The role of lipids and protein kinase Cs in the pathogenesis of diabetic retinopathy

Diabetic retinopathy is one of the most common complications of diabetes and is a major cause of new blindness in the working-age population of developed countries. While the exact pathogenic basis of this condition remains ill defined, it is clear that hyperglycaemia is a critical factor in its aetiology. Protein kinase C (PKC) activation is one of the sequelae of hyperglycaemia and it is thought to play an important role in the development of diabetic complications. This review questions the currently held dogma that PKC stimulation in diabetes is solely mediated through the overproduction of palmitate and oleate enriched diacylglycerols. Blood glucose concentrations are closely tracked by changes in the levels of free fatty acids and these, in addition to oxidative stress, may account for the aberrant activation of PKCs in diabetes. Little is known about why PKCs fail to downregulate in diabetes and efforts should be directed towards acquiring such information. Considerable evidence implicates the PKCbeta isoform in the pathogenesis of diabetic retinopathy, but other isoforms may also be of relevance. In addition to PKCs, it is evident that novel diacyglycerol-activated non-kinase receptors could also play a role in the development of diabetic complications. Therapeutic agents have been developed to inhibit specific PKC isoforms and PKCbeta antagonists are currently undergoing clinical trials to test their toxicity and efficacy in suppressing diabetic complications. The likely impact of these drugs in the treatment of diabetic patients is considered.

Analysis of alteration of p75NTR processing and signalling by PS2 mutation and gamma-secretase inhibition

The presenilins (PSs) were identified as causative genes in cases of early-onset familial Alzheimer's disease (AD) and current evidence indicates that PSs are part of the gamma-secretase complex responsible for proteolytic processing of type I membrane proteins. p75NTR, a common neurotrophin receptor, was shown to be subject to gamma-secretase processing. However, it is not clear if the p75NTR downstream signal is altered in response to gamma-secretase cleavage, and further there is a possibility that AD-related PS mutations may affect this cleavage, resulting in pathogenic alterations in signal transduction. In this study, we confirmed that p75NTR downstream signalling is altered by PS2 mutation or gamma-secretase inhibition in SHSY-5Y cells. The activity of the small GTPase RhoA is strongly affected by these treatments. This study demonstrates that gamma-secretase and PS2 play an important role in regulating neurotrophin signal transduction and either mutation of PS2 or inhibition of gamma-secretase disturbs this function.

Risk of Alzheimer's disease is associated with a very low-density lipoprotein receptor genotype in Northern Ireland

The epsilon-4 allele of apolipoprotein E (APOE) is associated with increased risk of Alzheimer's disease (AD), but the pathogenic mechanism is unknown. The 5-repeat allele of a CGG repeat polymorphism in the 5' untranslated region of the very low-density lipoprotein receptor (VLDL-R) gene, a receptor for apoE, has been found to be associated with increased risk of AD in a Japanese population. Other groups have been unable to replicate this in American Caucasian populations. A case-control study utilizing a clinically well-defined group of late-onset AD patients (n = 108) and age- and sex-matched control subjects (n = 108) from Northern Ireland was performed to test this association in a relatively homogeneous population. The 9,9 genotype of the VLDL-R was found to be significantly increased in patients compared to controls (P = 0.003; Pcorr = 0.035), leading to an increased risk of AD to subjects with this genotype (OR = 3.9; 95% CI, 1.52-11.25). In contrast to results from the Japanese study, the 5-repeat allele was found to be significantly reduced in the patient group when compared to controls (P = 0.008; Pcorr = 0.047). The results from this study suggest that individuals who have the 9,9 genotype of the VLDL-R gene are at increased risk of AD in Northern Ireland.

A whole-genome massively parallel sequencing analysis of BRCA1 mutant oestrogen receptor-negative and -positive breast cancers

BRCA1 encodes a tumour suppressor protein that plays pivotal roles in homologous recombination (HR) DNA repair, cell-cycle checkpoints, and transcriptional regulation. BRCA1 germline mutations confer a high risk of early-onset breast and ovarian cancer. In more than 80% of cases, tumours arising in BRCA1 germline mutation carriers are oestrogen receptor (ER)-negative; however, up to 15% are ER-positive. It has been suggested that BRCA1 ER-positive breast cancers constitute sporadic cancers arising in the context of a BRCA1 germline mutation rather than being causally related to BRCA1 loss-of-function. Whole-genome massively parallel sequencing of ER-positive and ER-negative BRCA1 breast cancers, and their respective germline DNAs, was used to characterize the genetic landscape of BRCA1 cancers at base-pair resolution. Only BRCA1 germline mutations, somatic loss of the wild-type allele, and TP53 somatic mutations were recurrently found in the index cases. BRCA1 breast cancers displayed a mutational signature consistent with that caused by lack of HR DNA repair in both ER-positive and ER-negative cases. Sequencing analysis of independent cohorts of hereditary BRCA1 and sporadic non-BRCA1 breast cancers for the presence of recurrent pathogenic mutations and/or homozygous deletions found in the index cases revealed that DAPK3, TMEM135, KIAA1797, PDE4D, and GATA4 are potential additional drivers of breast cancers. This study demonstrates that BRCA1 pathogenic germline mutations coupled with somatic loss of the wild-type allele are not sufficient for hereditary breast cancers to display an ER-negative phenotype, and has led to the identification of three potential novel breast cancer genes (ie DAPK3, TMEM135, and GATA4).

Defense peptides secreted by helminth pathogens: antimicrobial and/or immunomodulator molecules?

Host defense peptides (HDPs) are an evolutionarily conserved component of the innate immune response found in all living species. They possess antimicrobial activities against a broad range of organisms including bacteria, fungi, eukaryotic parasites, and viruses. HDPs also have the ability to enhance immune responses by acting as immunomodulators. We discovered a new family of HDPs derived from pathogenic helminth (worms) that cause enormous disease in animals and humans worldwide. The discovery of these peptides was based on their similar biochemical and functional characteristics to the human defense peptide LL-37. We propose that these new peptides modulate the immune response via molecular mimicry of mammalian HDPs thus providing a mechanism behind the anti-inflammatory properties of helminth infections.

Immune reactions to Bacteroides fragilis populations with three different types of capsule in a model of infection

The survival and growth of populations of the obligately anaerobic pathogenic bacterium Bacteroides fragilis enriched for large capsules (LCs), small capsules (SCs) or an electron-dense layer (EDL; non-capsulate by light microscopy) were examined in a mouse model of infection over a minimum period of 20 d. Chambers which allowed the influx of leukocytes, but not the efflux of bacteria, were implanted in the mouse peritoneal cavity. The LC and EDL populations consistently attained viable cell densities of the order of 10(8)-10(9) c.f.u. ml-1 within 24 h, whereas the SC population did not. However, after 3 d, all three bacterial populations maintained total viable numbers of 10(8)-10(9) c.f.u. ml-1 within the chambers. LC expression was selected against within 24 h in the model, the populations becoming non-capsulate by light microscopy, whereas in the SC population expression of the SC was retained by approximately 90% of the population. The EDL population remained non-capsulate by light microscopy throughout. Lymphocytes infiltrated the chambers to an equal extent for all three B. fragilis populations and at approximately 1000 times higher concentration than chambers which contained only quarter-strength Ringer's solution. The presence of neutrophils within the chambers did not cause a decrease in the total viable bacterial count. Each population elicited antibodies specific for outer-membrane proteins and polysaccharide, as detected by immunoblotting, which cross-reacted with the other populations. Differences were observed in the immunogenicity of the outer-membrane proteins within the three populations. Neutrophils were initially the predominant cell type in the chambers, but as the total leukocyte count increased with incubation time, neutrophils were outnumbered by other leukocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

A comparison of the haemagglutinating and enzymic activities of Bacteroides fragilis whole cells and outer membrane vesicles

The haemagglutinating and enzymic activities of the obligately anaerobic pathogenic bacterium Bacteroides fragilis were examined. Outer membrane vesicles are released from the surface of B. fragilis. They can be detected by electron microscopy in ultrathin sections and bacterial suspensions after negative staining. Electron microscopy and immunogold labelling with a MAb specific for surface polysaccharide of B. fragilis confirmed that the vesicles carried outer membrane associated epitopes. The haemagglutinating activity of whole cells from populations of B. fragilis strains NCTC9343, BE3 and LS66 enriched by Percoll density gradient centrifugation for a large capsule (LC), electron dense layer (EDL); non-capsulate by light microscopy) and outer membrane vesicles (OMV) which had been purified by centrifugation from EDL-enriched populations were compared using human and horse erythrocytes. The enzymic activity of OMV, LC- and EDL-enriched populations, as detected by the API ZYM kit, was compared for strains NCTC 9343 and BE3. Purified OMV from the strains examined exhibited both haemagglutinating and enzymatic activity. Haemagglutination by the EDL-enriched population was sensitive to treatment with sodium periodate. The LC-enriched population haemagglutinated only after ultrasonic removal of the capsule. This indicates that the LC masks a haemagglutinin. The results suggest a potential role for OMV in the virulence of B. fragilis.

The unexpected discovery of a novel low-oxygen-activated locus for the anoxic persistence of Burkholderia cenocepacia

Burkholderia cenocepacia is a Gram-negative aerobic bacterium that belongs to a group of opportunistic pathogens displaying diverse environmental and pathogenic lifestyles. B. cenocepacia is known for its ability to cause lung infections in people with cystic fibrosis and it possesses a large 8?Mb multireplicon genome encoding a wide array of pathogenicity and fitness genes. Transcriptomic profiling across nine growth conditions was performed to identify the global gene expression changes made when B. cenocepacia changes niches from an environmental lifestyle to infection. In comparison to exponential growth, the results demonstrated that B. cenocepacia changes expression of over one-quarter of its genome during conditions of growth arrest, stationary phase and surprisingly, under reduced oxygen concentrations (6% instead of 20.9% normal atmospheric conditions). Multiple virulence factors are upregulated during these growth arrest conditions. A unique discovery from the comparative expression analysis was the identification of a distinct, co-regulated 50-gene cluster that was significantly upregulated during growth under low oxygen conditions. This gene cluster was designated the low-oxygen-activated (lxa) locus and encodes six universal stress proteins and proteins predicted to be involved in metabolism, transport, electron transfer and regulation. Deletion of the lxa locus resulted in B. cenocepacia mutants with aerobic growth deficiencies in minimal medium and compromised viability after prolonged incubation in the absence of oxygen. In summary, transcriptomic profiling of B. cenocepacia revealed an unexpected ability of aerobic Burkholderia to persist in the absence of oxygen and identified the novel lxa locus as key determinant of this important ecophysiological trait.

Deciphering the Acylation Pattern of Yersinia enterocolitica Lipid A

Pathogenic bacteria may modify their surface to evade the host innate immune response. Yersinia enterocolitica modulates its lipopolysaccharide (LPS) lipid A structure, and the key regulatory signal is temperature. At 21°C, lipid A is hexa-acylated and may be modified with aminoarabinose or palmitate. At 37°C, Y. enterocolitica expresses a tetra-acylated lipid A consistent with the 3'-O-deacylation of the molecule. In this work, by combining genetic and mass spectrometric analysis, we establish that Y. enterocolitica encodes a lipid A deacylase, LpxR, responsible for the lipid A structure observed at 37°C. Western blot analyses indicate that LpxR exhibits latency at 21°C, deacylation of lipid A is not observed despite the expression of LpxR in the membrane. Aminoarabinose-modified lipid A is involved in the latency. 3-D modelling, docking and site-directed mutagenesis experiments showed that LpxR D31 reduces the active site cavity volume so that aminoarabinose containing Kdo(2)-lipid A cannot be accommodated and, therefore, not deacylated. Our data revealed that the expression of lpxR is negatively controlled by RovA and PhoPQ which are necessary for the lipid A modification with aminoarabinose. Next, we investigated the role of lipid A structural plasticity conferred by LpxR on the expression/function of Y. enterocolitica virulence factors. We present evidence that motility and invasion of eukaryotic cells were reduced in the lpxR mutant grown at 21°C. Mechanistically, our data revealed that the expressions of flhDC and rovA, regulators controlling the flagellar regulon and invasin respectively, were down-regulated in the mutant. In contrast, the levels of the virulence plasmid (pYV)-encoded virulence factors Yops and YadA were not affected in the lpxR mutant. Finally, we establish that the low inflammatory response associated to Y. enterocolitica infections is the sum of the anti-inflammatory action exerted by pYV-encoded YopP and the reduced activation of the LPS receptor by a LpxR-dependent deacylated LPS.

The lipopolysaccharide core of Brucella abortus acts as a shield against innate immunity recognition

Innate immunity recognizes bacterial molecules bearing pathogen-associated molecular patterns to launch inflammatory responses leading to the activation of adaptive immunity. However, the lipopolysaccharide (LPS) of the gram-negative bacterium Brucella lacks a marked pathogen-associated molecular pattern, and it has been postulated that this delays the development of immunity, creating a gap that is critical for the bacterium to reach the intracellular replicative niche. We found that a B. abortus mutant in the wadC gene displayed a disrupted LPS core while keeping both the LPS O-polysaccharide and lipid A. In mice, the wadC mutant induced proinflammatory responses and was attenuated. In addition, it was sensitive to killing by non-immune serum and bactericidal peptides and did not multiply in dendritic cells being targeted to lysosomal compartments. In contrast to wild type B. abortus, the wadC mutant induced dendritic cell maturation and secretion of pro-inflammatory cytokines. All these properties were reproduced by the wadC mutant purified LPS in a TLR4-dependent manner. Moreover, the core-mutated LPS displayed an increased binding to MD-2, the TLR4 co-receptor leading to subsequent increase in intracellular signaling. Here we show that Brucella escapes recognition in early stages of infection by expressing a shield against recognition by innate immunity in its LPS core and identify a novel virulence mechanism in intracellular pathogenic gram-negative bacteria. These results also encourage for an improvement in the generation of novel bacterial vaccines.

Experimental pig yersiniosis to assess attenuation of Yersinia enterocolitica O:8 mutant strains

An experimental oral pig model was used to assess the pathogenic and immunogenic potential of Yersinia enterocolitica serotype O:8 wild-type strain 8081-L2 and its lipopolysaccharide (LPS) mutant derivatives: a spontaneous rough mutant 8081-R2, strain 8081-DeltawzzGB expressing O-antigen with uncontrolled chain lengths, and strain 8081-wbcEGB expressing semirough LPS with only one O-unit. Microbiological and immunological parameters of the infected pigs were followed from day 7 to 60 postinfection. The wild-type and all LPS mutant strains persisted in the lymphoid tissue of tonsils and small intestines, causing asymptomatic infection without any pathological changes. Although the pig is known as a reservoir of Yersiniae, a precise analysis of pathogenic and immunogenic parameters based on different in vitro tests (hematological response, killing ability of leukocytes and blood sera, antibody response, hydrogen peroxide production by macrophages, classical and alternative pathways of complement activation), revealed significant attenuation in the pathogenicity of the LPS mutant strains but not the loss of immunogenic potential. In comparison with the other strains, strain 8081-DeltawzzGB demonstrated more continuous leucocytosis with monocytosis, higher invasive potential, significant activation of hydrogen peroxide production by macrophages and an effective immunoglobulin G immune response accompanied by relevant histological immunomorphological rearrangements.

Capsule polysaccharide mediates bacterial resistance to antimicrobial peptides

The innate immune system plays a critical role in the defense of areas exposed to microorganisms. There is an increasing body of evidence indicating that antimicrobial peptides and proteins (APs) are one of the most important weapons of this system and that they make up the protective front for the respiratory tract. On the other hand, it is known that pathogenic organisms have developed countermeasures to resist these agents such as reducing the net negative charge of the bacterial membranes. Here we report the characterization of a novel mechanism of resistance to APs that is dependent on the bacterial capsule polysaccharide (CPS). Klebsiella pneumoniae CPS mutant was more sensitive than the wild type to human neutrophil defensin 1, beta-defensin 1, lactoferrin, protamine sulfate, and polymyxin B. K. pneumoniae lipopolysaccharide O antigen did not play an important role in AP resistance, and CPS was the only factor conferring protection against polymyxin B in strains lacking O antigen. In addition, we found a significant correlation between the amount of CPS expressed by a given strain and the resistance to polymyxin B. We also showed that K. pneumoniae CPS mutant bound more polymyxin B than the wild-type strain with a concomitant increased in the self-promoted pathway. Taken together, our results suggest that CPS protects bacteria by limiting the interaction of APs with the surface. Finally, we report that K. pneumoniae increased the amount of CPS and upregulated cps transcription when grown in the presence of polymyxin B and lactoferrin.

Yersinia pseudotuberculosis and Yersinia pestis show increased outer membrane permeability to hydrophobic agents which correlates with lipopolysaccharide acyl-chain fluidity

The hydrophobic probe N-phenyl-1-naphthylamine accumulated less in non-pathogenic Yersinia spp. and non-pathogenic and pathogenic Yersinia enterocolitica than in Yersinia pseudotuberculosis or Yersinia pestis. This was largely due to differences in the activity of efflux systems, but also to differences in outer membrane permeability because uptake of the probe in KCN/arsenate-poisoned cells was slower in the former group than in Y. pseudotuberculosis and Y. pestis. The probe accumulation rate was higher in Y. pseudotuberculosis and Y. pestis grown at 37 degrees C than at 26 degrees C and was always highest in Y. pestis. These yersiniae had LPSs with shorter polysaccharides than Y. enterocolitica, particularly when grown at 37 degrees C. Gelliquid-crystalline phase transitions (Tc 28-31 degrees C) were observed in LPS aggregates of Y. enterocolitica grown at 26 and 37 degrees C, with no differences between non-pathogenic and pathogenic strains. Y. pseudotuberculosis and Y. pestis LPSs showed no phase transitions and, although the fluidity of LPSs of Y. pseudotuberculosis and Y. enterocolitica grown at 26 degrees C were close below the Tc of the latter, they were always in a more fluid state than Y. enterocolitica LPS. Comparison with previous studies of Salmonella choleraesuis subsp. choleraesuis serotype minnesota rough LPS showed that the increased fluidity and absence of transition of Y. pseudotuberculosis and Y. pestis LPSs cannot be explained by their shorter polysaccharides and suggested differences at the lipid A/core level. It is proposed that differences in LPS-LPS interactions and efflux activity explain the above observations and reflect the adaptation of Yersinia spp. to different habitats.

Yersinia pseudotuberculosis and Yersinia pestis are more resistant to bactericidal cationic peptides than Yersinia enterocolitica

The action of bactericidal polycationic peptides was compared in Yersinia spp. by testing peptide binding to live cells and changes in outer membrane (OM) morphology and permeability. Moreover, polycation interaction with LPS was studied by measuring the dependence of dansylcadaverine displacement and zeta potential on polycation concentration. When growth at 37 degrees C, Yersinia pestis and Yersinia pseudotuberculosis bound less polymyxin B (PMB) than pathogenic or non-pathogenic Yersinia enterocolitica, regardless of virulence plasmid expression. Y. pseudotuberculosis OMs were unharmed by PMB concentrations causing extensive OM blebbing in Y. enterocolitica. The permeability to lysozyme caused by PMB was greater in Y. enterocolitica than in Y. pseudotuberculosis or Y. pestis and differences increased at 37 degrees C. Similar observations were made with other polycations using a polymyxin/novobiocin permeability assay. With LPS of cells grown at 26 degrees C, polycation binding was highest for Y. pseudotuberculosis and lowest for Y. pestis, with Y. enterocolitica yielding intermediate results which were lower for pathogenic than for non-pathogenic strains. With LPS of cells grown at 37 degrees C, polycation binding remained unchanged for Y. pestis and pathogenic Y. enterocolitica, increased for non-pathogenic Y. enterocolitica and decreased for Y. pseudotuberculosis to Y. pestis levels. Polycation binding related in part to differences in charge density (zeta potential) of LPS aggregates, suggesting similar effects at bacterial surfaces. It is suggested that species and temperature differences in polycation resistance relate to infection route, invasiveness and intracellular multiplication of Yersinia spp.

Enhanced antimicrobial activities of 1-alkyl-3-methyl imidazolium ionic liquids based on silver or copper containing anions

We have developed a series of 1-alkyl-3-methylimidazolium tetrachlorocuprate(II) and dibromoargentate(I) ionic liquids with enhanced antimicrobial activity when compared with 1-alkyl-3-methylimidazolium chloride ionic liquids. These new ionic liquids proved to be effective against a range of pathogenic bacteria and fungi.