Heterotelechelic homopolymers mimicking high χ - ultralow N block copolymers with sub-2 nm domain size.

Three fluorinated, hydrophobic initiators have been utilised for the synthesis of low molecular mass fluoro-poly(acrylic acid) heterotelechelic homopolymers to mimic high chi (χ)-low N diblock copolymers with ultrafine domains of sub-2 nm length scale. Polymers were obtained by a simple photoinduced copper(ii)-mediated reversible-deactivation radical polymerisation (Cu-RDRP) affording low molecular mass (<3 kDa) and low dispersity (D = 1.04-1.21) homopolymers. Heating/cooling ramps were performed on bulk samples (ca. 250 µm thick) to obtain thermodynamically stable nanomorpologies of lamellar (LAM) or hexagonally packed cylinders (HEX), as deduced by small-angle X-ray scattering (SAXS). Construction of the experimental phase diagram alongside a detailed theoretical model demonstrated typical rod-coil block copolymer phase behaviour for these fluoro-poly(acrylic acid) homopolymers, where the fluorinated initiator-derived segment acts as a rod and the poly(acrylic acid) as a coil. This work reveals that these telechelic homopolymers mimic high χ-ultralow N diblock copolymers and enables reproducible targeting of nanomorphologies with incredibly small, tunable domain size.

An acoustic on-chip goniometer for room temperature macromolecular crystallography.

This paper describes the design, development and successful use of an on-chip goniometer for room-temperature macromolecular crystallography via acoustically induced rotations. We present for the first time a low cost, rate-tunable, acoustic actuator for gradual in-fluid sample reorientation about varying axes and its utilisation for protein structure determination on a synchrotron beamline. The device enables the efficient collection of diffraction data via a rotation method from a sample within a surface confined droplet. This method facilitates efficient macromolecular structural data acquisition in fluid environments for dynamical studies.

Induction treatment of previously undiagnosed ANCA-associated vasculitis in a renal transplant patient with Rituximab.

We report the case of a 40-year-old female transplant patient with undiagnosed ANCA-associated vasculitis (AAV) and renal allograft dysfunction who achieved disease remission with restoration of transplant function following induction therapy with rituximab. There are currently no trial data looking at the use of rituximab for induction of remission of renal transplant patients with AAV. Although recurrence of AAV following renal transplantation is rare, such patients have invariably had multiple previous exposures to induction and maintenance immunosuppressive regimens, often limiting treatment options post-transplantation. In this case, rituximab was well tolerated with no side effects, and was successful in salvaging transplant function. Optimal treatment regimens for relapsed AAV in the transplant population are not known, and clinical trials are needed to evaluate the efficacy and safety of rituximab at inducing and maintaining disease remission in relapsed AAV following transplantation.

The effect of albumin on podocytes: the role of the fatty acid moiety and the potential role of CD36 scavenger receptor.

Evidence is emerging that podocytes are able to endocytose proteins such as albumin using kinetics consistent with a receptor-mediated process. To date the role of the fatty acid moiety on albumin uptake kinetics has not been delineated and the receptor responsible for uptake is yet to be identified. Albumin uptake studies were carried out on cultured human podocytes exposed to FITC-labelled human serum albumin either carrying fatty acids (HSA+FA) or depleted of them (HSA-FA). Receptor-mediated endocytosis of FITC-HSA+FA over 60 min was 5 times greater than that of FITC-HSA-FA. 24h exposure of podocytes to albumin up-regulated nephrin expression and induced the activation of caspase-3. These effects were more pronounced in response to HSA-FA. Individually, anti-CD36 antibodies had no effect upon endocytosis of FITC-HSA. However, a cocktail of 2 antibodies reduced uptake by nearly 50%. Albumin endocytosis was enhanced in the presence of the CD36 specific inhibitor sulfo-N-succinimidyl oleate (SSO) while knock-down of CD36 using CD36siRNA had no effect on uptake. These data suggest that receptor-mediated endocytosis of albumin by podocytes is regulated by the fatty acid moiety, although, some of the detrimental effects are induced independently of it. CD36 does not play a direct role in the uptake of albumin.

The efficiency of encapsulation within surface rehydrated polymersomes.

The key to the use of polymersomes as effective molecular delivery systems is in the ability to design processing routes that can efficiently encapsulate the molecular payload. We have evaluated various surface rehydration mechanisms for encapsulation, in each case characterizing the morphologies formed using DLS and confocal microscopy as well as determining the encapsulation efficiency for the hydrophilic dye Rhodamine B. In contrast to bulk methods, where the encapsulation efficiencies are low, we find that higher efficiencies can be obtained by the rehydration of thin films. We relate these results to the non-equilibrium mechanisms that underlie vesicle formation and discuss how an understanding of these mechanisms can help optimize encapsulation efficiencies. Our conclusion is that, even considering the good encapsulation efficiency, surface methods are still unsuitable for the massive scale-up needed when applied to commercial "mass market" molecular delivery scenarios. However, targeting more specialized applications for high value ingredients (like pharmaceuticals) might be more feasible.

Responsive brushes and gels as components of soft nanotechnology.

Progress in the development of generic molecular devices based on responsive polymers is discussed. Characterisation of specially synthesised polyelectrolyte gels, "grafted from" brushes and triblock copolymers is reported. A Landolt pH-oscillator, based on bromate/ sulfite/ferrocyanide, with a room temperature period of 20 min and a range of 3.1 < pH < 7.0, has been used to drive periodic oscillations in volume in a pH responsive hydrogel. The gel is coupled to the reaction and changes volume by a factor of at least 6. A continuously stirred, constant volume, tank reactor was set-up on an optical microscope and the reaction pH and gel size monitored. The cyclic force generation of this system has been measured directly in a modified JKR experiment. The responsive nature of polyelectrolyte brushes, grown by surface initiated ATRP, have been characterised by scanning force microscopy, neutron reflectometry and single molecule force measurements. Triblock copolymers, based on hydrophobic end-blocks and either polyacid or polybase mid-block, have been used to produce polymer gels where the deformation of the molecules can be followed directly by SAXS and a correlation between molecular shape change and macroscopic deformation has been established. The three systems studied allow both the macroscopic and a molecular response to be investigated independently for the crosslinked gels and the brushes. The triblock copolymers demonstrate that the individual response of the polyelectrolyte molecules scale-up to give the macroscopic response of the system in an oscillating chemical reaction.

The use of direct susceptibility tests from BACTEC 9240 to Microscan Walkaway.

Using a straight-from-the-bottle into-the-instrument technique, a series of direct susceptibility tests were performed with the BACTEC 9240 and Microscan Walkaway systems. Although rapid, unacceptably high error rates were obtained when compared with a standardized method. This procedure is not sufficiently accurate for clinical use.

Aminopeptidase A: a nephritogenic target antigen of nephrotoxic serum.

BACKGROUND: We investigated potential targets of antibody-mediated glomerular injury induced with a noncomplement binding fraction of sheep anti-rat nephrotoxic serum (NTS). This model is characterized by severe complement- and leukocyte-independent proteinuria within 24 hours of NTS injection into rats. METHODS: NTS-reactive glomerular cell and matrix proteins were identified by immunoprecipitation, Western blot analysis, protein sequencing, cDNA library screening, and enzyme-linked immunosorbent assay. Proteinuria was measured in rats injected with NTS from which reactivity against type IV collagen had been removed by immunoadsorption, and antibodies were eluted from the glomeruli of proteinuric rats that had been injected with unabsorbed NTS. Having identified aminopeptidase A (APA) as a major target of NTS, we studied the effect of NTS and anti-APA on mouse glomerular epithelial cells in culture. RESULTS: NTS identified several podocyte and matrix proteins; however, APA was the only cell surface protein reactive with antibodies eluted from the glomeruli of rats injected with NTS. Although the eluate also contained reactivity to the noncollagenous domains of alpha1 and alpha3 chains of type IV collagen, immunodepletion of these antibodies did not diminish the ability of NTS to cause proteinuria. We also documented the surface expression of APA on mouse glomerular epithelial cells in culture, and found that NTS and specific anti-APA antibodies induce a time- and temperature-dependent redistribution of the antigen. CONCLUSIONS: APA, a type II integral membrane metallopeptidase, is a major target of NTS in vivo and is known to be present on the surface of podocytes. NTS-induced proteinuria is independent of reactivity to known nephritogenic matrix proteins. These findings, in combination with previous studies showing that monoclonal anti-APA antibodies induce severe proteinuria in mice, suggest that anti-APA antibodies are responsible for complement-independent proteinuria in this model.

Targeting of the chemokine receptor CCR1 suppresses development of acute and chronic cardiac allograft rejection.

Although mononuclear cell infiltration is a hallmark of cellular rejection of a vascularized allograft, efforts to inhibit rejection by blocking leukocyte-endothelial cell adhesion have proved largely unsuccessful, perhaps in part because of persistent generation of chemokines within rejecting grafts. We now provide, to our knowledge, the first evidence that in vivo blockade of specific chemokine receptors is of therapeutic significance in organ transplantation. Inbred mice with a targeted deletion of the chemokine receptor CCR1 showed significant prolongation of allograft survival in 4 models. First, cardiac allografts across a class II mismatch were rejected by CCR1(+/+) recipients but were accepted permanently by CCR1(-/-) recipients. Second, CCR1(-/-) mice rejected completely class I- and class II-mismatched BALB/c cardiac allografts more slowly than control mice. Third, levels of cyclosporin A that had marginal effects in CCR1(+/+) mice resulted in permanent allograft acceptance in CCR1(-/-) recipients. These latter allografts showed no sign of chronic rejection 50-200 days after transplantation, and transfer of CD4(+) splenic T cells from these mice to naive allograft recipients significantly prolonged allograft survival, whereas cells from CCR1(+/+) mice conferred no such benefit. Finally, both CCR1(+/+) and CCR1(-/-) allograft recipients, when treated with a mAb to CD4, showed permanent engraftment, but these allografts showed florid chronic rejection in the former strain and were normal in CCR1(-/-) mice. We conclude that therapies to block CCR1/ligand interactions may prove useful in preventing acute and chronic rejection clinically.

Lack of chemokine receptor CCR1 enhances Th1 responses and glomerular injury during nephrotoxic nephritis.

During the development of nephrotoxic nephritis (NTN) in the mouse, we find that a variety of chemokines and chemokine receptors are induced: CCR1 (RANTES, MIP-1alpha), CCR2 (MCP-1), CCR5 (RANTES, MIP-1alpha, MIP-1beta), CXCR2 (MIP-2), and CXCR3 (IP-10). Their timing of expression indicated that CXCR2 and CCR1 are probably important in the neutrophil-dependent heterologous phase of the disease, whereas CCR1, CCR2, CCR5, and CXCR3 accompany the subsequent mononuclear cell infiltration characteristic of autologous disease. We therefore assessed the role of CCR1 in NTN using CCR1(-/-) mice. We found that neutrophil accumulation in CCR1(-/-) mice was comparable to that in wild-type animals but that renal recruitment of CD4(+) and CD8(+) T cells and macrophages increased significantly. Moreover, CCR1(-/-) mice developed more severe glomerulonephritis than did controls, with greater proteinuria and blood urea nitrogen, as well as a higher frequency of crescent formation. In addition, CCR1(-/-) mice showed enhanced Th1 immune responses, including titers of antigen-specific IgG2a antibody, delayed-type hypersensitivity responses, and production of IFN-gamma and TNF-alpha. Lastly, using recombinant proteins and transfected cells that overexpressed CCR1, we demonstrated that MIP-1alpha, but not RANTES, bound CCR1 and induced cell chemotaxis. Thus, rather than simply promoting leukocyte recruitment during NTN, CCR1 expression profoundly alters the effector phase of glomerulonephritis. Therapeutic targeting of chemokine receptors may, on occasion, exacerbate underlying disease.

Nephritogenic mAb 5-1-6 is directed at the extracellular domain of rat nephrin.

mAb 5-1-6 identifies an antigen on rat podocyte slit-diaphragms and induces severe proteinuria when injected into rats. Nephrin, an Ig-like transmembrane protein that is mutated in congenital nephrotic syndrome of the Finnish type, has been localized to the slit-diaphragm on human podocytes. Here we document that the mAb 5-1-6 antigen is rat nephrin. After incubation of rat glomeruli with this mAb, the antibody/antigen complex was chemically cross-linked, extracted, and immunoprecipitated, prior to Western analysis. By mass spectrometry and 2D gel electrophoresis, we identified several peptides with complete identity to human nephrin. In addition, the 185-kDa protein immunoprecipitated by mAb 5-1-6 from rat glomerular extracts reacts with a rabbit anti-mouse nephrin antibody. Finally, nephrin and the mAb 5-1-6 antigen have identical glomerular localization patterns on immunofluorescence of rat kidney. These results demonstrate that the nephritogenic mAb 5-1-6 identifies the extracellular domain of nephrin, thereby documenting the importance of the slit-diaphragm and its component, nephrin, in the regulation of glomerular permselectivity.

Complement-mediated injury reversibly disrupts glomerular epithelial cell actin microfilaments and focal adhesions.

BACKGROUND: Foot process effacement and condensation of the glomerular epithelial cell (GEC) cytoskeleton are manifestations of passive Heymann nephritis, a model of complement-mediated membranous nephropathy. METHODS: To study the effects of complement on the actin cytoskeleton in this model, we have used an in vitro system in which GECs are sublethally injured using a combination of complement-fixing anti-Fx1A IgG and human serum as a source of complement. We examined the effects of this injury on the organization of the cytoskeleton and focal contacts using immunohistology and immunochemistry. RESULTS: By immunofluorescence, sublethal complement-mediated injury was accompanied by a loss of actin stress fibers and focal contacts but retention of matrix-associated integrins. Full recovery was seen after 18 hours. Western blot analysis showed no change in the cellular content of the focal contact proteins. Inhibition of the calcium-dependent protease calpain did not prevent injury. In addition, cycloheximide during recovery did not inhibit the reassembly of stress fibers or focal contacts. Injury was associated with a reduction in tyrosine phosphorylation of paxillin and a currently unidentified 200 kDa protein, but inhibition of tyrosine phosphatase activity with sodium vanadate did not prevent injury. Cellular adenosine triphosphate content was significantly reduced in injured cells. CONCLUSION: These results document reversible, complement-dependent disruption of actin microfilaments and focal contacts leading to the dissociation of the cytoskeleton from matrix-attached integrins. This may explain the altered cell-matrix relationship accompanying podocyte effacement in membranous nephropathy.

Isolated microscopic haematuria in the genitourinary clinic: the value of renal biopsy.

Isolated microscopic haematuria is a common finding in the genitourinary clinic. The conventional approach to investigation includes urological referral for cystourethroscopy if renal imaging is normal. However the diagnostic yield is very low; in particular urothelial malignancy at age < 40 years is rare. Glomerular disease is increasingly recognized as a common cause of microscopic haematuria. In this study 50 patients with persistent microscopic haematuria detected at a genitourinary clinic underwent renal biopsy. Twelve (24%) had an abnormal biopsy--IgA nephropathy 6 (12%), thin membrane nephropathy 3 (6%), other glomerulonephritis 3 (6%). In 7 others no abnormality was found but information was incomplete as electron microscopy was unavailable. It is important to establish these diagnoses since some patients will develop progressive renal disease. In this clinical setting renal biopsy will give diagnostic and prognostic information, protects from repeated urological investigation, and allows reassurance if renal histology is normal. Renal biopsy is recommended for patients age < 40 years with persistent microscopic haematuria. An algorithm for the investigation of microscopic haematuria is presented.

Leucocyte beta 1,3 galactosyltransferase activity in IgA nephropathy.

BACKGROUND: Reduced galactosylation of the O-linked glycans of the IgA1 hinge region in IgAN has recently been described. To investigate the underlying defect resulting in this abnormality, we have measured the activity of beta 1,3 galactosyltransferase, the enzyme responsible for galactosylation of O-linked sugars. METHODS: A galactose-acceptor substrate was prepared from degalactosylated hinge region fragments of normal IgA1, and incubated with the T cell, B cell, and monocyte lysates from patients with IgAN and controls for acceptor regalactosylation. The extent of acceptor galactosylation was then measured with biotinylated Vicia villosa lectin (VV), which is specific for ungalactosylated moieties. Lectin binding of serum IgA from the same subjects was also measured. RESULTS: T cell and monocyte beta 1,3 galactosyltransferase activities did not differ between IgAN and control, but B cell lysates in IgAN showed significantly lower beta 1,3 galactosyltransferase activity than control (6.2 +/- 0.71 vs. 9.5 +/- 1.03 AU/microgram, P = 0.018). Furthermore, B cell beta 1,3 galactosyltransferase activity showed a negative correlation (r = -0.87, P = 0.002) with VV lectin binding of serum IgA in IgAN, but not controls. CONCLUSIONS: These data indicate that altered IgA1 O-galactosylation in IgAN results from a B cell-restricted reduction of beta 1,3 galactosyltransferase activity. This enzyme defect may be a fundamental pathogenic abnormality in IgAN.

Slit diaphragm-reactive nephritogenic MAb 5-1-6 alters expression of ZO-1 in rat podocytes.

Monoclonal antibody (MAb) 5-1-6 identifies a 51-kDa protein (p51) on rat podocyte foot processes and causes severe complement- and leukocyte-independent proteinuria when injected into rats. In the studies reported here, we used various immunohistological techniques to define the precise location of p51 and its relationship to ZO-1, a known component of the podocyte slit diaphragm in adult rat glomeruli. Our results demonstrate that p51 and ZO-1 lie close to each other on opposite sides of the podocyte plasma membrane at the point of insertion of the slit diaphragm: ZO-1 on the cytoplasmic face and p51 on the slit diaphragm and adjoining outer leaflet of the plasma membrane bordering the filtration slits. In addition to their geographic proximity, there appears to be a relationship between p51 and ZO-1. After MAb 5-1-6 injection, there was a progressive decline in stainable ZO-1 in the podocytes of heavily proteinuric rats. In addition, Western blot analysis of glomerular lysates showed that the decline in staining was due to a loss of immunoreactive ZO-1 rather than redistribution or diffusion of the protein. Simultaneously, the distribution of glomerular-bound MAb 5-1-6 became more clumped, apparently because of partial endocytosis into a lysosomal compartment, while the slit diaphragms remained morphologically intact. These findings suggest that MAb 5-1-6 alters the molecular composition of the slit diaphragm and thereby affects the glomerular permeability barrier.

Glomerular disease as a cause of isolated microscopic haematuria.

Microscopic haematuria is a common clinical finding, with reported prevalences of up to 22%. The role of renal biopsy in the investigation of this condition is still debated. Currently urological investigation including cystourethroscopy is often regarded as adequate. We investigated 165 patients (94 male, 71 female; mean age 37.5 years, range 10-71) referred with isolated microscopic haematuria, using renal biopsy and cystourethroscopy. All patients were normotensive with normal serum creatinine, no proteinuria, sterile urine and a normal IVU. Renal biopsy abnormalities were found in 77/165 (46.6%): IgA nephropathy (49), global or segmental mesangial proliferative glomerulonephritis without IgA deposits (16), thin membrane nephropathy (7), vascular changes suggestive of hypertension (3), interstitial nephritis (1), and membranous nephropathy (1). Only five abnormalities were found on cystourethroscopy (cystitis 3, urethral stricture 1, bladder stone 1). Two patients with cystitis also had IgA nephropathy. Biopsy abnormalities were commonest under the age of 20 (69.2%), but 40% of biopsies were abnormal even in the seventh decade of life. Because renal biopsy abnormalities are very frequent in patients with isolated haematuria, renal biopsy is indicated in patients over 45 years of age if renal imaging and cystoscopy are normal. In those under 45 years, renal biopsy should replace cystoscopy as the investigation to follow normal renal imaging.

The use of Taylor's power law to describe the aggregated distribution of gastro-intestinal nematodes of sheep.

The distribution of gastro-intestinal nematodes collected from lambs was investigated using the Taylor's power law index of aggregation beta, which is known to be independent of the mean population size. For 12 out of the 15 nematode species investigated the estimate of beta was not significantly different from 2.0. For these species a logarithmic transformation would stabilize the variance of the distributions prior to further statistical analysis. The remaining species had values of beta which were significantly lower than 2.0. For these species a variance-stabilizing transformation is given by z = x1-beta/2.

Spatial distribution on pasture of infective larvae of the gastro-intestinal nematode parasites of sheep.

The horizontal distributions of infective larvae on pasture grazed by sheep have been investigated. Using Taylor's Power Law it was found that larvae had a more aggregated distribution in September than August, the Law index of aggregation being 1.97 and 1.89 for the 2 months, respectively. However, at each time the degree of aggregation remained fairly constant for a range of spacings between points from 5 to 30 m. These results suggest that Taylor's Power Law could be used as a basis for devising an efficient pasture sampling strategy. More data are required, however, to determine the extent to which aggregation of the larvae varies with time of the year.