Membrane potentials, oxidative stress and the dispersal response of bacterial biofilms to 405 nm light.

The majority of chronic infections are caused by biofilms, which have higher levels of antibiotic resistance than planktonic growth. Violet-blue 405 nm light has recently emerged as a novel bactericide, but limited studies have been conducted on its effectiveness against biofilms. We found that in response to 405 nm light both Pseudomonas aeruginosa and Bacillus subtilis biofilms exhibited cell dispersal and membrane potential hyperpolarisations. The response to 405 nm light depended on the stage of biofilm growth. The use of reactive oxygen species scavengers reduced membrane hyperpolarisation and biofilm dispersal in response to 405 nm light. This is the first time that membrane potential hyperpolarisations have been linked with photooxidative stress in bacteria and with biofilm dispersal. These results provide a new insight into the role of membrane potentials in the bacterial stress response and could be used in the development of 405 nm light based biofilm treatments.

An unusual presentation of propylthiouracil-induced anti-MPO and PR3 positive ANCA vasculitis with associated anti-GBM antibodies, IgA nephropathy and an IgG4 interstitial infiltrate: a case report.

BACKGROUND: A number of disease processes can culminate in rapidly progressive glomerulonephritis, including pauci-immune focal segmental necrotising glomerulonephritis, usually seen with positive serum antineutrophil cytoplasmic antibodies (ANCA). Propylthiouracil (PTU) has been associated with drug-induced ANCA-associated vasculitis (AAV), with antibodies against myeloperoxidase (MPO) and proteinase 3 (PR3) present individually and together having been recognised. 'Double-positive' vasculitis with ANCA and anti-glomerular basement membrane (GBM) antibodies has also been reported in association with PTU treatment. We present a case of PTU-induced anti-MPO and PR3 positive ANCA vasculitis with associated anti-GBM antibodies, IgA nephropathy and an IgG4 interstitial infiltrate. CASE PRESENTATION: A 51-year-old man presented 2 weeks after re-commencing propylthiouracil (PTU) treatment for Graves' disease, with a severe acute kidney injury and haemato-proteinuria. He demonstrated positive titres for autoantibodies to PR3 (76.9 IU/mL), MPO (28.8 IU/mL) and GBM (94 IU/mL). Renal biopsy demonstrated numerous glomerular crescents, widespread IgG4-positive lymphoplasmacytic infiltrate and mesangial positivity for IgA. PTU was stopped and he was treated with steroids, plasma exchange and cyclophosphamide with sustained improvement in his renal function. CONCLUSIONS: This case of drug-induced AAV presented a unique and intriguing collection of serological and histological features. We propose that the PTU-induced AAV resulted in epiphenomena of anti-GBM antibody production and an IgG4-cell-rich tubulointerstitial infiltrate. It is uncertain whether the mesangial IgA deposition preceded or resulted from the AAV.

Microrheology and Spatial Heterogeneity of Staphylococcus aureus Biofilms Modulated by Hydrodynamic Shear and Biofilm-Degrading Enzymes.

Particle tracking microrheology was used to investigate the viscoelasticity of Staphylococcus aureus biofilms grown in microfluidic cells at various flow rates and when subjected to biofilm-degrading enzymes. Biofilm viscoelasticity was found to harden as a function of shear rate but soften with increasing height away from the attachment surface in good agreement with previous bulk results. Ripley's K-function was used to quantify the spatial distribution of the bacteria within the biofilm. For all conditions, biofilms would cluster as a function of height during growth. The effects of proteinase K and DNase-1 on the viscoelasticity of biofilms were also investigated. Proteinase K caused an order of magnitude change in the compliances, softening the biofilms. However, DNase-1 was found to have no significant effects over the first 6 h of development, indicating that DNA is less important in biofilm maintenance during the initial stages of growth. Our results demonstrate that during the preliminary stages of Staphylococcus aureus biofilm development, column-like structures with a vertical gradient of viscoelasticity are established and modulated by the hydrodynamic shear caused by fluid flow in the surrounding environment. An understanding of these mechanical properties will provide more accurate insights for removal strategies of early-stage biofilms.

Brain injury induces specific changes in the caecal microbiota of mice via altered autonomic activity and mucoprotein production.

Intestinal microbiota are critical for health with changes associated with diverse human diseases. Research suggests that altered intestinal microbiota can profoundly affect brain function. However, whether altering brain function directly affects the microbiota is unknown. Since it is currently unclear how brain injury induces clinical complications such as infections or paralytic ileus, key contributors to prolonged hospitalization and death post-stroke, we tested in mice the hypothesis that brain damage induced changes in the intestinal microbiota. Experimental stroke altered the composition of caecal microbiota, with specific changes in Peptococcaceae and Prevotellaceae correlating with the extent of injury. These effects are mediated by noradrenaline release from the autonomic nervous system with altered caecal mucoprotein production and goblet cell numbers. Traumatic brain injury also caused changes in the gut microbiota, confirming brain injury effects gut microbiota. Changes in intestinal microbiota after brain injury may affect recovery and treatment of patients should appreciate such changes.

Fatal case of sorafenib-associated idiosyncratic hepatotoxicity in the adjuvant treatment of a patient with renal cell carcinoma.

BACKGROUND: Sorafenib is an orally available kinase inhibitor with activity at Raf, PDGFß and VEGF receptors that is licensed for the treatment of advanced renal cell carcinoma (RCC) and hepatocellular carcinoma (HCC). Current evidence-based post-nephrectomy management of individuals with localized RCC consists of surveillance-based follow up. The SORCE trial is designed to investigate whether treatment with adjuvant sorafenib can reduce recurrence rates in this cohort. CASE PRESENTATION: Here we report an idiosyncratic reaction to sorafenib resulting in fatal hepatotoxicity and associated renal failure in a 62 year-old man treated with sorafenib within the SORCE trial. CONCLUSION: This is the first reported case of sorafenib exposure associated fatal toxicity in the adjuvant setting and highlights the unpredictable adverse effects of novel adjuvant therapies.

Spatial propagation of electrical signals in circular biofilms: A combined experimental and agent-based fire-diffuse-fire study.

Bacterial biofilms are a risk to human health, playing critical roles in persistent infections. Recent studies have observed electrical signaling in biofilms and thus biofilms represent a new class of active excitable matter in which cell division is the active process and the spiking of the individual bacterial cells is the excitable process. Electrophysiological models have predominantly been developed to describe eukaryotic systems, but we demonstrate their use in understanding bacterial biofilms. Our agent-based fire-diffuse-fire (ABFDF) model successfully simulates the propagation of both centrifugal (away from the center) and centripetal (toward the center) electrical signals through biofilms of Bacillus subtilis. Furthermore, the ABFDF model allows realistic spatial positioning of the bacteria in two dimensions to be included in the fire-diffuse-fire model and this is the crucial factor that improves agreement with experiments. The speed of propagation is not constant and depends on the radius of the propagating electrical wave front. Centripetal waves are observed to move faster than centrifugal waves, which is a curvature driven effect and is correctly captured by our simulations.

The VHL tumor suppressor inhibits expression of the IGF1R and its loss induces IGF1R upregulation in human clear cell renal carcinoma.

Clear cell renal cell cancer (CC-RCC) is a highly chemoresistant tumor characterized by frequent inactivation of the von Hippel-Lindau (VHL) gene. The prognosis is reportedly worse in patients whose tumors express immunoreactive type I insulin-like growth factor receptor (IGF1R), a key mediator of tumor cell survival. We aimed to investigate how IGF1R expression is regulated, and found that IGF1R protein levels were unaffected by hypoxia, but were higher in CC-RCC cells harboring mutant inactive VHL than in isogenic cells expressing wild-type (WT) VHL. IGF1R mRNA and promoter activities were significantly lower in CC-RCC cells expressing WT VHL, consistent with a transcriptional effect. In Sp1-null Drosophila Schneider cells, IGF1R promoter activity was dependent on exogenous Sp1, and was suppressed by full-length VHL protein (pVHL) but only partially by truncated VHL lacking the Sp1-binding motif. pVHL also reduced the stability of IGF1R mRNA via sequestration of HuR protein. Finally, IGF1R mRNA levels were significantly higher in CC-RCC biopsies than benign kidney, confirming the clinical relevance of these findings. Thus, we have identified a new hypoxia-independent role for VHL in suppressing IGF1R transcription and mRNA stability. VHL inactivation leads to IGF1R upregulation, contributing to renal tumorigenesis and potentially also to chemoresistance.

Polyoma virus infection and urothelial carcinoma of the bladder following renal transplantation.

Renal transplant recipients are at increased risk of bladder carcinoma. The aetiology is unknown but a polyoma virus (PV), BK virus (BKV), may play a role; urinary reactivation of this virus is common post-renal transplantation and PV large T-antigen (T-Ag) has transforming activity. In this study, we investigate the potential role of BKV in post-transplant urothelial carcinoma by immunostaining tumour tissue for PV T-Ag. There was no positivity for PV T-Ag in urothelial carcinomas from 20 non-transplant patients. Since 1990, 10 transplant recipients in our unit have developed urothelial carcinoma, and tumour tissue was available in eight recipients. Two patients were transplanted since the first case of PV nephropathy (PVN) was diagnosed in our unit in 2000 and both showed PV reactivation post-transplantation. In one of these patients, there was strong nuclear staining for PV T-Ag in tumour cells, with no staining of non-neoplastic urothelium. We conclude that PV infection is not associated with urothelial carcinoma in non-transplant patients, and is uncommon in transplant-associated tumours. Its presence in all tumour cells in one patient transplanted in the PVN era might suggest a possible role in tumorigenesis in that case.

Peritubular capillaritis in renal allografts: prevalence, scoring system, reproducibility and clinicopathological correlates.

While glomerulitis is graded according to the Banff classification, no criteria for scoring peritubular capillaritis (PTC) have been established. We retrospectively applied PTC-scoring criteria to 688 renal allograft (46 preimplantation, 461 protocol, 181 indication) biopsies. A total of 26.3% of all analyzed biopsies had peritubular capillaritis (implant 0%, protocol 17.6%, indication 45.5%; p < 0.0001). The most common capillaritis pattern was of moderate severity (5-10 luminal cells), focal in extent (10-50% of PTC), with a minority of neutrophils. A total of 24% of C4d- compared with 75% of C4d+ biopsies showed capillaritis (p < 0.0001). More than 80% of biopsies with glomerulitis had peritubular capillaritis. A total of 50.4% of biopsies with borderline or T-cell mediated rejection (TCMR) and 14.1% of biopsies without TCMR or antibody-mediated rejection (ABMR) showed capillaritis (p < 0.0001). The inter-observer reproducibility of the PTC-scoring features was fair to moderate. Diffuse capillaritis detected in early protocol biopsies had significant negative prognostic impact in terms of glomerular filtration rate 2 years posttransplantation. Indication biopsies show a significantly higher prevalence of capillaritis than protocol biopsies (45.5% vs. 17.6%; p < 0.0001). Capillaritis is more frequent and pronounced in ABMR, but can be observed in TCMR cases. Thus, scoring of peritubular capillaritis is feasible and can provide prognostic and diagnostic information in renal allograft biopsies.

Apoptosis in podocytes induced by TGF-beta and Smad7.

Primary and secondary forms of focal segmental glomerulosclerosis (FSGS) are characterized by depletion of podocytes and constitute a central manifestation of chronic progressive glomerular diseases. Here we report that podocytes undergo apoptosis at early stages in the course of progressive glomerulosclerosis in TGF-beta1 transgenic mice. Apoptosis is associated with progressive depletion of podocytes and precedes mesangial expansion. Smad7 protein expression is strongly induced specifically in damaged podocytes of transgenic mice and in cultured murine podocytes treated with TGF-beta. TGF-beta1 and Smad7 each induce apoptosis in podocytes, and their coexpression has an additive effect. Activation of p38 MAP kinase and caspase-3 is required for TGF-beta-mediated apoptosis, but not for apoptosis induced by Smad7. Unlike TGF-beta, Smad7 inhibits nuclear translocation and transcriptional activity of the cell survival factor NF-kappaB. Our results suggest a novel functional role for Smad7 as amplifier of TGF-beta-induced apoptosis in podocytes and a new pathomechanism for podocyte depletion in progressive glomerulosclerosis.

Assessment of the Cardiff nephrectomy cut-up protocol with total blocking of the renal sinus: effect on tumour staging and practical issues.

AIM: To evaluate the effects on detection of vascular invasion and workload of a new standard dissection protocol for examining nephrectomy specimens for renal cell carcinoma. METHODS: Using 192 consecutive renal cell carcinoma nephrectomy specimens, the incidence of vascular invasion and number of tissue blocks per tumour were compared before and after introduction of the new protocol. RESULTS: The Cardiff protocol increased the percentage of tumours staged as T3b (renal sinus or hilar vein invasion) from 37.7% to 55.7% cases (p<0.001), with an increase from 9.1% to 21.7% of those staged as T3b due to renal sinus vein invasion alone (p<0.01). A small, but significant, permanent increase in workload was observed from an average of 11.7 to 13.4 blocks per case (p<0.001). CONCLUSIONS: This protocol is suitable for use in routine practice to evaluate pathological prognostic determinants important for clinical management, while causing only a small increase in workload.

Prevention of chronic rejection by donor-specific blood transfusion in a new model of chronic cardiac allograft rejection.

Chronic graft rejection is now a major barrier to the long-term survival of cardiac transplants. A major hallmark of chronic rejection is intimal thickening of arteries in the graft leading to vascular occlusion. Current animal models of chronic rejection generally utilize immunosuppression to prevent acute rejection of grafts disparate at major histocompatibility antigens or graft disparities involving minor histocompatibility antigens alone. In the present communication we describe a new model of chronic rejection involving grafting of PVG-R23 hearts into PVG-RT1(u) recipients. The R23 hearts, which differ from the RT1(u) recipients at class II MHC, are rejected with a chronic time course and demonstrate extensive severe vascular myointimal proliferation within the coronary arteries. Furthermore we demonstrate for the first time that donor-specific blood transfusion can prevent chronic rejection and the intimal thickening of the coronary arteries.

Mast cells: the forgotten cells of renal fibrosis.

BACKGROUND/AIMS: Mast cells, when activated, secrete a large number of fibrogenic factors and have been implicated in the development of fibrotic conditions of the liver, lung, and skin. There is evidence that renal fibrosis is closely linked with a chronic inflammatory cell infiltrate within the interstitium, but a potential role for mast cells in this process has yet to be defined. Therefore, the numbers of mast cells in normal and fibrotic kidneys with various pathologies were investigated. METHODS: Mast cells were quantified in renal transplants showing acute and chronic rejection and cyclosporin toxicity, kidneys removed for chronic pyelonephritis, and renal biopsies from patients with IgA nephropathy, membranous nephropathy, and diabetic nephropathy. Mast cells were stained using two methods: acid toluidine blue detected less than 30% of the mast cells revealed by immunohistochemistry for mast cell tryptase. RESULTS: Mast cells were scarce or absent in normal kidney (median, 1.6 mast cells/mm2) but numerous throughout the cortex and medulla in all specimens that showed fibrosis. They were almost entirely confined to the renal interstitium. Mast cells were present in large numbers in biopsies from patients with membranous nephropathy (median, 21.7 mast cells/mm2) and diabetic nephropathy (median, 29.2 mast cells/mm2), which were selected on the basis of showing chronic injury. In 24 unselected IgA nephropathy biopsies there was a close correlation between numbers of mast cells and the extent of interstitial fibrosis (r = 0.771; p < 0.0001). In renal transplant biopsies, mast cells were associated with allograft fibrosis in chronic rejection (median, 27.1 mast cells/mm2) and chronic cyclosporin toxicity (median, 10.6 mast cells/mm2) but not acute rejection (median, 2.7 mast cells/mm2) or acute cyclosporin toxicity (median, 2.0 mast cells/mm2). There was no detectable increase in mast cell numbers during acute rejection in those transplants that subsequently progressed to chronic rejection. In some biopsies the mast cells were largely intact, but in most cases some or all were degranulated. CONCLUSIONS: An increased number of mast cells is a consistent feature of renal fibrosis, whatever the underlying pathology, and the number of mast cells correlates with the extent of interstitial fibrosis. This suggests that mast cells might play a pathogenetic role in the fibrotic process.

Postmortem findings after fatal anaphylactic reactions.

AIMS: To determine the frequency at which classic manifestations of anaphylaxis are present at necropsy after fatal anaphylactic reactions. METHODS: A register has been established of fatal anaphylactic reactions in the UK since 1992, traced from the certified cause of death and other sources. Details of the previous medical history and the reaction suggest anaphylaxis as the cause of death for 130 cases; a postmortem report was available for 56. RESULTS: The 56 deaths studied included 19 reactions to bee or wasp venom, 16 to foods, and 21 to drugs or contrast media. Death occurred within one hour of anaphylaxis in 39 cases. Macroscopic findings included signs of asthma (mucous plugging and/or hyper-inflated lungs) (15 of 56), petechial haemorrhages (10 of 56), pharyngeal/laryngeal oedema (23 of 56), but for 23 of 56 there was nothing indicative of an allergic death. Mast cell tryptase was raised in 14 of 16 cases tested; three of three tested had detectable IgE specific for the suspected allergen. CONCLUSIONS: In many cases of fatal anaphylaxis no specific macroscopic findings are present at postmortem examination. This reflects the rapidity and mode of death, which is often the result of shock rather than asphyxia. Investigations that might help determine whether anaphylaxis was the cause of death had rarely been performed. In the presence of a typical clinical history, absence of postmortem findings does not exclude the diagnosis of anaphylaxis.

What is a natural cause of death? A survey of how coroners in England and Wales approach borderline cases.

AIM: Many deaths fall in the "grey" area between those that are clearly natural and those that are unnatural. There are no guidelines to help doctors in dealing with such cases and death certification is often arbitrary and inconsistent. In an attempt to initiate debate on these difficult areas, and with the ultimate aim of achieving national consensus, the views of coroners in England and Wales were sought. METHODS: Sixteen clinical scenarios, with causes of death, were circulated to all coroners in England and Wales. For each case they were asked to provide a verdict, with explanation. The deaths fell into three groups: (1) postoperative, (2) a combination of trauma and natural disease, and (3) infectious disease. RESULTS: Sixty four questionnaires were returned. There was near consensus (> 80% concordance) in only two of the 16 cases. In five, there was no significant agreement between coroners in the verdicts returned ("natural causes" versus "misadventure/accidental"). These included all three cases in which death resulted from a combination of trauma and natural disease (a fall after a grand mal fit; falls resulting in fractures of bones affected by metastatic carcinoma and osteoporosis), bronchopneumonia after hip replacement for osteoarthritis, and new variant Creutzfeldt-Jakob disease. The comments made for each case indicate that the variation between coroners in whether or not to hold an inquest, and the verdict arrived at, reflect the lack of a definition for natural causes, together with differences in the personal attitudes of each coroner. CONCLUSIONS: There is considerable variation in the way in which coroners approach these borderline cases, many of which are common in clinical practice. This study indicates a need for discussion, working towards a national consensus on such issues. It highlights the importance of good communication between coroners and medical staff at a local level.

Interstitial myofibroblasts: predictors of progression in membranous nephropathy.

AIMS: To determine the role of interstitial myofibroblasts in the progression of membranous nephropathy; and to assess the predictive value of quantifying myofibroblasts in determining long term renal outcome. METHODS: All cases of membranous nephropathy, diagnosed by renal biopsy at University Hospital of South Manchester between 1984 and 1987, were studied retrospectively. The biopsy specimens (n = 26) were reviewed and analysed morphometrically to measure interstitial volume as a proportion of the total volume of renal cortex, and numbers of interstitial myofibroblasts (cells positive for alpha-smooth muscle actin within the interstitium). Clinical data, with a follow up of seven to eight years, was available for 24 patients, and renal outcome was correlated with pathological changes in the initial diagnostic biopsy specimen. RESULTS: The number of myofibroblasts and interstitial volume were inversely correlated with creatinine clearance at the initial biopsy, and at the end of follow up. Percentage sclerosed glomeruli or stage of glomerular disease, assessed by electron microscopy, did not correlate with renal function at initial biopsy or during follow up. The number of myofibroblasts, but not interstitial volume, correlated with severity of proteinuria at initial biopsy. Of 15 biopsy specimens showing no or mild interstitial fibrosis, four showed a notable increase in the number of interstitial myofibroblasts. All of these patients developed chronic renal failure, compared with three of 11 patients whose specimens showed no or a mild increase in myofibroblast numbers. CONCLUSIONS: Interstitial myofibroblasts play a role in the development of interstitial fibrosis and progressive renal failure in membranous nephropathy. Increased numbers of myofibroblasts in biopsy specimens showing only mild fibrosis may predict subsequent chronic renal failure.

Small intestinal infarction: a fatal complication of systemic oxalosis.

Primary hyperoxaluria is a rare genetic disorder characterised by calcium oxalate nephrolithiasis and nephrocalcinosis leading to renal failure, often with extra-renal oxalate deposition (systemic oxalosis). Although ischaemic complications of crystal deposition in vessel walls are well recognised clinically, these usually take the form of peripheral limb or cutaneous ischaemia. This paper documents the first reported case of fatal intestinal infarction in a 49 year old woman with systemic oxalosis and advocates its consideration in the differential diagnosis of an acute abdomen in such patients.

Characterisation of IS1126 from Porphyromonas gingivalis W83: a new member of the IS4 family of insertion sequence elements.

The nucleotide sequence of IS1126, the only insertion sequence so far isolated from the oral pathogen Porphyromonas gingivalis, has been determined. It had a nucleotide sequence of 1338 base pair (bp) flanked by 12 bp perfect inverted repeats and generated a 5 bp target site duplication. The single major open reading frame encoded a predicted protein of 361 amino acids and molecular mass of 41 kDa. The gene encoding the transposase was subcloned into pUC18 and the transposase expressed in Escherichia coli minicells. The predicted amino acid sequence of the transposase had homology to putative transposases of IS1106 and IS1186 both of which belong to the IS5 group within the IS4 super-family of insertion elements. On the basis of this homology we propose that IS1126 should also be included in the IS5 group. Southern-blot analysis of a number of P. gingivalis strains using IS1126 as a probe revealed a unique pattern of hybridisation for each strain and the absence of IS1126 from other closely related Porphyromonas species. This should allow IS1126 to be used as a rapid epidemiological tool in studying oral infections by P. gingivalis.

Isolation from recombinant Escherichia coli and characterization of CMP-Kdo synthetase, involved in the expression of the capsular K5 polysaccharide (K-CKS).

In Escherichia coli with group II capsules, the synthesis of capsular polysaccharide and its cellular expression are encoded by the kps gene cluster, which is composed of three regions. The central region 2 encodes proteins involved in polysaccharide synthesis, and the flanking regions 1 and 3 direct the translocation of the finished polysaccharide across the cytoplasmic membrane and its surface expression. The kps genes of E. coli with the group II capsular K5 polysaccharide, have been cloned and sequenced. Region 1 contains the kpsE, D, U, C and S genes. In this communication we describe the overexpression of the kpsD and kpsU genes as well as the isolation of the KpsU protein from the recombinant bacteria by chloroform treatment. The purified KpsU protein exhibited CMP-Kdo-synthetase activity. The N-terminal sequence and two internal peptide sequences of the isolated protein are in agreement with that previously predicted from the DNA sequence of the kpsU gene. The kinetic data of the CMP-Kdo-synthetase participating in K5 capsule expression (K-CMP-Kdo-synthetase) differ from those described for the CMP-Kdo-synthetase, participating in lipopolysaccharide synthesis (L-CMP-Kdo-synthetase).