Sherief El-Shazly

Increased expression of pro-inflammatory cytokines in placentas of women undergoing spontaneous preterm delivery or premature rupture of membranes

Problem:  The objective of this study was to determine the levels of cytokines in the placentas of women undergoing preterm delivery (PTD) or premature rupture of membranes (PROM) as compared with women undergoing normal delivery at term.

Pro‐inflammatory Maternal Cytokine Profile in Preterm Delivery

PROBLEM:  The objective of this study was to determine the levels of cytokines produced by maternal peripheral blood mononuclear cells (PBMC) upon stimulation with a mitogen, with autologous placental cells and with a trophoblast antigen extract.

Mammalian Cell-Entry Proteins Encoded by the mce3 Operon of Mycobacterium tuberculosis are Expressed During Natural Infection in Humans

The mammalian cell‐entry (mce)3 operon is one of four homologous mce operons on Mycobacterium tuberculosis genome that encodes six putative invasin/ adhesin‐like proteins (Mce3A–F) possibly involved in the entry and survival of this bacterium inside macrophages. To study the in vivo expression of the mce3 operon‐encoded proteins during natural human infection, the genes encoding Mce3A–F were cloned and expressed in Escherichia coli as fusion proteins with glutathione‐S‐transferase (GST) at the N‐terminal and a ×6 histidine (His) tag at the C‐terminal end. The recombinant proteins appeared as major cellular proteins in SDS‐PAGE gels and reacted with anti‐GST and antipenta‐His antibodies at the expected molecular mass of 72, 61, 78, 80, 66 and 78 kDa for GST‐Mce3A, GST‐Mce3B, GST‐Mce3C, GST‐Mce3D, GST‐Mce3E and GST‐Mce3F, respectively. In Western immunoblots, all the six fusion proteins, particularly GST‐Mce3A, GST‐Mce3C, GST‐Mce3D and GST‐Mce3E, reacted with antibodies in combined human serum from 11 tuberculosis (TB) patients. Pure Mce3A, Mce3D and Mce3E could be isolated by specific proteolytic cleavage by thrombin protease of the respective purified fusion protein followed by preparative SDS‐PAGE. The pure Mce3A, Mce3D and Mce3E reacted to various extents with antibodies in serum samples from TB patients. The Mce3E reacted with 51 of 55 (93%) and all the three proteins reacted with 34 of 55 (62%) serum samples. The Mce3A, Mce3D and Mce3E proteins also reacted, albeit at lower frequency, with one of 23 (4%) serum sample obtained from M. bovis bacillus Calmette–Guérin‐vaccinated healthy subjects and four of 18 (22%) serum samples from long‐term contacts of TB patients showing reactivity with all the three Mce3 proteins. The data show that Mce3A, Mce3D and Mce3E encoded by mce3 operon of M. tuberculosis are expressed and elicit antibody responses in humans during natural infection with this pathogen.

The six mammalian cell entry proteins (Mce3A-F) encoded by the mce3 operon are expressed during in vitro growth of Mycobacterium tuberculosis.

The pathogenesis of Mycobacterium tuberculosis is largely due to its ability to enter and survive within human macrophages. The mammalian cell entry (mce)3 operon is one of four homologous mce operons that encodes six putative invasin‐like exported proteins (Mce3A–F), possibly involved in entry and survival of M. tuberculosis inside macrophages. We have recently shown that Mce3A, Mce3D and Mce3E are expressed and elicit antibody responses in a majority of human subjects during natural infection with M. tuberculosis. In this study, we demonstrate the expression of Mce3A–F proteins and their mRNA during in vitro growth of M. tuberculosis. To demonstrate the expression of mce3A–F proteins, the antibodies were raised in rabbits against three pure proteins (Mce3A, Mce3D and Mce3E), and their specificity was checked by immunoblotting with recombinant Mce1A–F proteins encoded by mce1 operon. The antibodies were also generated against all the six Mce3 proteins, which were expressed and purified as fusion proteins with glutathione S‐transferase (GST) as the fusion partner (GST‐Mce3A–F). The antibodies reacted, in each case, with a protein of expected molecular mass (Mr) for the corresponding Mce3 protein in the cell wall fraction but not in the soluble fraction of in vitro‐grown M. tuberculosis cells. The presence of mRNA for mce3A–F genes was also shown by using mce3A–F gene‐specific primers, and total RNA isolated from in vitro‐grown M. tuberculosis cells by reverse transcription‐polymerase chain reaction (RT‐PCR). Pretreatment of the RNA preparation with RNase A abolished amplification in RT‐PCR confirming that mce3A–F mRNA rather than genomic DNA was being amplified. The data show that Mce3A–F encoded by the mce3 operon are expressed during in vitro growth of M. tuberculosis.

Cytokine patterns in maternal blood after premature rupture of membranes

OBJECTIVE To compare two types of cytokines, type 1, which activate cell‐mediated reactions and are important in cytotoxic and delayed‐type hypersensitivity reactions, and type 2, which encourage vigorous antibody production and are commonly found in association with humoral immune responses, in blood of women with premature rupture of membranes (PROM). METHODS Forty‐four women with histories of at least three successful pregnancies and who currently delivered normally served as controls. The PROM group consisted of 30 women with spontaneous rupture of fetal membranes at term. Peripheral blood mononuclear cells were stimulated separately with a mitogen, placental cells, and a trophoblast antigen extract, and the supernatants examined for type 1 and type 2 cytokines. RESULTS Mitogen‐stimulated blood cells produced significantly higher levels of type 1 cytokines in PROM women than in normal controls. Higher levels of the type 1 cytokine interferon‐γ were produced by PROM samples stimulated with autologous placental cells and with trophoblast antigens. Ratios of type 1 to type 2 cytokines were higher in PROM compared with normal pregnancy, and in some cases as much as 25‐fold higher. CONCLUSION Women in the PROM group had a stronger type 1 reactivity whereas normal women were more predisposed to type 2 immunity; thus, PROM appears to be associated with a maternal type 1 bias.

Molecular epidemiology and characterization of multiple drug-resistant (MDR) clinical isolates of Acinetobacter baumannii

OBJECTIVES The aim of this study was to identify the genetic relatedness of multiple drug-resistant (MDR) Acinetobacter baumannii clinical isolates recovered from a hospital in Los Angeles. METHODS Twenty-one MDR A. baumannii isolates were collected and their antibiotic susceptibilities determined according to Clinical and Laboratory Standards Institute guidelines. Genes coding for antibiotic resistance were identified by PCR, and their identities were confirmed by DNA sequencing. Clonal relationships were studied by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). RESULTS MDR consistently correlated with the presence of oxacillinases, mostly in the form of the plasmid-mediated OXA-23 enzyme, which was detected in 12 (57.1%) isolates. GES-type carbapenemases were found in 20 (95.2%) strains, AAC in all 21 (100%) strains, and PER in seven (33.3%) strains, and ISAba1 was detected in 16 (76.2%) isolates. The association between ISAba1 and resistance genes confirms insertion elements as a source of β-lactamase production. Of the 21 clinical isolates, five were found to be related to sequence type 1 (ST1) and 16 to ST2, as analyzed by MLST. PFGE demonstrated that the majority of clinical isolates were highly related (>85%). CONCLUSIONS This study supports a more complete understanding of genotyping of antibiotic resistance for better assessment of MDR strain transmission.

The characterization and antibiotic resistance profiles of clinical Escherichia coli O25b-B2-ST131 isolates in Kuwait

BackgroundEscherichia coli O25b-B2-ST131 are considered virulent extra-intestinal pathogens causing serious clinical complications such as urinary tract infection and bacteraemia. Our main objectives in this study were to characterise the multi-drug resistant (MDR) isolates of this lineage in Kuwait, and to demonstrate whether reduced susceptibility is spread clonally.ResultsA subset of 83 (10%) non-duplicate and non-selective E. coli O25b-B2-ST131 out of 832 MDR E. coli was identified and collected. Minimum inhibitory concentrations of the isolates were determined and pulsed-field gel electrophoresis was used for typing.The majority (95.2%) of the 83 E. coli O25b-B2-ST131 harboured at least one bla gene with blaCTX-M-15 being the most prevalent. blaCTX-M-2 was present in one isolate. Also one isolate harboured blaCTX-M-56, qnr B1 and blaCMY-2 genes and carried IncF1 plasmids of about 97kb and160 kb. qnr B and qnr S were found in 8 other blaCTX-M-15 containing isolates. The blaNDM, blaIMP, blaVIM and qnr A were not detected, however, the blaOXA-48 was present in two (2.4%).ConclusionsThe majority of isolates harbouring qnr genes demonstrated relatedness (≥85%) by PFGE. However, the diversity in PFGE profiles for the other MDR isolates reflected the changes in population genetics of E. coli O25b-B2-ST131. We identified for the first time the appearance of blaCTX-M-2 in the Middle East and blaCTX-M-56 outside the Latin American countries. The isolate harbouring blaCTX-M-56 also contained qnr B1 and blaCMY-2 genes and carried IncF1 plasmids. The appearance of a highly virulent E. coli O25b-ST131 that is resistant to penicillins, most cephalosproins, β-lactamase inhibitors as well as fluoroquinolones is a cause for concern.

The emergence of carbapenem resistance in ESBL-producing Escherirchia coli O25B-ST131 strain from community acquired infection in Kuwait

In this study we investigated a multi-drug resistant E.coli recovered from ascetic fluid of a haemodialysis patient with community-onset urinary tract infection from Al-Amiri hospital in Kuwait. The patient was suffering from advanced liver disease with portal hypertension and multiple current inter abdominal abscesses.

First report of QNRA isolated from extended spectrum B-lactamase producing hospital-acquired Klebsiella pheumoniae in Kuwait

Extended Spectrum beta-lactamase (ESBL)-mediated resistant Klebsiella pneumoniae are important opportunistic pathogens. In this study we investigated the prevalence of plasmid-mediated fluoroquinolone resistance in ESBL-producing K. pneumoniae in nosocomial infections in Kuwait.

Correlation between heat shock proteins, adiponectin, and T lymphocyte cytokine expression in type 2 diabetics

Type 2 diabetes mellitus (T2DM) features insulin resistance, hyperglycemia, dyslipidemia, overproduction of inflammatory cytokines, and systemic oxidative stress. Here, heat shock proteins Hsp70 and Hsp 90, adiponectin, and heme oxygenase-1 (HO-1, Hsp32) are profiled in peripheral blood mononuclear cells (PBMC) and serum from 25 T2DM patients and 25 healthy control subjects. Cells cultured with phorbol 12-myristate 13-acetate/ionomycin were evaluated by three-color flow cytometry for immunophenotypic biomarkers. Plasma HO-1, Hsp, and adiponectin levels were assayed by enzyme-linked immunosorbent assay (ELISA). Relative to healthy controls, T2DM patients exhibited significantly elevated plasma Hsp70, and representation of T helper immunophenotypes activated to express inflammatory cytokines, including CD4+ IFN-γ+, CD4+ TNF-α+, CD4+ IL-6+, CD4+ IL-1β+ T cells, significantly lower representation of CD4+ IL-10+ T cells, plasma adiponectin and cell-associated HO-1 expression—with no significant differences in plasma Hsp90 between T2DM and healthy controls. Plasma HO-1 and adiponectin in T2DM patients inversely correlated with TNF-α and showed inverse correlation between serum LDL and plasma HO-1. Moreover, TNF-α and Hsp90 in T2DM patients correlated positively with fasting blood glucose (FBG). These results demonstrate correlation between potentially pathogenic T cells, HO-1, and adiponectin, additionally revealing a T helper (Th)1-related character of T2DM immunopathogenesis, suggesting potential for novel T cell-related management strategies for T2DM and related co-morbidities.