Ramadan A. Eldomany

Inflammasome Priming by Lipopolysaccharide Is Dependent upon ERK Signaling and Proteasome Function

Caspase-1 activation is a central event in innate immune responses to many pathogenic infections and tissue damage. The NLRP3 inflammasome, a multiprotein scaffolding complex that assembles in response to two distinct steps, priming and activation, is required for caspase-1 activation. However, the detailed mechanisms of these steps remain poorly characterized. To investigate the process of LPS-mediated NLRP3 inflammasome priming, we used constitutively present pro–IL-18 as the caspase-1–specific substrate to allow study of the early events. We analyzed human monocyte caspase-1 activity in response to LPS priming, followed by activation with ATP. Within minutes of endotoxin priming, the NLRP3 inflammasome is licensed for ATP-induced release of processed IL-18, apoptosis-associated speck-forming complex containing CARD, and active caspase-1, independent of new mRNA or protein synthesis. Moreover, extracellular signal–regulated kinase 1 (ERK1) phosphorylation is central to the priming process. ERK inhibition and small interfering RNA–mediated ERK1 knockdown profoundly impair priming. In addition, proteasome inhibition prevents ERK phosphorylation and blocks priming. Scavenging reactive oxygen species with diphenylene iodonium also blocks both priming and ERK phosphorylation. These findings suggest that ERK1-mediated posttranslational modifications license the NLRP3 inflammasome to respond to the second signal ATP by inducing posttranslational events that are independent of new production of pro–IL-1β and NOD-like receptor components.

OXA-163-Producing Klebsiella pneumoniae in Cairo, Egypt, in 2009 and 2010

ABSTRACT Two genetically unrelated OXA-163-carrying Klebsiella pneumoniae strains were identified from two infection cases in June 2009 and May 2010 in Cairo, Egypt. OXA-163-producing Enterobacteriaceae had been previously reported in Argentina only. Both patients had no history of travel abroad. The emergence of this newly recognized OXA-48-related β-lactamase able to hydrolyze cephalosporins and carbapenems is especially worrying in a geographic area where OXA-48 is endemic and effective surveillance for antibiotic resistance is largely unaffordable.

Evaluation of immunomodulatory effect of three herbal plants growing in Egypt.

A group of medicinal plant including Silene (Silene nocturna), Black seed (Nigella sativa) and Chamomile (Matricaria chamomilla) growing in Egypt were examined for their immunomodulatory effect in Balb/c mice. Treatment (intraperitoneal injection) with five doses of methanolic extract for each plant was found to enhance the total white blood cells count (up to 1.2 × 104 cells/mm3). Bone marrow cellularity also increased significantly (P < 0.01) after the administration of the extract of each of three test plants. Furthermore, spleen weight of the treated groups was significantly increased (P < 0.01). Two groups of mice were immunosuppressed with cyclophosphamide, the one which pretreated with the plants extracts significantly (P < 0.01) restored their resistance against lethal infection with the predominately granulocyte-dependant Candida albicans. These results confirm the immunomodulatory activity of Silene, Black seed, and Chamomile extracts and may have therapeutical implications in prophylactic treatment of opportunistic infections and as supportive treatment in oncogenic cases.

Antimicrobial Susceptibility Profile of Pseudomonas aeruginosa Isolates in Egypt

PURPOSE Pseudomonas aeruginosa is a leading cause of nosocomial respiratory tract, urinary tract and skin infections. Data are sparse on the antimicrobial resistance of P. aeruginosa in Egypt. We sought to detect and compare the antimicrobial susceptibility of P. aeruginosa isolates from respiratory tract, urinary tract and skin infections at 3 Egyptian hospitals. MATERIALS AND METHODS Minimum inhibitory concentrations of antibiotics were determined by the agar dilution method. RESULTS P. aeruginosa respiratory tract infections isolates were 100% resistant to ampicillin, ampicillin/sulbactam, amoxicillin, amoxicillin/clavulanate and chloramphenicol, highly resistant to cefuroxime (89%), tetracycline (89%) and azithromycin (84%), and susceptible to norfloxacin (89%), amikacin (84%) and meropenem (68%). P. aeruginosa urinary tract infection isolates were 100% resistant to ampicillin, amoxicillin, chloramphenicol, cefuroxime and tetracycline, highly resistant to amoxicillin/clavulanate (95%), azithromycin (95%), cefalexin (91%) and ampicillin/sulbactam (82%), and susceptible to amikacin (82%), meropenem (73%) and norfloxacin (64%). P. aeruginosa skin infection isolates were 100% resistant to ampicillin and amoxicillin, highly resistant to tetracycline (95%), amoxicillin/clavulanate (95%), cefalexin (87%) and azithromycin (84%), and susceptible to amikacin (87%), norfloxacin (71%) and meropenem (68%). The anti-pseudomonal effect of antibiotics varied among different infection sites only for ampicillin/sulbactam, cefoperazone or chloramphenicol but not with the other tested antibiotics. CONCLUSIONS Norfloxacin and amikacin could be used for initial therapy for P. aeruginosa mediated respiratory tract infections. Amikacin, meropenem and norfloxacin could be used for P. aeruginosa mediated urinary tract and skin infections. Such studies are essential to determine the current guidelines for empirical therapy regimens, which vary by location, and help with the establishment of effective infection control measures.

The Multivalent Adhesion Molecule SSO1327 plays a key role in Shigella sonnei pathogenesis

Shigella sonnei is a bacterial pathogen and causative agent of bacillary dysentery. It deploys a type III secretion system to inject effector proteins into host epithelial cells and macrophages, an essential step for tissue invasion and immune evasion. Although the arsenal of bacterial effectors and their cellular targets have been studied extensively, little is known about the prerequisites for deployment of type III secreted proteins during infection. Here, we describe a novel S. sonnei adhesin, SSO1327 which is a multivalent adhesion molecule (MAM) required for invasion of epithelial cells and macrophages and for infection in vivo. The S. sonnei MAM mediates intimate attachment to host cells, which is required for efficient translocation of type III effectors into host cells. SSO1327 is non‐redundant to IcsA; its activity is independent of type III secretion. In contrast to the up‐regulation of IcsA‐dependent and independent attachment and invasion by deoxycholate in Shigella flexneri, deoxycholate negatively regulates IcsA and MAM in S. sonnei resulting in reduction in attachment and invasion and virulence attenuation in vivo. A strain deficient for SSO1327 is avirulent in vivo, but still elicits a host immune response.

Epidemiology and antimicrobial resistance of staphylococci isolated from different infectious diseases.

A total of 187 isolates from 470 clinical specimens were collected from three hospitals in El-Minia governorate and identified as 132 Staphylococcus aureus strains and 55 coagulase-negative staphylococci (CoNS) strains. Susceptibility of isolates to antimicrobial agents was tested by the agar dilution method. The isolated S. aureus strains showed low resistance to vancomycin (1.5%), amikacin (2.3%) and gatifloxacin (3.8%). Vancomycin was the most effective antibiotic against CoNS. The ampicillin-resistant isolates were tested for β-lactamase production where, 61.7% of S. aureus and 42.9% of CoNS were positive for β-lactamase enzyme. Beta-lactamase producing strains were screened for their plasmid profile using alkaline lysis method. Some of these strains carried at least one plasmid suggesting plasmid-mediated antibiotic resistance. When cells of these strains were exposed to curing agent ethidium bromide, the production of the β-lactamase was lost. Resistance by efflux was studied by a modified fluorometric assay. Addition of uncoupler carbonyl cyanide m-chlorophenylhydrazone (CCCP) increased norfloxacin accumulation in quinolone resistant S. aureus strains, suggesting endogenous energy-dependent efflux. Combinations of ciprofloxacin with four antimicrobial agents against methicillin resistant S.aureus (MRSA) strains were investigated using decimal assay for additivity (DAA) technique. Synergistic interaction was observed between ciprofloxacin and oxacillin. ciprofloxacin plus cefepime and gentamicin appeared to be additive, while ciprofloxacin plus erythromycin was antagonistic.

Inflammasome Priming Is Similar for Francisella Species That Differentially Induce Inflammasome Activation

Inflammasome activation is a two-step process where step one, priming, prepares the inflammasome for its subsequent activation, by step two. Classically step one can be induced by LPS priming followed by step two, high dose ATP. Furthermore, when IL-18 processing is used as the inflammasome readout, priming occurs before new protein synthesis. In this context, how intracellular pathogens such as Francisella activate the inflammasome is incompletely understood, particularly regarding the relative importance of priming versus activation steps. To better understand these events we compared Francisella strains that differ in virulence and ability to induce inflammasome activation for their relative effects on step one vs. step two. When using the rapid priming model, i.e., 30 min priming by live or heat killed Francisella strains (step 1), followed by ATP (step 2), we found no difference in IL-18 release, p20 caspase-1 release and ASC oligomerization between Francisella strains (F. novicida, F. holarctica –LVS and F. tularensis Schu S4). This priming is fast, independent of bacteria viability, internalization and phagosome escape, but requires TLR2-mediated ERK phosphorylation. In contrast to their efficient priming capacity, Francisella strains LVS and Schu S4 were impaired in inflammasome triggering compared to F. novicida. Thus, observed differences in inflammasome activation by F. novicida, LVS and Schu S4 depend not on differences in priming but rather on their propensity to trigger the primed inflammasome.

Inhibition of quorum sensing-mediated biofilm formation in Pseudomonas aeruginosa by a locally isolated Bacillus cereus

Quorum sensing has been shown to play a crucial role in Pseudomonas aeruginosa pathogenesis where it activates expression of myriad genes that regulate the production of important virulence factors such as biofilm formation. Antagonism of quorum sensing is an excellent target for antimicrobial therapy and represents a novel approach to combat drug resistance. In this study, Chromobacterium violaceum biosensor strain was employed as a fast, sensitive, reliable, and easy to use tool for rapid screening of soil samples for Quorum Sensing Inhibitors (QSI) and the optimal conditions for maximal QSI production were scrutinized. Screening of 127 soil isolates showed that 43 isolates were able to breakdown the HHL signal. Out of the 43 isolates, 38 isolates were able to inhibit the violet color of the biosensor and to form easily detectable zones of color inhibition around their growth. A confirmatory bioassay was carried out after concentrating the putative positive cell‐free lysates. Three different isolates that belonged to Bacillus cereus group were shown to have QSI activities and their QSI activities were optimized by changing their culture conditions. Further experiments revealed that the cell‐free lysates of these isolates were able to inhibit biofilm formation by P. aeruginosa clinical isolates.

Immunomodulation of RAW 264.7 Murine Macrophage Functions and Antioxidant Activities of 11 Plant Extracts

A group of 11 medicinal plants, including Lavandula pubescens, Trigonella foenugricium, Salsola schweinforthi, Calligonum comosum, Silene succulenta, Silene villosa, Bogonvillea glabra, Cakile maritime, Gomphrene celesoids, Mirabilis jalaba, and Silene nocturna growing in Egypt, were extracted and examined for their immunomodulatory and antioxidant activities. RAW 264.7 cells were recruited to investigate the immunomodulatory effect through multiple parameters analysis. First, the proliferation index of macrophages cells was evaluated revealing that Trigonella foenugricium, Silene succulenta and Silene villosa have a significant cytotoxic effect on RAW cells. Interestingly, we observed enhancement of macrophages phagocytic function of by all extracts except Cakile maritime, Gomphrena celosioides and Silene nocturna. Afterwards, macrophages were challenged by incubation with LPS and the effect of various extracts on inflammatory responses was investigated; the generation of NO from activated macrophage was substantially suppressed by 7 extracts namely, Trigonella foenugricium, Calligonum comosum, Silene succulenta, Bougainvillea glabra, Mirabilis jalaba, Gomphrena celosioides and Silene nocturna. TNF-α was decreased by percentage range from 3.8 to 85.8% and Trigonella foenugricium extract showed the highest inhibition of TNF-α release. All extracts except Trigonella foenugricium, Salsola schweinforthi, Silene succulenta and Mirabilis jalaba significantly inhibited COX-2 production from stimulated macrophage. Moreover, evaluating the potential antioxidant activity of these extracts showed that Trigonella foenugricium, Salsola schweinforthi, Calligonum comosum, Bogonvillea glabra and Mirabilis jalaba exhibited some antioxidant activities. Taken together, our results suggest that some of these extracts may have a considerable antinflammatory and antioxidant effects and may be a potential therapeutic choice in the treatment of inflammatory diseases.

Tyrosine Phosphatase Inhibition Induces an ASC-dependent Pyroptosis

Pyroptosis is a type of cell death in which danger associated molecular patterns (DAMPs) and pathogen associated molecular patterns (PAMPs) induce mononuclear phagocytes to activate caspase-1 and release mature IL-1β. Because the tyrosine kinase inhibitor AG126 can prevent DAMP/PAMP induced activation of caspase-1, we hypothesized that tipping the tyrosine kinase/phosphatase balance toward phosphorylation would promote caspase-1 activation and cell death. THP-1 derived macrophages were therefore treated with the potent specific tyrosine phosphatase inhibitor, sodium orthovanadate (OVN) and analyzed for caspase-1 activation and cell death. OVN induced generalized increase in phosphorylated proteins, IL-1β release and cell death in a time and dose dependent pattern. This OVN induced pyroptosis correlated with speck formations that contained the apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC). Culturing the cells in the presence of extracellular K(+) (known to inhibit ATP dependent pyroptosis), a caspase inhibitor (ZVAD) or down regulating the expression of ASC with stable expression of siASC prevented the OVN induced pyroptosis. These data demonstrate that pyroptotic death is linked to tyrosine phosphatase activity providing novel targets for future pharmacologic interventions.