Pre-existing diabetes is a risk factor for increased rates of cellular rejection after kidney transplantation: an observational cohort study.

AIM: To investigate whether people with diabetes have an elevated risk of kidney allograft rejection in a well characterized clinical cohort in the setting of contemporary immunosuppression. METHODS: We conducted a retrospective cohort study including all kidney allograft recipients at a single centre between 2007 and 2015, linking clinical, biochemical and histopathological data from electronic patient records. RESULTS: Data were analysed for 1140 kidney transplant recipients. The median follow-up was 4.4 years post-transplantation, and 117 of the kidney transplant recipients (10.2%) had diabetes at time of transplantation. Kidney allograft recipients with vs without diabetes were older (53 vs 45 years; P<0.001) and more likely to be non-white (41.0% vs 26.4%; P=0.001). Kidney allograft recipients with vs without diabetes had a higher risk of cellular rejection (19.7% vs 12.4%; P=0.024), but not of antibody-mediated rejection (3.4% vs 3.7%; P=0.564). Graft function and risk of death-censored graft loss were similar in the two groups, but kidney allograft recipients with diabetes had a higher risk of death and overall graft loss than those without diabetes. In a Cox regression model of non-modifiable risk factors at time of transplantation, diabetes was found to be an independent risk factor for cellular rejection (hazard ratio 1.445, 95% CI 1.023-1.945; P=0.042). CONCLUSIONS: Kidney allograft recipients with diabetes at transplantation should be counselled regarding their increased risk of cellular rejection but reassured regarding the lack of any adverse impact on short-to-medium term allograft function or survival.

Effect of cold ischaemia time on outcome after living donor renal transplantation.

BACKGROUND: The aim of the present study was to determine the effects of cold ischaemia time (CIT) on living donor kidney transplant recipients in a large national data set. METHODS: Data from the National Health Service Blood and Transplant and UK Renal Registry databases for all patients receiving a living donor kidney transplant in the UK between January 2001 and December 2014 were analysed. Patients were divided into three groups depending on CIT (less than 2 h, 2-4 h, 4-8 h). Risk-adjusted outcomes were assessed by multivariable analysis adjusting for discordance in both donor and recipient characteristics. RESULTS: Outcomes of 9156 transplants were analysed (CIT less than 2 h in 2662, 2-4 h in 4652, and 4-8 h in 1842). After adjusting for confounders, there was no significant difference in patient survival between CIT groups. Recipients of kidneys with a CIT of 4-8 h had excellent graft outcomes, although these were slightly inferior to outcomes in those with a CIT of less than 2 h, with risk-adjusted rates of delayed graft function of 8·6 versus 4·3 per cent, and 1-year graft survival rates of 96·2 versus 97·1 per cent, respectively. CONCLUSION: The detrimental effect of prolonging CIT for up to 8 h in living donation kidney transplantation is marginal.

Systematic detection and association of Entamoeba species in stool samples from selected sites in India.

This study developed a fast and high throughput dot-blot technique to evaluate the presence of Entamoeba in stool samples (n = 643) followed by a PCR-based method to validate and differentiate the two species E. histolytica and E. dispar. The prevalence rate of the parasite has been detected in a cross-sectional study carried out in the population of the Eastern and Northern parts of India. Of the various demographic features, prevalence was highest in the monsoon season (P = 0·017), in the <15 years age group (P = 0·015). In HIV-positive individuals, the prevalence rate was significantly high (P = 0·008) in patients with a CD4 cell count <200 as well as in patients without antiretroviral therapy (ART) (P = 0·011). Our analysis further confirmed that risk factors such as toilet facilities, living conditions, hygienic practices, drinking water source, occupation and level of education are important predictors as they were found to contribute significantly in the prevalence of the parasite.

Pseudotumoral presentation of fungating mycetoma caused by Phaeoacremonium fuscum in a renal transplant patient.

Eumycetoma is an unusual infection in immunocompromised patients outside the tropics, caused by a variety of fungal pathogens. We describe the case of a 51-year-old renal transplant recipient who presented with a large pseudotumoral foot lesion necessitating complete surgical excision of the lesion. Cultures and molecular diagnosis confirmed Phaeoacremonium fuscum. This is the first case, to our knowledge, of fungating mycetoma caused by this fungal species in a solid organ transplant recipient.

Understanding flux switching in metabolic networks through an analysis of synthetic lethals.

Biological systems are robust and redundant. The redundancy can manifest as alternative metabolic pathways. Synthetic double lethals are pairs of reactions that, when deleted simultaneously, abrogate cell growth. However, removing one reaction allows the rerouting of metabolites through alternative pathways. Little is known about these hidden linkages between pathways. Understanding them in the context of pathogens is useful for therapeutic innovations. We propose a constraint-based optimisation approach to identify inter-dependencies between metabolic pathways. It minimises rerouting between two reaction deletions, corresponding to a synthetic lethal pair, and outputs the set of reactions vital for metabolic rewiring, known as the synthetic lethal cluster. We depict the results for different pathogens and show that the reactions span across metabolic modules, illustrating the complexity of metabolism. Finally, we demonstrate how the two classes of synthetic lethals play a role in metabolic networks and influence the different properties of a synthetic lethal cluster.

Two-particle time-domain interferometry in the fractional quantum Hall effect regime.

Quasi-particles are elementary excitations of condensed matter quantum phases. Demonstrating that they keep quantum coherence while propagating is a fundamental issue for their manipulation for quantum information tasks. Here, we consider anyons, the fractionally charged quasi-particles of the Fractional Quantum Hall Effect occurring in two-dimensional electronic conductors in high magnetic fields. They obey anyonic statistics, intermediate between fermionic and bosonic. Surprisingly, anyons show large quantum coherence when transmitted through the localized states of electronic Fabry-Pérot interferometers, but almost no quantum interference when transmitted via the propagating states of Mach-Zehnder interferometers. Here, using a novel interferometric approach, we demonstrate that anyons do keep quantum coherence while propagating. Performing two-particle time-domain interference measurements sensitive to the two-particle Hanbury Brown Twiss phase, we find 53 and 60% visibilities for anyons with charges e/5 and e/3. Our results give a positive message for the challenge of performing controlled quantum coherent braiding of anyons.

Uptake, translocation, weathering and speciation of gold nanoparticles in potato, radish, carrot and lettuce crops.

Extensive use of nanomaterials in agriculture will inevitably lead to their release to the environment in significant loads. Thus, understanding the fate of nanoparticles in the soil-plant environment, and potential presence and consequent implication of nanoparticles in food and feed products, is required. We study plant uptake of gold nanoparticles from soil, and their distribution, translocation and speciation (in terms of particle size change and release of ionic Au) in the different plant tissues of four important crops (potato, radish, carrot and lettuce). Our new analytical protocol and experiments show the feasibility of determining the presence, concentration and distribution of nanoparticles in different plant parts, which differ from plant to plant. Critically, we identify the evident capacity of plants to break down (or substantially change the properties of) nanoparticles in the rhizosphere prior to uptake, as well as the evident capacity of plants to reorganize ionic metals as nanoparticles in their tissues. This could lead to nanoparticle exposure through consumption of crops.

Effects of caffeine and other methylxanthines on the development and metabolism of sea urchin eggs. Involvement of NADP and glutathione.

Methylxanthines (MX) inhibit cell division in sea urchin and clam eggs. This inhibitory effect is not mediated via cAMP. MX also inhibit respiration in marine eggs, at concentrations which inhibit cleavage. Studies showed that no changes occurred in ATP and ADP levels in the presence of inhibitory concentrations of MX, indicating an extra-mitochondrial site of action for the drug. Subsequent studies revealed decreased levels of NADP+ and NADPH, when eggs were incubated with inhibitory concentrations of MX, but no change in levels of NAD+ and NADH. MX did not affect the pentose phosphate shunt pathway and did not have any effect on the enzyme NAD+ -kinase. Further studies showed a marked inhibitory effect on the glutathione reductase activity of MX-treated eggs. Reduced glutathione (GSH) could reverse the cleavage inhibitory effect of MX. Moreover, diamide, a thiol-oxidizing agent specific for GSH in living cells, caused inhibition of cell division in sea urchin eggs. Diamide added to eggs containing mitotic apparatus (MA) could prevent cleavage by causing a dissolution of the formed MA. Both MX and diamide inhibit a Ca2+-activated ATPase in whole eggs. The enzyme can be reactivated by sulfhydryl reducing agents added in the assay mixture. In addition, diamide causes an inhibition of microtubule polymerization, reversible with dithioerythritol. All experimental evidence so far suggests that inhibition of mitosis in sea urchin eggs by MX is mediated by perturbations of the in vivo thiol-disulfide status of target systems, with a primary effect on glutathione levels.

Stimulation of tubulin tyrosinolation in rabbit leukocytes evoked by the chemoattractant formyl-methionyl-leucyl-phenylalanine.

Cellular tubulin is subject to a posttranslational modification involving the reversible addition to tyrosine through peptide linkage to the C-terminal glutamate of the alpha-chain. The synthetic peptide chemoattractant, N-formyl-methionyl-leucyl-phenylalanine, causes a specific, dose-dependent stimulation of tubulin tyrosinolation in rabbit leukocytes. This stimulation is prevented by carbobenzoxy-phenylalanyl-methionine, benzoyl-tyrosine ethylester, and nordihydroguaiaretic acid, which are all inhibitors of chemotaxis presumed to act via membrane-associated events. The combination of 3-deazaadenosine and homocysteine thiolactone, which inhibits phospholipid methylation, and quinacrine, an inhibitor of phospholipase A2, also abolishes the response to the peptide. Colchicine, however, which causes a marked disassembly of cellular microtubules in these cells and also inhibits chemotaxis, does not have any inhibitory effect on the basal or peptide-stimulated rate of tubulin tyrosinolation. In contrast, taxol, a microtubule-stabilizing agent, has an inhibitory effect on both the basal and peptide-stimulated tyrosine incorporation. Taxol also inhibits chemotaxis in rabbit leukocytes. The results strongly suggest the role of closely linked membrane-cytoskeleton interactions in leukocyte chemotaxis, in which tyrosinolation of tubulin may be functionally involved.

Tubulin tyrosinolation in human polymorphonuclear leukocytes: studies in normal subjects and in patients with the Chediak-Higashi syndrome.

We have recently reported a specific dose-dependent stimulation of posttranslational incorporation of tyrosine into tubulin alpha-chains of rabbit peritoneal leukocytes as induced by the synthetic peptide chemoattractant formyl-methionyl-leucyl-phenylalanine (FMLP). The present study reports a similar, specific stimulation of tubulin tyrosinolation in human polymorphonuclear leukocytes (PMN). When compared to normal PMN, both the resting and FMLP-stimulated levels of posttranslational tyrosine incorporation were two- to threefold higher in PMN of three patients with the Chediak-Higashi syndrome (CHS). The concentration of cellular tubulin and the specific activity of tubulin tyrosine ligase were similar in PMN of CHS patients and normal donors and resembled that of other non-neuronal cells. The high levels of tyrosine incorporation in PMN of CHS patients were normalized by the administration of ascorbate, both in vitro and in in vivo experiments. In vitro addition of ascorbate also inhibited the FMLP-induced stimulation of tyrosine incorporation in both normal and CHS cells. Normalization of higher levels of tyrosine incorporation in PMN of CHS patients and the inhibition of FMLP-induced stimulation of tubulin tyrosinolation in normal and CHS cells as observed with ascorbate could also be affected by other reducing agents such as reduced glutathione, cysteine, or dithiothreitol. These results suggest a possible relationship between cellular redox and tubulin tyrosinolation in PMN.

Interactions of cytoplasmic granules with microtubules in human neutrophils.

Ultrastructural and functional studies of degranulation responses by human neutrophils have suggested that microtubules (MTs) have a role in the intracellular transport of neutrophil granules. We have found that granule-MT complexes can be isolated from disrupted taxol-treated (1.0 microM) neutrophils, visualized by electron microscopy, and quantified in terms of granules per MT length. After incubation of neutrophils with the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP), granule-MT complex formation was found to be increased two- to threefold. Enhanced binding of granules to MTs was detectable within 30 s of fMLP stimulation and was dependent on the concentration of fMLP. Incubation of cells with dibutyryl cAMP inhibited this fMLP-stimulated granule-MT complex formation in a dose-responsive fashion. These granule-MT interactions could be reproduced in a cell-free system with neutrophil granules isolated by density gradient centrifugation and MTs polymerized from phosphocellulose-purified tubulin. Furthermore, reconstituted granule-MT interactions were found to be modulated by ATPase inhibitors. Sodium orthovanadate increased granule-MT interactions in a concentration-dependent manner, while AMP-PNP, a nonhydrolyzable ATP analogue, and N-ethylmaleimide decreased or eliminated these interactions. In addition, we found that a MT-activated ATPase could be recovered from intact neutrophil granules by salt extraction, and that extracts enriched in this ATPase contained a polypeptide of between 115 and 120 kD which binds ATP and is immunologically related to kinesin. These studies demonstrate that cytoplasmic granules interact with MTs in human neutrophils in a regulated stimulus-responsive manner, and they suggest that such interactions may involve an MT-based, ATPase-dependent, vesicle translocation system as has been demonstrated in other types of cells.

Rectal cancer in young adults: a series of 102 patients at a tertiary care centre in India.

OBJECTIVE: Rectal cancer in young patients is uncommon. There is little information on rectal cancer in young adults in India. The aim of this study was to determine the relative incidence of rectal cancer in young patients in India and identify any differences in histological grade and pathological stage between younger and older cohorts. METHOD: All adult patients presenting at a tertiary colorectal unit with primary rectal adenocarcinoma between September 2003 and August 2007 were included. Patients were divided into two groups: 40 years and younger, and older than 40 years. Details regarding patient demographics, preoperative assessment, management and tumour grade and stage were obtained from a prospectively maintained database. RESULTS: One hundred and two of 287 patients (35.5%) were 40 or younger at presentation. Younger patients were more likely to present with less favourable histological features (52.0% vs 20.5% (P < 0.001)) and low rectal tumours (63.0% vs 50.0%) (P = 0.043), but were equally likely to undergo curative surgery compared to the older group (P = 0.629). Younger patients undergoing surgery had a higher pathological T stage (T0-2 18.9%, T3 62.3%, T4 19.7% vs 34.5%, 56.0%, 9.5%) (P = 0.027) and more advanced pathological N stage (N0 31.1%, N1 41.0%, N2 27.9% vs 53.4%, 26.7%, 17.2%) (P = 0.014). CONCLUSION: The relative number of young patients with rectal cancer in this Indian series is higher than figures reported in western populations. The reasons for this are not clear. The histopathological features of rectal tumours in young patients in this study are consistent with similar studies in Western populations.

A simple ex vivo model of human renal allograft preservation using the gonadal vein.

INTRODUCTION: Hypothermic machine perfusion, an organ preservation modality, involves flow of chilled preservation fluid through an allograft's vasculature. This study describes a simple, reproducible, human model that allows for interrogation of flow effects during ex vivo organ perfusion. MATERIALS AND METHODS: Gonadal veins from deceased human renal allografts were subjected to either static cold storage or hypothermic machine perfusion for up to 24 hours. Caspase-3, Krüppel-like factor 2 expression and electron microscopic analysis were compared between 'flow' and 'no-flow' conditions, with living donor gonadal vein sections serving as negative controls. RESULTS: The increase in caspase-3 expression was less pronounced for hypothermic machine-perfused veins compared with static cold storage (median-fold increase 1.2 vs 2.3; P < 0.05). Transmission electron microscopy provided ultrastructural corroboration of endothelial cell apoptosis in static cold storage conditions. For static cold storage preserved veins, Krüppel-like factor 2 expression diminished in a time-dependent manner between baseline and 12 hours (P < 0.05) but was abrogated and reversed by hypothermic machine perfusion (P < 0.05). CONCLUSIONS: Our methodology is a simple, reproducible and successful model of ex vivo perfusion in the context of human organ preservation. To demonstrate the model's utility, we establish that two widely used markers of endothelial health (caspase-3 and Krüppel-like factor 2) differ between the flow and no-flow conditions of the two predominant kidney preservation modalities. These findings suggest that ex vivo perfusion may mediate the induction of a biochemically favourable endothelial niche which may contribute tohypothermic machine perfusion's association with improved renal transplantation outcomes.

Peritoneal lavage cytology in patients with oesophagogastric adenocarcinoma.

BACKGROUND: The aim of the study was to determine the value of performing peritoneal lavage cytology during laparoscopy in the management of oesophagogastric adenocarcinoma. METHODS: Laparoscopy combined with peritoneal cytology was performed in patients with potentially resectable oesophagogastric adenocarcinoma. Macroscopic peritoneal findings at laparoscopy and the presence of free peritoneal tumour cells were recorded. All patients were followed to death or the census point. Patients with overt peritoneal disease or positive cytology were offered palliative chemotherapy, subject to performance status. RESULTS: Forty-eight (18.8 per cent) of 255 patients had overt peritoneal metastases at staging laparoscopy. Fifteen (7.2 per cent) of the remaining 207 patients had positive cytology; these patients had a median (95 per cent confidence interval) survival of 13 (3.1 to 22.9) months, versus 9 (7.4 to 10.6) months for those with overt peritoneal metastases (P = 0.517). Of patients receiving chemotherapy, those without overt metastases had a slight survival advantage over patients with metastases (median 15 (10.8 to 19.2) versus 9 (7.4 to 10.7) months; P = 0.045). CONCLUSION: Positive peritoneal cytology in the absence of overt peritoneal metastases is not uncommon in oesophagogastric adenocarcinoma. It is a marker of poor prognosis even in the absence of overt peritoneal metastases.

Studies in normal and chronic granulomatous disease neutrophils indicate a correlation of tubulin tyrosinolation with the cellular redox state.

A specific stimulation of tubulin tyrosinolation in human polymorphonuclear leukocytes (PMN) is induced by the synthetic peptide chemoattractant, N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe), and this stimulation of tyrosinolation in PMN is completely inhibited in the presence of various reducing agents. Further studies to characterize the mechanism of stimulation of tyrosinolation in PMN have revealed that conditions that inhibited the respiratory burst in stimulated PMN, e.g., an anaerobic atmosphere, or addition of antioxidants such as cysteamine, azide, or 2,3-dihydroxybenzoic acid, also inhibited the peptide-induced stimulation of tyrosinolation in these cells. Moreover, the sulfhydryl reagent, N-ethylmaleimide, depressed tyrosinolation in resting PMN and completely inhibited the fMet-Leu-Phe-induced stimulation. In contrast, addition of diamide, which preferentially oxidizes cellular glutathione, significantly stimulated tyrosinolation both in resting and fMet-Leu-Phe-stimulated PMN. Furthermore, resting levels of tyrosinolation in seven patients with chronic granulomatous disease (CGD), whose oxidative metabolism is severely depressed, were 35-45% lower (P less than 0.01). Most strikingly, PMN from CGD patients failed to respond to fMet-Leu-Phe or the Ca2+-ionophore A23187, which also induced stimulation of tyrosinolation in normal resting PMN. Methylene blue normalized the depressed tyrosinolation in resting CGD PMN, although it did not increase tyrosinolation in stimulated PMN. These results are consistent with the idea that the characteristic activation of the oxidative metabolism and the associated changes in the redox state in stimulated PMN are coupled to the induction of stimulation of tubulin tyrosinolation in these cells.

Detection of Focal Longitudinal Changes in the Brain by Subtraction of MR Images.

BACKGROUND AND PURPOSE: The detection of new subtle brain pathology on MR imaging is a time-consuming and error-prone task for the radiologist. This article introduces and evaluates an image-registration and subtraction method for highlighting small changes in the brain with a view to minimizing the risk of missed pathology and reducing fatigue. MATERIALS AND METHODS: We present a fully automated algorithm for highlighting subtle changes between multiple serially acquired brain MR images with a novel approach to registration and MR imaging bias field correction. The method was evaluated for the detection of new lesions in 77 patients undergoing cardiac surgery, by using pairs of fluid-attenuated inversion recovery MR images acquired 1-2 weeks before the operation and 6-8 weeks postoperatively. Three radiologists reviewed the images. RESULTS: On the basis of qualitative comparison of pre- and postsurgery FLAIR images, radiologists identified 37 new ischemic lesions in 22 patients. When these images were accompanied by a subtraction image, 46 new ischemic lesions were identified in 26 patients. After we accounted for interpatient and interradiologist variability using a multilevel statistical model, the likelihood of detecting a lesion was 2.59 (95% CI, 1.18-5.67) times greater when aided by the subtraction algorithm (P = .017). Radiologists also reviewed the images significantly faster (P < .001) by using the subtraction image (mean, 42 seconds; 95% CI, 29-60 seconds) than through qualitative assessment alone (mean, 66 seconds; 95% CI, 46-96 seconds). CONCLUSIONS: Use of this new subtraction algorithm would result in considerable savings in the time required to review images and in improved sensitivity to subtle focal pathology.

Inhibition of cyclophosphamide and mitomycin C-induced sister chromatid exchanges in mice by vitamin C.

Ascorbic acid (vitamin C) is known to act as an antimutagen and anticarcinogen in several test systems. However, there is no report of its effect on carcinogen-induced chromosomal damage in vivo in animals. The present study was performed to determine whether or not ascorbic acid affects sister chromatid exchanges (SCEs) induced by cyclophosphamide (CPA) and mitomycin C (MMC) in bone marrow and spleen cells in mice. The results indicate that ascorbic acid per se did not cause a significant increase in SCEs in mice. However, increasing concentrations of ascorbic acid caused decreasing levels of CPA- and MMC-induced SCEs in both cell types in vivo. At the highest concentration of ascorbic acid, 6.68 g/kg, approximately 75 and 40% SCE inhibition in both cell types was noted for CPA and MMC, respectively. Likewise, under in vivo/in vitro conditions (exposure of animals to experimental chemicals followed by culturing of cells), ascorbic acid caused a dose-related decrease in CPA- and MMC-induced SCEs, up to a dose of 3.34 g/kg At this concentration, approximately 50% CPA- and MMC-induced SCE inhibition was observed in both cell types studied. Thus, ascorbic acid acts as an anti-SCE agent in both in vivo and in vivo/in vitro conditions in mice.

Tubulin tyrosinolated in vivo can be different from that tyrosinolated in vitro.

Tubulin can be tyrosinolated, in the presence of ATP, by tubulin-tyrosine ligase, and tyrosine can be released by the same enzyme in the presence of ADP plus inorganic phosphate. There is however a 'non-substrate' component of tubulin which can not be tyrosinolated or detyrosinolated by this enzyme. Tubulin tyrosinolated in vivo was found to be the non-substrate species in HeLa cells, and the substrate species in cultured neuronal cells. In this respect HeLa tubulin resembled membrane-associated tubulin from brain, and neuronal cell tubulin resembled brain cytosolic tubulin.

Purification of bovine adrenocortical and brain tubulin. A comparative study.

Microtubules from the cow adrenal cortex and brain were purified by three cycles of the temperature-dependent polymerization-depolymerization procedure. Whereas tubulin comprised approximately 8--10% of soluble brain protein, it comprised only 0.5-1.0% of the soluble adrenocortical protein. The partially purified tubulin from both sources gave similar results in the following studies: (1) [3H]colchicine binding examined by Scatchard analysis revealed an apparent Ka of 1 . 10(6) M-1 and a colchicine/tubulin molar binding ratio of 0.4-0.6; (2) tyrosylation studies using a specific tubulin-tyrosine ligase (which adds a tyrosine residue to the C-terminal glutamate or glutamine of the alpha-chain) in conjunction with carboxypeptidase A (which recovers the tyrosine) and (3) amino acid analysis. Examination of protein bands, in addition to the tubulin doublet of 55 000 molecular weight, on sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed a difference between the two tubulin preparations. The adrenocortical preparation had protein bands corresponding to apparent molecular weight of 36 000, 60 000, and 68 000. In contrast the brain preparation had only proteins of molecular weights greater than 200 000 (these bands were absent in all adrenal preparations). It would thus appear that if proteins which copurify with tubulin through repeated cycles of polymerization-depolymerization play a role in either microtubule formation or function there is a distinct difference between neural and non-neural tissue.