1 was considered seropositive. Ethical issues.  Written informed consent was obtained from parents/guardians who consented on behalf of their children. All laboratory procedures were carried out within the guide lines of good laboratory practice. Ethical clearance to conduct the study was sought from both KCMC Research Ethics Committee and from the National Institute for Medical Research and assigned ethical clearance certificate number NIMR/HQ/R.8a/Vol.

IX/759. Data analysis.  Analysis of data was carried out using Statistical Package for Social Sciences (spss) version 16.0 (SPSS Inc., Chicago, IL, Roxadustat datasheet USA). Categorical data were analysed by using Pearson χ2 test or Fisher’s exact test in the case of counts <5. Student’s t-test statistic was used to determine statistical differences of continuous data across

the genotypes: malaria incidence data were analysed in association with antibody seropositivity, OD readings and genotypes. A P-value < 0.05 was as the cut-off point for statistical significance. Of 747 children genotyped for the c.1264 T>G CD36 mutation, nine (1.2%) were homozygous for the mutation and 27 (3.6%) heterozygous, whereas 711 (95.2%) had the wild-type allele (Table 1). During the 1 year follow-up, only 55 of the 747 study participants (7.4%) had malaria, at least once. Genotype-specific malaria incidence showed higher malaria incidences in homozygous and heterozygous children (44.4% and 55.6%, respectively), compared to children having the wild type who had the lowest incidence of 5.1% (Table 1 and Fig. 1). The difference in malaria incidence between normal children Akt inhibitor and those with either homozygous or heterozygous CD36 polymorphism was statistically significant (χ2 = 115.59; P < 0.01). Overall, seropositivity to MSP-119 increased from 22.5% at baseline to 47.7% after 1 year. Seropositivity Benzatropine to MSP-119 in wild-type and heterozygous children increased from baseline to the final survey, and the increase from baseline to 12 months later was statistically significant (P < 0.05), but declined in CD36 homozygous deficient children slightly from 33.3% to 22.2%. This

drop was not statistically significant. The mean anti-MSP-119 IgG levels (ODs) showed an overall increase across genotypes from baseline to final survey from 36 ± 0.4 to 47 ± 0.4, respectively. Stratified by genotypes, the mean OD levels increased from the baseline to the final survey in normal and heterozygous children from 36 ± 0.5 to 47 ± 0.4 and from 33 ± 3 to 51 ± 0.9, respectively. The increase from baseline to 12 months later was statistically significant (P < 0.05). There was an insignificant decrease in antibody levels from 38 ± 1.4 to 35 ± 2 at the final survey in CD36 deficient children. Results presented in Fig. 2 indicate that four of nine (44.4%) in the homozygous mutant children had malaria, two of which (22.2%) had two malaria attacks. Fifteen children of 27 heterozygous children (55.

IL-10 levels in infected pregnant B6 mice were statistically significantly reduced relative to infected pregnant A/J mice 3-MA solubility dmso on experiment day 9 (median (IQR): 36 (0–46) pg/mL for B6 vs. 550 (431–735) pg/mL

for A/J; P = 0·001), but this pattern was reversed on experiment day 10 (Figure 4a). In both strains, IL-10 levels were enhanced at experiment day 11 in infected pregnant relative to uninfected pregnant mice. Levels of sTNFRII did not differ between infected pregnant A/J and B6 mice at any of the tested time points, although the levels were consistently statistically significantly higher in the infected mice relative to their within strain uninfected counterparts (Figure 4c, d and data not shown). At none of the time points were the differences in IL-10 or sTNFRII observed between infected pregnant and infected non-pregnant mice of either strain nor were across-strain differences between infected non-pregnant mice found (Figure 4). To further evaluate immune changes associated with P. chabaudi AS infection and pregnancy loss in A/J and B6 mice, phenotypes RG7204 mouse and levels of splenic leucocyte subsets were analysed flow cytometrically at experiment days 9 and 10, time points at which mice of both strains retain a proportion of viable conceptuses. No statistically significant differences in B-, natural killer (NK) or T-cell counts, including T-cell subsets, were observed between infected pregnant

A/J and B6 mice (Figure 5). However, malarial infection clearly stimulated expansion of all of these cell types in pregnant A/J mice, in whom splenocyte numbers for all subsets (except T cells at experiment day 9) were statistically significantly higher relative to their uninfected pregnant counterparts (Figure 5).

Similar differences in B6 mice were noted only for T, CD8+ T, B and NK cells on experiment day 9, but not on 10 (Figure 5). The total number of lymphocytes and lymphocyte subsets in general did not differ between infected pregnant and infected non-pregnant mice within each strain; only CD4+ T cells on experiment day 9 were significantly expanded in infected pregnant Histone demethylase relative to infected non-pregnant A/J mice (Figure 5c). Similar to the lymphocyte subsets, numbers of neutrophils, monocytes and monocytes with an inflammatory phenotype (CD11b+/CD115+/Gr1high) were similar in the infected pregnant B6 and A/J mouse spleens on experiment days 9 and 10 (Figure 6). Neutrophil levels were enhanced in infected B6 mice at experiment day 9 relative to uninfected pregnant B6 mice (Figure 6a), a difference that did not reach statistical significance on experiment day 10 (Figure 6b: Kruskal–Wallis, P = 0·0024; Dunn’s pairwise comparisons, all P > 0·05). Monocytes levels were increased in infected pregnant B6 mice compared to their uninfected counterparts on experiment days 9 and 10, and on the former day were also higher than in infected non-pregnant mice (Figure 6c, d).

8 In a study by Schreiber et al. 44% of 85 patients had progression of ARVD on mean follow up of 52 months. A total of 16% progressed to total occlusion. Half the patients with less than 50% stenosis

demonstrated no change in the sequential angiogram. The rate of progression to complete occlusion was 39% in the ‘75–99% stenosis’ group compared with 5% in the ‘<50%’ group. The average monthly rate of progression in the three patient groups (<50%, 50–75%, 75–99%) were 1.59, 1.37 and 2.01, respectively.9 Dean et al. performed a subset analysis of a prospective randomized study and reported progression in patients designated to the medical management arm. The method of randomization was not specified. Over a mean follow-up period of 28 months, progression to Selleckchem Antiinfection Compound Library total occlusion occurred in

four patients (12%). No data were provided regarding the baseline degree of stenosis in these arteries.10 Renal duplex sonography (RDS), although fraught with drawbacks of reproducibility and availability of technical expertise, is currently considered a useful tool for monitoring ARVD when optimal sonographic conditions can be ensured. A number of studies have looked at the stenosis progression with RDS. A large prospective observational study by Caps et al. looked at 295 renal arteries in 170 patients over a 5-year period using RDS. They used the principle that blood flow velocity across the stenosis was proportional to the degree of vessel diameter reduction. An increase in peak BVD-523 in vivo systolic velocity (PSV) of ≥100 cm/s was derived as being significant based on the between-observer variability for renal artery PSV measurements. Disease progression was defined as any detectable increase in the degree diameter reduction in the renal artery, including renal artery occlusion. The 3-year cumulative incidence

of renal artery disease progression was 18%, 28% and 49% for renal arteries initially classified as normal, <60% stenosis and ≥60% stenosis, respectively. Systolic blood pressure (BP) ≥ 160 mmHg, diabetes mellitus, ipsilateral or contralateral stenosis ≥ 60%, and occlusion of contralateral Carbohydrate renal artery were identified as independent risk factors for stenosis progression in a stepwise Cox proportional hazard analysis.11 Study limitations, apart from being observational included: selected patients had hypertension or reduced kidney function. Patients with ARVD and normal BP and renal function were not included. Despite these limitations, this study provides insight into the risk factors associated with the progression of stenosis. The first population-based prospective study looking at incident RAS and its progression was reported by Pearce et al. in 2006.

). A band with a molecular weight of about 46 000 with a faint band underneath appeared, which was greatly enhanced when B cells were activated with CD40L + IL-4 (Fig. 1c). AID and A3G mRNA expression were then evaluated by real-time PCR. All results were expressed as mean (± SEM) relative to unstimulated cells, which were accorded an arbitrary value of 100. CD40L induced an increase in AID mRNA from 100 to 258 (± 131) and to a lesser extent in A3G mRNA to 128 (± 13), these failed to reach the 5% level of significance (Table 1). However, IL-4 significantly 5-Fluoracil research buy up-regulated AID (P = 0·037) but not A3G (P = 0·29). The combined CD40L + IL-4 B-cell agonists up-regulated significantly both

AID and A3G mRNA (P < 0·05), and this was much greater for AID than A3G mRNA (Table 1). CD40L + HLA class II antibodies (177 ± 25) was more effective for A3G mRNA (P = 0·027) than that for AID mRNA (295 ± 128, P = 0·11). As with immunofluorescence the other B-cell agonists were not pursued further. The results suggest that CD40L + IL-4 H 89 yielded the most consistent and significant increases in both mRNA and protein of AID and A3G. We have evaluated a major function of AID in B cells by demonstrating a significant

increase in the cell-surface expressions of IgG (P < 0·001) and IgA (P < 0·0001) (Fig. 2b,c) when stimulated with CD40L + HLA-II mAb and to a lesser extent with CD40L + IL-4 (P < 0·03). The IgM also increased but to a greater extent with CD40L + IL-4 than CD40L + HLA-II mAb (Fig. 2a). These studies were then extended to the culture supernatants of the B-cell-agonist-stimulated cells. Using the Luminex bead technology confirmed the increase in IgA antibodies in the 4-day culture supernatants

of CD40L + IL-4-stimulated B cells (Fig. 3). The 6·4-fold higher concentration of IgA compared with IgG1 was surprising as the reverse is normally found Ribonucleotide reductase in serum. This might be related to the shorter half-life of IgA (about 9 days) compared with that of IgG (about 21 days). After 7 days of culture, the supernatants showed a significant increase in IgG4 by stimulation with CD40L + IL-4 (P = 0·01), though the total concentration was moderate (12·6 ± 5·4 ng/ml) (Fig. 3b). A functional effect of up-regulation of A3G by stimulating primary B cells with the selected agonists was studied in HIV-1 (BaL) infectivity of autologous CD4+ T cells. Isolated B cells were stimulated with CD40L + IL-4 or HLA-II mAb for 3 days, followed by co-culturing the B cells with autologous CD4+ T cells (activated with phytohaemagglutinin and IL-2 for 3 days) and infected with serial dilution HIV-1 (BaL) for 9 days. The results showed dose-dependent inhibition of HIV-1 replication with the B cells pre-treated with either of the B-cell agonists, compared with the untreated B cells (Fig. 4a,b).

Escherichia coli-derived rat MOG1–125 was produced as previously described [21]. MOG consists of aa 1–125 of the extracellular part of native MOG and a histidin tag at the C terminus. For in vivo ablation of DCs, CD11c-DTR mice that carry a transgene encoding a simian DTR-GFP fusion protein under the control of the murine CD11c PD-332991 promoter were generated as described [1] and obtained from Jackson Laboratory (Bar Harbor, ME, USA). C57BL/6 female

mice, obtained from Taconic (Denmark), were bred at the animal house at Rudbeck laboratories, Uppsala University. All animals were kept at specific pathogen-free conditions and all studies have been reviewed and approved by the local ethical committee and all experiments were carried out in accordance with EU Directive 2010/63/EU. Femur and tibiae this website bones were removed from euthanized CD11c-DTR female mice. Bone marrow was flushed out with DMEM supplemented with 10% FCS, 100 U/mL penicillin, 100 μg/mL streptomycin, and 292 μg/mL L-glutamine (DMEM complete) (all from Invitrogen, Carlsbad, CA, USA). Ten million bone marrow cells were injected i.v. into lethally irradiated (8 Gy) 6-week-old C57BL/6 female mice (Taconic). The bone marrow chimeras rested for 6 weeks before the experiments commenced. Age and sex-matched 9- to 17-week-old female mice were immunized with 200–260 μg of MOG in CFA containing 0.5 mg M.tb H37RA (Difco, BD Diagnostic

systems, Sparks, MD, USA) in IFA (Sigma-Aldrich, St. Louis, MO, USA)

s.c. at the day of immunization and 2 days after, mice were injected with 200 ng of pertussis toxin (Sigma-Aldrich) in 200 μL PBS i.p. Clinical symptoms of EAE were scored daily as follows: 1, tail weakness or tail paralysis; 2, hind leg paraparesis; 3, partial hind leg paralysis; 4, complete hind leg paralysis; 5, tetraplegia, moribund state or death caused by EAE. To deplete DC in vivo, CD11c-DTR mice or bone marrow chimeras were injected i.p. with 100 ng DTx (Sigma-Aldrich) in 100 μL as previously described [1]. Injection of CD11c-DTR mice or bone marrow chimeras with the same amount of PBS served as a control. To determine the efficiency of the ablation, DCs in dermis (Langerin− CD11c+ MHC II+ or Langerin+), Interleukin-2 receptor skin-draining inguinal LN (CD11chi MHC II+), and spleen (CD11chi MHC II+) from DTx-treated mice were measured by flow cytometry 24 h after DTx injection or 3, 10, or 13 days after MOG immunization. To test whether pDC were also depleted, CD11clo B220+ PDCA-1+ cells in the spleen from DTx-treated mice were measured by flow cytometry 24 h after DTx injection. Spleens were harvested 10 days after MOG immunization or from unimmunized mice, cells were resuspended in DMEM (SVA, Uppsala, Sweden) and filtered through a 40 μm cellstrainer (Falcon BD). Splenocytes were cultured in DMEM complete with or without 5 μg/mL MOG or 5 μg/mL M.tb for 48 h at 37°C and 5% CO2.

A high urinary albumin-to-creatinine ratio (UACR) and low estimated glomerular filtration rate (eGFR) have been believed to be predictors for diabetic end stage kidney disease. However, relationship between clinical manifestation selleck and pathological characteristics of type 2 diabetes is not fully elucidated. We would like to discuss these points in this presentation. Clinical manifestations in progression of diabetic kidney disease

in type 2 diabetes were diverse. Decreasing GFR and increasing UACR are more heterogeneous in type 2 diabetes than type 1 diabetes. Many types of variances of reduced eGFR and/or increased UACR were observed in type 2 diabetes. Our historical cohort study of 4328 Japanese participants with type 2 diabetes from 10 centers (median

follow-up period 7.0 years, interquartile range 3.0–8.0 years) revealed that Selleckchem PI3K Inhibitor Library increased UACR levels were closely related to the increase in risks for renal, cardiovascular events and all-cause mortality. Moreover, an association between high levels of UACR and reduced eGFR was a strong predictor for renal events. These findings reinforced the importance of increased UACR levels and reduced eGFR as prognosis predictors in type 2 diabetes. These clinical manifestations of reduced eGFR and/or increased UACR should depend on pathological changes in kidney. In type 1 diabetes, pathological natural history of diabetic kidney disease, such as basement membrane thickening and increased mesangial matrix, were observed accompanied with reduced GFR and increased UACR. However, pathological changes in kidney, as well as clinical manifestation, are thought to be more heterogeneous in

type 2 diabetes acetylcholine than type 1 diabetes. Although pathological changes should affect on UACR and/or eGFR, particulars of clinic-pathological relationship were unclear so far in type 2 diabetes. To clarify the relation of two major clinical factors (UACR and eGFR) and pathological changes, we are now collecting and evaluating more than two hundred kidney biopsy findings and clinical data from twelve centers in Japan. These data will reveal that some characteristic pathological changes in diabetic kidney disease would participate clinical manifestations of reduced eGFR and/or increased UACR. In addition to the relationship between clinical manifestations and pathological changes, these pathological changes might contribute to kidney prognosis and/or cardiovascular events. Recent our study revealed the relation of pathological changes in diabetic kidney disease and kidney prognosis, cardiovascular events, and all-cause mortality. Kidney biopsy findings and clinical data of 260 Japanese type 2 diabetic patients (mean follow-up period 8.1 years) revealed that glomerular lesions, IFTA, and arteriosclerosis were predictors for renal events, arteriosclerosis for cardiovascular events, and IFTA for all-cause mortality.

Indeed, there is growing evidence that the innate immune system is activated in the maternal–fetal interface. For instance, innate immune cells such as natural Hedgehog inhibitor killer (NK) cells, macrophages and dendritic cells are known to infiltrate the decidua and accumulate around the invading trophoblasts.5–8 In addition

to a population increase, these immune cells acquire an activated phenotype during pregnancy.7,9 Cells of the innate immune system express a series of receptors known as pattern recognition receptors (PRRs) which recognize and bind to sequences know as pathogen-associated molecular patterns (PAMPs), which are unique to, and expressed on, the surface learn more of microorganisms. In addition, non-immune cells such as epithelial cells also express PRRs that allow these cells to respond to PAMPs. The ligation of PRRs by PAMPs results in an inflammatory response generated against the invading pathogen.9 There are a number of different PRRs including the mannose-binding receptor and the scavenger receptor;10 however, this review will focus on the major family of PRRs, the Toll-like receptors (TLRs). We will discuss the expression and function of TLRs at the maternal–fetal interface and their roles in the interaction between the trophoblast and the maternal immune system. Toll-like receptors (TLR) are transmembrane

proteins with extracellular domains of leucine-rich repeat motifs, which are evolutionarily conserved to recognize PAMPs in bacteria, viruses, fungi and parasites. Eleven mammalian TLRs have been identified to date (TLR1 to TLR11);11,12

however, no functional TLR11 proteins have been documented in humans.13,14 Each receptor differs in its specificity (Table I). TLR4 is crucial for effective host cell responses to gram-negative bacterial lipopolysaccharide (LPS).15 TLR2 has the widest specificity, recognizing bacterial Glutathione peroxidase lipoproteins, gram-positive bacterial peptidoglycan (PDG), lipoteichoic acid (LTA) and fungal zymosan.16–18 The range of ligands to which TLR2 responds appears to be broadened by its heterodimerization with other TLRs, so that TLR1/2 heterodimers respond to a panel of lipoproteins different from those recognized by TLR2/6.19,20 TLRs 3, 7 and 8 appear to play important roles in response to viruses. TLR3 is known to bind viral double-stranded RNA,21 while TLRs 7 and 8 interact with single-stranded RNA.22,23 TLR9 mediates cell responses to bacterial DNA through recognition of cytosine–guanine pairs (‘CpG’ motifs)24 and can also be activated by Herpes virus.23,25 In addition to detecting pathogen-derived ligands, TLRs interact with the hosts’ other endogenous molecules, typically in response to danger.

Together, they may affect the antigenic determinant of the C-terminal part of the ZnT8. Comparing Rucaparib in vitro the results on the human patient sera between the short

ZnT8 peptide and the long ZnT8 protein suggests that it should be possible to identify the minimum requirement for the conformational epitope by deletion mutants followed by, for example, alanine replacement scanning. Whether the difference of the binding affinity between the R and W protein in the ZnT8WAb-specific patient, P5-W, may be a result from lack of epitope spreading due to early diabetes-onset (2.3 years) needs to be clarified. Age-specific antibody affinity was previously reported for IAAb in children with high T1D risk [28]. In addition,

it is important to take the HLA-DQ genotype into account as ZnT8WAb and ZnT8QAb were more often found in newly diagnosed patients with HLA-DQ8, while all three ZnT8Ab variants were more often associated with DQ6.4 [29]. Future studies of children at risk of T1D such as the TEDDY [30], DiPiS [31], DAISY [32] and BABY-DIAB CX-5461 cost [33] should therefore take into account not only the HLA genotype but also the SLC30A8 gene polymorphism and the ZnT8Ab variant specificity and affinity in the attempts to predict the clinical onset of autoimmune diabetes. Six patients were selected for the present investigation. The patients are unique as they showed monospecificity to either ZnT8RAb or ZnT8WAb. The analysis required significant volumes of serum which was not always available from patients tested at the time of clinical diagnosis. In our previous study, we have found that 15.6% of the patients had monospecific

ZnT8RAb and 10.3% monospecific ZnT8WAb [16]. A strength to the present study is the novel approach to combine the ZnT8tripleAb screening [16] to first identify subjects with any ZnT8Ab with the monospecific ZnT8 autoantibody assays to be followed by competition analysis with cold protein. In future studies, known amounts of recombinant proteins will be needed to reliably why determine affinity at the 325-associated epitope. Our study should prove useful for further studies of the contribution of epitope-specific ZnT8Ab in the pathogenesis of T1D. We believe that the epitope analysis should be combined with affinity determinations to better define ZnT8Ab-positive subjects at risk of diabetes [30, 31]. In conclusion, the 325-epitope is likely to be dependent on the amino acid residues extending from the short (318–331) peptide. This suggests that the ZnT8Ab are directed against a broader epitope represented than a single amino acid. Further analyses of epitope-specific sera both before and at the clinical diagnosis of diabetes are warranted to dissect the possible importance of ZnT8 epitope-specific autoantibodies and loss of beta cells. We thank Anita Nilsson and Ingrid Wigheden for expert advice.

1% sodium azide (FACS buffer) for 1 h at 4 °C, resuspended in 300 μl of FACS buffer and then analysed by flow cytometry. The data were analysed with CellQuest software (Becton

Dickinson, San Jose, CA, USA). MSC were seeded in a 6-well plate at 5 × 103/cm2 in DMEM containing 10% FCS. After overnight incubation, the medium was replaced with DMEM supplemented with 10% FCS with or without TLR2 [Pam3CS(K)4, 10 μg/ml] or NOD1 ligand (iE-DAP, 10 μg/ml). After 18 h of incubation, culture supernatants were collected and cytokine levels were measured by ELISA according to the manufacturer’s instructions. Human peripheral blood mononuclear cells (PBMCs) https://www.selleckchem.com/products/Everolimus(RAD001).html were prepared by density gradient centrifugation (Lymphoprep) from buffy coats obtained from healthy adult donors. Cells were washed and then resuspended in RPMI-1640 medium containing 10% fetal calf serum (FCS) and antibiotics. To study the effect of MSC on T-cell activation, mixed lymphocyte reaction (MLR) assays were performed in the presence of irradiated allogeneic

MSC. The cells were cocultured in 96-well U-bottom microtiter plates for 5 days. T-cell proliferation was evaluated by incubating cells EPZ015666 mouse with [3H]-thymidine for additional 16 h. Cells were harvested, and 3H- thymidine uptake was measured. All experiments were run in triplicate. Total protein lysates (30–60 μg) were resolved on 10% SDS–polyacrylamide gels and subsequently transferred to nitrocellulose by electrophoresis. Membranes were blocked with 5% non-fat dried milk in PBS containing 0.1% Tween overnight. Subsequent from to washing, membranes were incubated with antibodies against the selected proteins, followed by HRP-conjugated rabbit or mouse secondary antibodies. Antibody–protein complexes were visualized after

exposure to X-ray film by enhanced chemiluminescence reagent. To control for protein loading, the blots were stripped and reprobed with anti-β actin polyclonal antibodies (Santa Cruz Biotech, Santa Cruz, CA, USA). MSC (3 × 106 cells per sample) were treated with TLR-2 [Pam3CS(K)4; 10 μg/ml] or NOD-1 ligand (iE-DAP, 10 μg/ml) for 18 h. Subsequently, they were harvested and total RNA was prepared from controls and treated cells. Each treatment was performed in triplicates, and cells were collected prior total RNA preparation. Control cells were treated with a control peptide (iE-Lys). Total RNA (500 ng per sample) was used to generate complementary biotin UTP-labelled DNA using the Illumina TotalPrep RNA Amplification Kit. Around 1.5 μg of labelled transcripts were used for hybridization to an array according to the Illumina Sentrix humanref-6 beadchip protocol. Following hybridization, the samples were washed and scanned with a BeadArray Reader (Illumina). Expression values were extracted and normalized by the BeadStudio software. Freshly isolated human monocytes were transfected with siRNA using the BTX electroporation apparatus as described previously [16].

The sample remains frozen during Selleckchem XL765 imaging on the cold stage, which is cooled by liquid nitrogen. Even though the frozen state stabilizes the soft material and liquids of the biofilm (which would otherwise be impossible to examine at high magnification, because

of sample movement or beam damage), the cryo-SEM images (Fig. 3) appear to be of a lower resolution compared to conventional SEM. This is partly attributable to a lower conductivity of the frozen surface compared to the dehydrated gold-sputtered surface we employ in conventional SEM. Another downside of cryo-SEM is that the frozen surface melts and cracks at high magnifications because of the heat generated by the focused electron beam. However, we were able to produce images of the biofilm that clearly show that the bacteria are enveloped in a gel-like matrix. We were not able to

obtain high-magnification images showing details of the matrix. Cryo-SEM also allows for freeze-fracture, which exposes the internal structure of the biofilm and may thus reveal how the bacteria are interconnected. The ESEM was developed in the late ATM/ATR inhibitor drugs 1980s (Danilatos, 1988). The ESEM retains many of the advantages of a conventional SEM, without the high vacuum requirement by varying the sample environment through a range of pressures, temperatures, and gas compositions. Wet and nonconductive samples may be examined in their natural state without modification or preparation. The ESEM offers high-resolution secondary electron imaging in a gaseous environment. The obvious advantage of ESEM is the total lack of preparation. The sample is placed directly on a stub and placed in the SEM chamber. However, with ESEM, we had problems obtaining high-resolution images of the biofilm because of the lack of conductivity in the wet sample. We experienced that close to magnifications of 10 000× and more, where the beam current is locally very Erythromycin high, the focused electron beam seemed to destroy the 3D biofilm structure. We also observed that during prepumping, the sample also slightly dehydrates, but not to near the same extent as the dehydration used in conventional

SEM (Fig. 4). A superior, yet more sophisticated alternative to the conventional SEM and CLSM is the FIB–SEM. Similar to confocal scanning microscopy, it is possible with FIB–SEM to create 3D reconstructions. With a process termed ‘slice and view’, the FIB can sequentially mill away down to 10-nm-thick sections from the surface of a resin embedded specimen and subsequently record a SEM image (Fig. 5a) of the exposed block surface using a back scattered electron detector (BSED). Following acquisition of the successive image slices, the image data are processed to perform a 3D volume reconstruction (Fig. 5b and c). We were able to produce stunning 3D reconstructions of the spatial interaction of bacteria down through the 3-day-old biofilm (Supporting information, Movie S1).