Treatment-limiting renal tubulopathy in patients treated with tenofovir disoproxil fumarate.

OBJECTIVES: Tenofovir disoproxil fumarate (TDF) is widely used in the treatment or prevention of HIV and hepatitis B infection. TDF may cause renal tubulopathy in a small proportion of recipients. We aimed to study the risk factors for developing severe renal tubulopathy. METHODS: We conducted an observational cohort study with retrospective identification of cases of treatment-limiting tubulopathy during TDF exposure. We used multivariate Poisson regression analysis to identify risk factors for tubulopathy, and mixed effects models to analyse adjusted estimated glomerular filtration rate (eGFR) slopes. RESULTS: Between October 2002 and June 2013, 60 (0.4%) of 15,983 patients who had received TDF developed tubulopathy after a median exposure of 44.1 (IQR 20.4, 64.4) months. Tubulopathy cases were predominantly male (92%), of white ethnicity (93%), and exposed to antiretroviral regimens that contained boosted protease inhibitors (PI, 90%). In multivariate analysis, age, ethnicity, CD4 cell count and use of didanosine or PI were significantly associated with tubulopathy. Tubulopathy cases experienced significantly greater eGFR decline while receiving TDF than the comparator group (-6.60 [-7.70, -5.50] vs. -0.34 [-0.43, -0.26] mL/min/1.73 m2/year, p < 0.0001). CONCLUSIONS: Older age, white ethnicity, immunodeficiency and co-administration of ddI and PI were risk factors for tubulopathy in patients who received TDF-containing antiretroviral therapy. The presence of rapid eGFR decline identified TDF recipients at increased risk of tubulopathy.

Prolonging microtubule dysruption enhances the immunogenicity of chronic lymphocytic leukaemia cells.

Cytotoxic chemotherapies do not usually mediate the expression of an immunogenic gene programme in tumours, despite activating many of the signalling pathways employed by highly immunogenic cells. Concomitant use of agents that modulate and complement stress-signalling pathways activated by chemotherapeutic agents may then enhance the immunogenicity of cancer cells, increase their susceptibility to T cell-mediated controls and lead to higher clinical remission rates. Consistent with this hypothesis, the microtubule inhibitor, vincristine, caused chronic lymphocytic leukaemia (CLL) cells to die rapidly, without increasing their immunogenicity. Protein kinase C (PKC) agonists (such as bryostatin) delayed the death of vincristine-treated CLL cells and made them highly immunogenic, with increased stimulatory abilities in mixed lymphocyte responses, production of proinflammatory cytokines, expression of co-stimulatory molecules and activation of c-Jun N-terminal kinase (JNK), p38 and nuclear factor kappa B (NF-kappaB) signalling pathways. This phenotype was similar to the result of activating CLL cells through Toll-like receptors (TLRs), which communicate 'danger' signals from infectious pathogens. Use of PKC agonists and microtubule inhibitors to mimic TLR-signalling, and increase the immunogenicity of CLL cells, has implications for the design of chemo-immunotherapeutic strategies.

Membrane dynamics in phagocytosis.

Engulfment of particles by phagocytes involves remodeling of the plasma membrane. We review recent work that suggests that focal exocytosis of endomembranes plays an important role in pseudopod extension during phagocytosis.

Restricted accumulation of phosphatidylinositol 3-kinase products in a plasmalemmal subdomain during Fc gamma receptor-mediated phagocytosis.

Phagocytosis is a highly localized and rapid event, requiring the generation of spatially and temporally restricted signals. Because phosphatidylinositol 3-kinase (PI3K) plays an important role in the innate immune response, we studied the generation and distribution of 3' phosphoinositides (3'PIs) in macrophages during the course of phagocytosis. The presence of 3'PI was monitored noninvasively in cells transfected with chimeras of green fluorescent protein and the pleckstrin homology domain of either Akt, Btk, or Gab1. Although virtually undetectable in unstimulated cells, 3'PI rapidly accumulated at sites of phagocytosis. This accumulation was sharply restricted to the phagosomal cup, with little 3'PI detectable in the immediately adjacent areas of the plasmalemma. Measurements of fluorescence recovery after photobleaching were made to estimate the mobility of lipids in the cytosolic monolayer of the phagosomal membrane. Stimulation of phagocytic receptors induced a marked reduction of lipid mobility that likely contributes to the restricted distribution of 3'PI at the cup. 3'PI accumulation during phagocytosis was transient, terminating shortly after sealing of the phagosomal vacuole. Two factors contribute to the rapid disappearance of 3'PI: the dissociation of the type I PI3K from the phagosomal membrane and the persistent accumulation of phosphoinositide phosphatases.

The yeast nucleoporin Nup2p is involved in nuclear export of importin alpha/Srp1p.

The importin alpha.beta heterodimer mediates nuclear import of proteins containing classical nuclear localization signals. After carrying its cargo into the nucleus, the importin dimer dissociates, and Srp1p (the yeast importin alpha subunit) is recycled to the cytoplasm in a complex with Cse1p and RanGTP. Nup2p is a yeast FXFG nucleoporin that contains a Ran-binding domain. We find that export of Srp1p from the nucleus is impaired in Deltanup2 mutants. Also, Srp1p fusion proteins accumulate at the nuclear rim in wild-type cells but accumulate in the nuclear interior in Deltanup2 cells. A deletion of NUP2 shows genetic interactions with mutants in SRP1 and PRP20, which encodes the Ran nucleotide exchange factor. Srp1p binds directly to an N-terminal domain of Nup2p. This region of Nup2p is sufficient to allow accumulation of an Srp1p fusion protein at the nuclear rim, but the C-terminal Ran-binding domain of Nup2p is required for efficient Srp1p export. Formation of the Srp1p.Cse1p. RanGTP export complex releases Srp1p from its binding site in Nup2p. We propose that Nup2p may act as a scaffold that facilitates formation of the Srp1p export complex.

Phosphate transport in yeast vacuoles.

The vacuole of the yeast Saccharomyces cerevisiae is a major storage compartment for phosphate. We have measured phosphate transport across the vacuolar membrane. Isolated intact vacuoles take up large amounts of added [32P]phosphate by counterflow exchange with phosphate present in the vacuoles at the time of their isolation. The bidirectional phosphate transporter has an intrinsic dissociation constant for phosphate of 0.4 mM. Exchange mediated by this carrier is faster than unidirectional efflux of phosphate from the vacuoles. The transporter is highly selective for phosphate; of other anions tested, only arsenate is also a substrate. Transport is strongly pH-dependent with increasing activity at lower pH. Similar phosphate transport behavior was observed in right-side-out vacuolar membrane vesicles.

An alleged yeast polyphosphate kinase is actually diadenosine-5', 5"'-P1,P4-tetraphosphate alpha,beta-phosphorylase.

Polyphosphates are a major constituent of the yeast Saccharomyces cerevisiae. A purification of the enzyme polyphosphate kinase (E.C. 2.7.4.1) from this organism has been reported (Felter, S., and Stahl, A.J.C. (1973) Biochimie (Paris) 55, 245-251). The assay for activity used in this purification was the production of 32P-labeled nucleotide, presumed to be ATP, in the presence of [32P]polyphosphate and ADP. We have found that this assay does not reflect the activity of a polyphosphate kinase but rather the combination of an exopolyphosphatase, releasing free [32P]phosphate from the added [32P]polyphosphate, and the ADP-[32P]phosphate exchange activity of the enzyme diadenosine 5',5"'-P1, P4-tetraphosphate alpha, beta-phosphorylase (Ap4A phosphorylase). We also present direct evidence for the formation of an enzyme-AMP intermediate in the actin of Ap4A phosphorylase.

Explaining glomerular pores with fiber matrices. A visualization study based on computer modeling.

The extracellular space of the glomerular capillary wall is occupied by a complex meshwork of fibrous molecules. Little is understood about how the size, shape, and charge recognition properties of glomerular ultrafiltration arise from this space-filling fiber matrix. We studied the problem of size recognition by visualizing the void volume accessible to hard spheres in computer-generated three-dimensional homogeneous random fiber matrices. The spatial organization of the void volume followed a complex "blob-and-throat" pattern in which circumscribed cavities of free space within the matrix ("blobs") were joined to adjacent cavities by narrower throats of void space. For sufficiently small solutes, chains of blobs and throats traversed the matrix, providing pathways for trans-matrix permeation. The matrices showed threshold or gating properties with respect to permeation: solutes whose radius exceeded a critical value, at which a throat on the last connected trans-matrix pathway pinched off, could not cross, whereas smaller solutes had nonzero permeability. The thresholds may give the glomerular fiber matrix porelike response properties and explain why pore models have been such a useful means of treating permselectivity.

Role of Norwalk virus in two foodborne outbreaks of gastroenteritis: definitive virus association.

Two separate food-associated outbreaks of gastroenteritis occurred among Erie County, New York residents in June 1986. In one outbreak, cases of illness were estimated to have occurred in 50% of the approximately 700 persons in 13 groups who ate at an out-of-county restaurant during a seven-day period, and, in the second outbreak, illness occurred in 26 (30%) of 87 persons who attended a graduation party held in a private home. Laboratory investigation included serology (blocking radioimmunoassay) to determine seroconversion to Norwalk virus and an enzyme immunoassay for detection of Norwalk virus antigen in stools, which the investigators have found to be more specific for Norwalk virus than serology. Seroconversion to Norwalk virus occurred in 11 (79%) of 14 restaurant-related cases and seven (100%) of seven graduation party cases. Seroconversion to Norwalk virus antigen was also found in four (40%) of 10 food handlers at the restaurant and in two (100%) of two food handlers at the graduation party. Antigen was detected in the stools of three (20%) of 15 restaurant-related cases and four (67%) of six graduation party cases. No stools for viral analyses were available for testing from food handlers. All seven of the patients with Norwalk virus-positive stools were also positive by seroconversion. Widespread availability of reagents for stool antigen detection would result in confirmation of more outbreaks due to Norwalk virus and in a more timely manner.

Uptake of iron from hemoglobin and the haptoglobin-hemoglobin complex by hemolytic bacteria.

The abilities of Staphylococcus aureus and Streptococcus pyogenes to remove iron from mouse 59Fe hemoglobin that was either in free form or complexed with human haptoglobin, were evaluated. 59Fe hemoglobin from the amphibian Taricha granulosa was also used in free form or complexed with the amphibian's hemoglobin-binding proteins. Contrary to what was reported from a study using pathogenic Escherichia coli, haptoglobin failed to exhibit a bacteriostatic influence when complexed with hemoglobin. In our study, more 59Fe was removed by the bacteria from the haptoglobin-hemoglobin complex than from free mouse hemoglobin. The hemoglobin and hemoglobin-plasma protein complexes of Taricha were stripped of 59Fe at similar rates and extents by both bacterial species.

[Inflammatory bowel disease in childhood]. / Malattia infiammatoria dell'intestino in età pediatrica.

Inflammatory bowel disease in childhood is becoming increasingly important as a result of a marked increase in incidence occurring over the past 20 years. In Crohn's disease the onset is commonly insidious, with symptoms such as growth failure, which do not immediately suggest intestinal disease. This may result in a correct diagnosis being delayed for several years. The role of paediatric colonoscopy is discussed, together with the macroscopic and microscopic appearances of the lesions. The indications and efficacy of drug therapy in Crohn's disease and ulcerative colitis are outlined and the importance of nutritional support, particularly in children with growth failure, is discussed. The indications for surgery are summarised and the need for a multidisciplinary approach, particularly in cases of fulminating colitis where early surgery is indicated, is emphasised. Most children with inflammatory bowel disease do reasonably well and even those children undergoing proctocolectomy adapt to a stoma provided they receive optimal psycho-social preparation and support.