Autoimmune Cyclopamine in vitro responses trigger demyelination in the CNS. Important examples of this phenomenon include multiple sclerosis (MS), neuromyelitis optica (NMO) and acute disseminated encephalomyelitis (ADEM). Although the direct role of inflammasomes in those diseases remains largely unknown, the use of experimental autoimmune encephalomyelitis (EAE), an animal model of MS, has made the impact of inflammasomes on CNS autoimmune demyelinating

diseases more apparent. Inflammasomes process interleukin-1β (IL-1β) and IL-18 maturation in myeloid cells, such as macrophages and dendritic cells (DCs); and, the basic biological function of inflammasomes is shared between humans and mice. Inflammasome is a multi-protein complex. Formation of the complex leads to pro-caspase-1 self-cleavage and generates active caspase-1, which processes pro-IL-1β and pro-IL-18 to mature IL-1β and IL-18, respectively, and induces cell death termed “pyroptosis”.

Pyroptosis is distinguished from apoptosis DNA/RNA Synthesis inhibitor and necrosis by cytoplasmic swelling and activation of caspase-1. Early plasma membrane rupture by pyroptosis[1-3] leads to the release of mature IL-1β and IL-18 and other cytoplasmic contents to the extracellular space.[4] Inflammasomes are known to sense and are activated by pathogen-associated molecular patterns (PAMPs), as well as damage-associated molecular patterns (DAMPs). The Nod-like receptor (NLR) family pyrin domain containing 3 (NLRP3, also known as NALP3 or CIAS1) inflammasome, is currently the most fully characterized inflammasome. It is known to sense bacteria, fungi, extracellular ATP, amyloid β and uric acid,[5-8] as well as various environmental irritants, such as silica, asbestos and alum.[7, 9-11] In addition to NLRP3, other NLR family members, including NLRP1, NLRC4 (IPAF) and AIM2, are known to have clear physiological functions in vivo upon inflammasome formation;[12] however, their involvement in CNS autoimmunity is not clear. Many excellent

reviews are available selleck chemicals llc in the literature that provide information on the detailed functions and structure of inflammasomes. Further discussion on inflammasomes themselves is therefore spared here. Rather, we look to briefly mention several basic features of inflammasomes below to provide a foundation for later discussions in this review, and to highlight selected recent findings considered crucial to the further study of inflammasomes in CNS autoimmune demyelinating diseases. The multi-protein complex of the NLRP3 inflammasome is comprised of three different proteins; NLRP3, ASC (apoptosis-associated speck like protein containing a caspase recruitment domain), and pro-caspase-1. Other types of inflammasomes have different compositions of proteins, but all have pro-caspase-1; therefore, the release of IL-1β and IL-18 from cells is a major common outcome by all inflammasomes.

Among the five peptides that failed to elicit a response in any subject, GAD201–220 and GAD369–388 were previously shown to be processed and presented by autologous monocytes. T cells that recognize these epitopes are apparently not prevalent or these epitopes are

not processed efficiently. Since none of our experimental results suggest that GAD1–20, GAD73–92 and GAD473–492 are able to be processed and presented, these may simply be cryptic epitopes that are not particularly relevant in GAD65 responses. The results summarized in Fig. 4(b) suggested that both healthy donors and subjects with T1D have GAD65-specific T-cell repertoires that recognize multiple epitopes. We wondered whether having a susceptible https://www.selleckchem.com/products/SP600125.html class II HLA such as DR0401 is sufficient to generate a diverse repertoire of GAD65-specific T cells. To address this question, we examined responses to each of the 15 putative GAD65 epitopes in 11 healthy DR0401 donors and six subjects with T1D diabetes using tetramers. Since our goal for these experiments was to examine the GAD-specific repertoire, irrespective of disease status, CD25+ T cells were depleted as previously described to remove Fludarabine molecular weight regulatory T cells.[19] A summary of the tetramer staining results for all of the subjects tested is shown in Table 2. In these experiments we used

more samples from healthy donors than from subjects with T1D, anticipating that a higher fraction of the healthy subjects might lack detectable T-cell Staurosporine research buy responses to GAD65. However, the positive response rates were not statistically different (9/11 for healthy versus 5/6 for T1D, P = 0·73 Fisher’s exact test). This lack of difference in response rate suggests that depletion of CD25+ cells enabled us to observe the repertoires of both healthy donors and subjects with T1D as intended. Not surprisingly, the number of epitopes detected in each subject varied. The number of responses to GAD65 epitopes

ranged from 0 to 5 in healthy donors, and from 0 to 3 in diabetic subjects (Table 2). There was no statistically significant difference in the number of epitopes detected in these two groups (unpaired Student;s t-test, P = 0·74). This would suggest that GAD65-specific repertoires were equally broad in subjects with T1D and healthy controls. The most commonly observed epitopes included GAD433–452 (six subjects), GAD553–572 (five subjects) and GAD305–324 (four subjects). Additional epitopes, such as GAD473–492, GAD265–284 and GAD113–132, were also positive in multiple subjects. The GAD65 T-cell repertoires selected by healthy and diabetic subjects appear to be similar. However, it has been previously documented that only patients with T1D have expanded memory populations of T cells that recognize β-cell antigens.[20] Therefore, GAD-specific T-cell responses in healthy and diabetic subjects could still differ significantly.

These cells carry an additional plasmid with exogenous BirA ligase under the lac promoter. Bacteria were grown in 1L cultures to mid-logarithmic phase (OD600 0.6–0.8) in Luria-Bertani broth containing ampicillin (100 μg/mL) at 37°C. Recombinant protein production was induced by the addition of 1 mM isopropyl-β-D-thiogalactoside and incubated overnight at 30°C. Biotinylated inclusion bodies containing RTLs were produced and purified using the principles described previously for rat 18 and human RTLs 49. DES TOPO DR-A1*0101/DR-B1*0401(HA-307-319) plasmids for inducible

expression in Schneider S2 cells, a gift from Dr. Lars Fugger, CX5461 were used for cloning of the DR-B1*0401(GAD-555-567) construct, transfection and expression of recombinant four-domain MHC-II as previously reported 45. The correct folding of the recombinant complexes was verified by recognition of anti-HLA-DR conformational sensitive mAb (clone L243, BD pharmingen) in an ELISA-binding assay. Selection of phage find more Abs on biotinylated complexes was performed according to principles described before 50. Briefly, a large human Fab library containing 3.7×1010 different Fab clones was used for the selection. Phages were first preincubated

with streptavidin-coated paramagnetic beads (200 μL; Dynal) to deplete the streptavidin binders. The remaining phages were subsequently used for panning SPTLC1 with decreasing amounts of biotinylated MHC-peptide complexes. The streptavidin-depleted library was incubated in solution with soluble biotinylated RTLs or four-domain DR4–GAD (500 nM for the first round, and 100 nM for the following rounds) for 30 min at room temperature. Streptavidin-coated magnetic beads (200 μL for the first round of selection and 100 μL for the following rounds) were added to the mixture and incubated for 10–15 min at room temperature. The beads were washed extensively 12 times with PBS/0.1% Tween 20 and an additional two washes were

with PBS. Bound phages were eluted with triethylamine (100 mM, 5 min at room temperature), followed by neutralization with Tris-HCl (1M, pH 7.4), and used to infect E. coli TG1 cells (OD=0.5) for 30 min at 37°C. The diversity of the selected Abs was determined by DNA fingerprinting using a restriction endonuclease (BstNI), which is a frequent cutter of Ab V gene sequences. Selected Fab Ab clones were expressed and purified as described before 50. Binding specificity of individual phage clone supernatants and soluble Fab fragments was determined by ELISA using biotinylated two- and four-domain MHC–peptide complexes. ELISA plates (Falcon) were coated overnight with BSA-biotin (1 μg/well). After being washed, the plates were incubated (1 h at room temperature) with streptavidin (10 μg/mL), washed extensively and further incubated (1 h at room temperature) with 5 μg/mL of MHC–peptide complexes.

, 1998). Here, we tested how different routes of immunization can be used to generate immune responses inducing a protection against CDI, with Cwp84 as an antigen. Immunizations by the intragastric route did not induce an increase of seric Cwp84-specific antibody levels and this result was correlated with the very low animal protection from CDI observed. Antigen degradation by gastric

and intestinal secretions, dilution in the intestinal fluids, poor sampling via Peyer’s patches, may all be factors that contribute to the limited efficiency of the oral route. It seems evident that this route requires that antigens ZVADFMK must be protected from degradation by digestive enzymes. The subcutaneous route was the best to induce a high systemic immune response with antibody titres more than twofold higher than that for the intrarectal route. However, in this study, serum Cwp84 antibody titres did not correlate with protection. The best animal protection was observed with the rectal route of immunization. Further studies are needed to specify the immune effectors induced by rectal immunization. Unfortunately, secondary antibodies directed to hamster IgA are not commercially available. This is why we were not able to determine more precisely the specific immune response at the intestinal level. We failed to find evidence of significant neutralization activity against the Cwp84 protease activity in the serum of hamster vaccinated with a protective intrarectal formula

vaccine. These results indicate Thiamine-diphosphate kinase see more that, in this model, protection is probably not only related to neutralizing antibodies and other factors may play an important role in the host immune response against CDI. Because survival correlated poorly with antibodies titres, it is possible that our immunization strategy generated a wider cell-based immunity that induces partial protection. Recent

data on Streptococcus pneumoniae have demonstrated that multiple immune cell types are required for the induction of a protective immunity in a murine model that lacks mature B cells and fails to produce antibody (Mizrachi-Nebenzahl et al., 2003; McCool & Weiser, 2004). Recently, surface proteins such as the SLPs, because they cover the cell almost completely, have been tested in a series of immunizations combined with different systemic and mucosal adjuvants and challenge experiments in Golden Syrian hamsters (Ni Eidhin et al., 2008). None of the immunization regimens conferred complete protection in the hamster model, and antibody stimulation was variable within regimens, and generally modest. Others have demonstrated the benefits of using a protease as components of vaccines against S. pneumoniae for example. Mucosal immunization with caseinolytic protease (ClpP) antigen induced both systemic and mucosal antibodies, and in this way, reduced lung colonization and also protected mice against death. ClpP has been found to be highly immunogenic and conserved among different strains of S.

However, insulin receptors and insulin signaling are not exclusively restricted to skeletal muscle, but can also be buy FK506 observed in vascular cells. Insulin directly targets the endothelial cell where it stimulates NO release from the vascular endothelium in a PI3K-dependent manner that involves the Akt-mediated phosphorylation of eNOS, which leads to vasodilatation [84]. Alternatively, insulin also activates the mitogen-activated protein kinase pathway in endothelial cells, which enhances the generation of the vasoconstrictor ET-1 via ERK1/2 signaling [84,96]. In healthy subjects,

the vasodilatory signal predominates, but if signaling from the insulin receptor to eNOS is inhibited pharmacologically or downregulated by insulin resistance, this can lead to impaired

insulin-mediated vasodilatation or even insulin-stimulated vasoconstriction. In this manner, vascular insulin resistance may contribute to the development of hypertension and impaired overall insulin-stimulated selleck inhibitor glucose uptake [64,73,97]. In obese rats, the insulin-signaling pathways are selectively impaired: insulin-mediated activation of PI3-kinase, Akt and eNOS is impaired, but insulin-mediated activation of ERK1/2 is intact [29,51]. Recently, it has been demonstrated that impaired insulin signaling in endothelial cells, due to reduced IRS2 expression and insulin-induced eNOS phosphorylation, caused attenuation of insulin-induced capillary recruitment and insulin delivery, which in turn reduced glucose uptake by skeletal muscle [64]. Moreover, restoration of insulin-induced eNOS phosphorylation in endothelial cells completely reversed Epothilone B (EPO906, Patupilone) the reduction in capillary recruitment and insulin delivery in

tissue-specific knockout mice lacking Irs2 in endothelial cells and fed a high-fat diet. As a result, glucose uptake by skeletal muscle was restored in these mice. These results show that insulin signaling in endothelial cells plays a pivotal role in the regulation of glucose uptake by skeletal muscle. Notably, during obesity induced by high fat feeding, inflammation and insulin resistance developed in the vasculature well before these responses were detected in the muscle, liver, or adipose tissue [61]. This observation suggests that the vasculature is more susceptible than other tissues to the deleterious effects of nutrient overload, and may play a pathophysiological role in inducing insulin resistance. The contribution of insulin signaling to the regulation of blood pressure in different states of insulin resistance is less unequivocal [108]. In healthy humans, insulin has also been shown to stimulate both ET-1 and NO at the level of the resistance vessels of forearm [11]. Moreover, obese, hypertensive humans show an insulin-induced vasoconstriction [37], as well as increased ET-1-dependent vasoconstrictor tone and decreased NO-dependent vasodilator tone at the level of the resistance arteries [10].

This study examined the ability of the host immune system to discriminate selleck chemicals llc commensal oral bacteria from pathogens at mucosal surfaces, i.e. oral cavity. Serum immunoglobulin (Ig)G antibody reactive with three pathogenic and five commensal oral bacteria in 301 current smokers

(age range 21–66 years) were examined by enzyme-linked immunosorbent assay. Clinical features of periodontal health were used as measures of periodontitis. Antibody to the pathogens and salivary cotinine levels were related positively to disease severity; however, the antibody levels were best described by the clinical disease unrelated to the amount of smoking. The data showed a greater immune response to pathogens than commensals that was related specifically buy BMS-354825 to disease extent, and most noted in black males. Significant correlations in individual patient responses to the pathogens and commensals were lost with an increasing extent of periodontitis and serum

antibody to the pathogens. Antibody to Porphyromonas gingivalis was particularly distinct with respect to the discriminatory nature of the immune responses in recognizing the pathogens. Antibody responses to selected pathogenic and commensal oral microorganisms differed among racial groups and genders. The antibody response to the pathogens was related to disease severity. The level of antibody to the pathogens, and in particular P. gingivalis, was correlated with disease severity in black and male subsets of patients. The amount of smoking did not appear to impact directly serum antibody levels to these oral bacteria. Successful colonization of the oral cavity depends upon the presence of bacterial

attachment sites on the conditioning layer derived from saliva and gingival crevicular fluid coating the oral hard and soft tissues surfaces [1] and microbial accumulation by autogenic and allogenic succession. Initial bacterial colonization by pioneering microorganisms alters the environment and enhances subsequent colonization by species more suited for the new environment (autogenic succession). Allogenic succession also occurs with environmental changes driven by a factor(s) other than those derived from the pioneer microorganisms, including those host-controlled factors Rebamipide [2,3]. The resulting microbial communities or biofilms are complex ecosystems of bacteria that develop over time and are somewhat unique to various ecological niches [2,4,5]. The ecology in an individual evolves over time at the level of the quantity and quality of phyla, genera and species [6–8], as well as the genomic profile of the individual species [9–12]. However, this evolution generally leads to equilibrium between the microbiota and the environment as a climax community. Climax biofilm communities are thought to be unique to each individual and ecological niche in the oral cavity [2,3].

“
“This comprehensive hardback book is divided into 17 sections and has 62 highly regarded contributors from around the world. Dinaciclib nmr The book is well bound with 360 tactile pages and a 15 page index. The introductory chapter neatly describes the internal structure of a muscle fibre. The first section is in turn introductory, with chapters on basic pathology, clinical features and neuromuscular genetics. The remaining 15 sections are focused upon different functional elements of the muscle fibre, as described in the introduction: for example, the sarcolemma, mitochondria and cytoplasmic proteins. Each section is then divided into chapters, usually multiple, such

that the sarcolemmal section, for example, contains 4 chapters on dystrophin and the associated glycoprotein complex, proteins of the extracellular matrix, plasma membrane and sarcolemmal ion channels. Each chapter produces a useful overview of structurally-related Ferroptosis cancer muscle diseases, including clinically-relevant information, and often MRI images. Histopathological and other images are clearly produced in colour

and well annotated, and there is a realistic representation of electron microscopy. Each chapter is authored by a relevant expert in the field, and the chapters are well edited, they feel like a cohesive body of work. The book certainly has met one of its challenges, to assemble ‘a coherent text that reflects the mood of this rapidly changing field of medical science’. Its purpose is to offer the reader ‘a modern view of the pathology and genetics of muscle disease’ that integrates across the relevant clinical

and scientific specialties. It certainly achieves this objective. By including chapters on myasthenic syndromes and the breadth of acquired inflammatory and toxic conditions, the editors have reflected the body of neuromuscular disease, and have been more inclusive than perhaps other texts. This book is useful as a supportive text or reference but I wouldn’t reach for it while trying to interpret a muscle biopsy. It is not a diagnostic manual. The rate of advancement of knowledge relating to neuromuscular disease is such that providing a printed, up-to-date tome is, in reality, no longer viable. Endonuclease This is acknowledged by the editors, who guide the reader to the best of online resources. It would be a brave (misguided) clinician or scientist who sought out these websites without first grappling with the foundations of neuromuscular genetics and pathology. This text performs this role admirably. “
“Polyglucosan (PG) is an abnormal polysaccharide that, compared to glycogen, has fewer branched points and excessively long peripheral chains that structurally resemble the plant polysaccharide “amylopectin”. Under electron microscopy, PG bodies are round, non-membrane-bound cytoplasmic particles with irregular branched filaments, which often displace myofibrils, leading to Z disk streaming.

The differences in the complexity of the CD8+ T-cell response or the influence

of background genes (e.g. extent of IFN-γ production) may account for the results. Using LCMV infection of naïve C57BL-6-PKO mice Lykens et al. recently showed that heightened antigenic stimulation is responsible for exaggerated T-cell activation [[49]]. They suggested that perforin-dependent cytotoxicity, in addition to promoting viral clearance, regulates T-cell activation by modulating Ag presentation [[49]]. Despite the differences in susceptibility of naïve BALB/c and C57BL/6 PKO mice to LCMV infection, we also observed massive CD8+ T-cell expansion and accelerated LCMV-induced mortality in GP33-vaccinated compared with naïve C57BL/6-PKO Lapatinib mice (data not shown). Thus, the vaccine-induced sensitization to mortality associated with PKO memory CD8+ T cells after LCMV infection is not restricted to BALB/c background. In addition, functional exhaustion of antigen-specific CD8+ T cells is not always associated with chronic infection [[50, 51]]. Chronic infection may be pathogen or host specific and it does not necessarily lead to Ag-specific CD8+ T-cell

exhaustion in all the cases. Although we observed lesser degree of “exhaustion” as characterized by TNF and PD-1 expression in GP283-specific CD8+ T cells compared with NP118-specific CD8+ T cells, viral control was not achieved in the absence of perforin in both cases (Fig. 5). In the absence of perforin, the phenotype of GP283-specific CD8+ T cells appeared “less exhausted” at the time we analyzed them could reflect the see more extent that these cells can regulate cytokine production. In addition, it remained to be elucidated whether encounter with antigen is similar between the NP118- and GP283-specific memory CD8+ T

cells in the PKO mice, not just initially, but throughout the infection course. Previous studies using different models of infection showed that protective immunity mediated by pathogen-specific CD8+ T cells did not correlate with immunodominance hierarchies after infection [[36, 37]]. Based on the results with PKO mice vaccinated with dominant NP118 epitope, we expected that massive antigen-specific memory CD8+ T-cell expansion contributed to the LCMV-induced mortality independent of epitope specificity. Interestingly, Afatinib solubility dmso PKO mice vaccinated with subdominant GP283 epitope survived the LCMV infection even though they contained similar starting memory CD8+ T-cell numbers and underwent similar expansion in numbers as NP118-specfic CD8+ T cells. These results suggested that epitope specificity dictates the LCMV-induced mortality in vaccinated PKO mice. Furthermore, we also observed less cytokine dysregulation, in particular IFN-γ, by GP283-specific CD8+ T cells following LCMV infection. It is unclear which specific parameter(s) influence the cytokine profile of these GP283-specific CD8+ T cells and subsequent vaccine-induced mortality in PKO mice.

This hypothesis is supported by the earlier finding that p53−/−RAG1−/−, p53−/−RAG2−/− and p53−/− SCID mice develop lymphomas at much higher frequency and at a faster rate with short latency than p53−/− mice 20, 32, 33. Some of the conditions that may favor the immune escape of lymphomas in p53−/− mice include (i) their initial development inside the immunologically privileged Selleck SCH727965 site (e.g. thymus microenvironment), (ii) absence of antigenic

epitopes from initial T-cell lymphomas due to negative selection of T cells against lymphoma (T-cell) specific Ag, and/or (iii) lower expression of MHC class I by double positive thymocytes. In the present study, we used a thymoma EG.7 that expresses high level of MHC class I (data not shown), is immunogenic 34, 35 and was inoculated outside of thymus, which may facilitate the generation of immune responses against them. In summary we have shown a previously unknown function of p53 in negative regulation of T-cell proliferation and generation of anti-tumor CTL responses. In addition to the roles described here, p53 may regulate apoptosis and/or cell cycle checkpoint of T cells under other conditions, e.g. during proliferation of immature double negative (CD4−CD8−) T cells, etc. and dysregulation of these mechanisms may lead to development of lymphomas

in p53−/− mice. Reactivation of p53 or p53 pathways by drugs has been sought Racecadotril as a therapeutic treatment toward tumors 28–31. Data presented herein suggest that systemic administration of BGB324 these drugs will negatively affect the T-cell responses against tumors. Since p53 has multiple downstream effector molecules, it may be possible that p53 effector molecule (s) in T cells differ from those

required for induction of apoptosis in tumor cells. Identification of such T-cell-specific p53 effector molecule(s) will help in designing better therapeutics in controlling tumors under a general systemic p53 activation conditions and/or in generation of better effector T cells against tumors. C57BL/6, p53+/− (backcrossed to C57BL/6) and BALB/c were obtained from Jackson Laboratory (Bar harbor, ME, USA). p53+/− mice were interbred to get p53−/− mice. Mice were handled according to procedures and guidelines approved by the Institutional Animal Care Use Committee. Functional grade or fluorochrome labeled antibodies against CD4 (clone GK1.5), CD8 (clone 53–6.7), CD25 (clone PC61), CD3-ε (clone 145-2C11), CD28 (clone 37.51), CD69 (clone H1.2F3) and anti-B7.1 (clone 16-10A1) and anti-B7.2 (clone GL1) were purchased from eBiosciences (San Diego, CA, USA). Annexin-V-PE and 7-AAD were from BD biosciences (San Diego, CA, USA).

Diameters were determined for n = 72 beads and were 136 µm (range 74–205 µm) for LB and 40 µm (range 15–85 µm) for SB (Fig. 1a). Using the formula for sphere volume = 4/3 ×π×r3, the LB were found to have a mean volume of 1 317 000 µm3 compared to 34 000 µm3 for the SB, giving a ratio difference in volume of 38·7 between LB and SB. Using the formula for sphere surface area = 4 × p ×r2, the LB were found to have a surface area of 58 107 mm2 compared to 5027 mm2 for the SB, giving a ratio difference in surface area of 11·6 between Neratinib order LB and SB. Because both groups received the same amount of bacteria and alginate, this provides a larger total surface area of the SB of 3·3 (38·7/11·6 = 3·3).

In addition, the volume of alginate in the two bead suspensions was adjusted to ensure equal volumes of alginate in the two groups. At day 1 after challenge, a significantly higher number of CFUs was observed in the lungs of SB group compared to the LB group (P < 0·003) (Fig. 2). At days 3, 5 and 6 no significant differences in quantitative bacteriology were observed between the two groups. P. aeruginosa could be cultured from the majority of mice at all time-points (Fig. 2). Four mice from each group were killed 2 h after infection, and lungs examined for

number of CFUs to confirm that the infection dose was equal in the two groups. No significant differences were observed in CFUs 2 h after challenge (Fig. 2). As expected, a PMN-dominated Vemurafenib supplier inflammation was observed in all mice at day 1 after infection (Table 1). However, in the SB group the inflammation was located exclusively endobronchially, in contrast to a partially mixed localization in the LB group (Table 1). In the SB group this shifted

significantly to a mixed localization or exclusively parenchymal localization on days 2/3 after challenge (P < 0·005, Table 1), and in general was paralleled by a more peripheral presence of the bacteria in the alveoli of the SB group. For the SB group, a significantly faster resolution of inflammation at days 5/6 compared to the LB group was observed (P < 0·03, Table 1). For both groups together, a significant increase in degree of inflammation from day 1 to days 2/3 was observed (P < 0·01, Table 1). However, the difference between the two groups for this observation did not reach significance. selleck inhibitor The area of the biofilm-like structures identified by Alcian blue staining were significantly smaller in the SB group compared to the LB group at day 1 and days 2/3 (P < 0·001, Figs 3 and 4). In accordance, the area of the airways in which biofilm-like structures were identified were significantly smaller in the SB group compared to the LB group at days 2/3 (P < 0·002, Figs 3 and 4). The number of identified biofilm-like structures was 137 in the LB group versus 308 in the SB group. PNA-FISH and DAPI staining confirmed the presence of P. aeruginosa in the biofilm-like structures (Fig. 5).