodiol as determined by (r-7,t-8,t-9,c-10)-benzo[a]pyrene tetraol formation, or such as the O-dealkylation of ethoxyresorufin and the 1'-hydroxylation of bufuralol. Molecular modelling suggests that amino acid residue 432 of CYP1B1 may be involved in the interaction between CYP1B1 and P450 reductase. Since 4-hydroxyestradiol has been implicated in hormonal carcinogenesis and CYP1B1 is expressed in target tissues, the data presented demonstrate that polymorphisms in CYP1B1 have the potential to affect disease susceptibility.

Creation versus destruction of T cell epitopes in the class II MHC pathway.

Proteases perform two key roles in the class II MHC antigen processing pathway. They initiate removal of the invariant chain chaperone for class II MHC and they generate peptides from foreign and self proteins for eventual capture and display to T cells. How a balance is achieved between generation of suitable peptides versus their complete destruction in an aggressive proteolytic environment is not known. Nor is it known in most cases which proteases are actually involved in antigen processing. Our recent studies have identified asparagine endopeptidase (AEP or legumain) as an enzyme that contributes to both productive and destructive antigen processing in the class II MHC pathway. The emerging consensus seems to be that individual proteolytic enzymes make clear and non-redundant contributions to antigen processing.

[Three lethal cases of Angiostrongylus cantonensis infected children].

OBJECTIVE: To offer approaches to diagnosis and treatment of Angiostrongylus cantonensis infection on the basis of analyzing three lethal cases of the disease. METHODS: Clinical manifestations, pathological changes and species identification were pursued. RESULTS: Angiostrongyliasis is usually neglected in medical departments. The three cases here reported were all misdiagnosed and had not receiced anthelmintic treatment hence fatality ensued. CONCLUSION: Early examination of CSF, bronchial lavage fluid and feces is urged so as to secure accurate diagnosis and conduct efficacious anthelmintic therapy to cure the patients.

A general strategy for the expression of recombinant human cytochrome P450s in Escherichia coli using bacterial signal peptides: expression of CYP3A4, CYP2A6, and CYP2E1.

Heterologous expression of unmodified recombinant human cytochrome P450 enzymes (P450s) in Escherichia coli has proved to be extremely difficult. To date, high-level expression has only been achieved after altering the 5'-end of the native cDNA, resulting in amino acid changes within the P450 protein chain. We have devised a strategy whereby unmodified P450s can be expressed to high levels in E. coli, by making NH2-terminal translational fusions to bacterial leader sequences. Using this approach, we initially tested two leader sequences, pelB and ompA, fused to CYP3A4. These were compared with an expression construct producing a conventional NH2-terminally modified CYP3A4 (17alpha-3A4). Both leader constructs produced spectrally active, functional protein. Furthermore, the ompA-3A4 fusion gave higher levels of expression, and a marked improvement in the recovery of active P450 in bacterial membrane fractions, when compared with 17alpha-3A4. We then tested the ompA leader with CYP2A6 and CYP2E1, again comparing with the conventional (17alpha-) approach. As before, the leader construct produced active enzyme, and, for CYP2E1 at least, gave a higher level of expression than the 17alpha-construct. The ompA fusion strategy thus appears to represent a significant advance for the expression of P450s in E. coli, circumventing the previous need for individual optimization of P450 sequences for expression.

Competition between cytochrome P-450 isozymes for NADPH-cytochrome P-450 oxidoreductase affects drug metabolism.

NADPH-cytochrome P-450 oxidoreductase (CPR) is essential for the catalytic activity of cytochrome P-450 (P-450). On a molar basis, the amount of P-450 exceeds that of CPR in human liver. In this study, we investigated whether drug-drug interactions can occur as a result of competition between P-450 isozymes for this ancillary protein. For this purpose, combinations of P-450 isozymes were coexpressed together with P-450 reductase in Escherichia coli. We show that testosterone inhibited the CYP2D6-mediated bufuralol 1'-hydroxylase activity in bacterial membranes containing both CYP2D6 and CYP3A4 but not in membranes containing CYP2D6 alone. Conversely, bufuralol inhibited the CYP3A4-mediated testosterone 6beta-hydroxylase activity in bacterial membranes containing both CYP3A4 and CYP2D6 but not in membranes containing only CYP3A4. In each case, inhibition was seen even at a P-450 to P-450 reductase ratio of 1.9:1, which is more favorable than the ratio of 4 reported for human liver. The physiological significance of this mechanism was demonstrated by the observation that testosterone inhibited several prototypical P-450 enzyme activities, such as bufuralol 1'-hydroxylase, coumarin 7-hydroxylase, and 7-ethoxyresorufin O-dealkylase, in human liver microsomes, but not if tested against a panel of bacterial membranes containing the human P-450 isozymes that mainly catalyze these reactions.