Safety of the use of gold nanoparticles conjugated with proinsulin peptide and administered by hollow microneedles as an immunotherapy in type 1 diabetes.

Antigen-specific immunotherapy is an immunomodulatory strategy for autoimmune diseases, such as type 1 diabetes, in which patients are treated with autoantigens to promote immune tolerance, stop autoimmune ß-cell destruction and prevent permanent dependence on exogenous insulin. In this study, human proinsulin peptide C19-A3 (known for its positive safety profile) was conjugated to ultrasmall gold nanoparticles (GNPs), an attractive drug delivery platform due to the potential anti-inflammatory properties of gold. We hypothesised that microneedle intradermal delivery of C19-A3 GNP may improve peptide pharmacokinetics and induce tolerogenic immunomodulation and proceeded to evaluate its safety and feasibility in a first-in-human trial. Allowing for the limitation of the small number of participants, intradermal administration of C19-A3 GNP appears safe and well tolerated in participants with type 1 diabetes. The associated prolonged skin retention of C19-A3 GNP after intradermal administration offers a number of possibilities to enhance its tolerogenic potential, which should be explored in future studies.

Application of an optimized marker amplification protocol in immunohistochemistry - a valuable tool for visualizing antigenic sites of low signal strength or sparse distribution.

Amplification of immunohistochemical markers received considerable attention during the 1980s and 1990s. The amplification approach was largely abandoned following the development of antigen retrieval and reporter amplification techniques, because the latter were incorporated more easily into high throughput automated procedures in industrial and diagnostic laboratories. There remain, however, a number of instances where marker amplification still has much to offer. Consequently, we examined experimentally the utility of an optimized marker amplification technique in diagnostically relevant tissue where either the original signal strength was low or positive sites were visible, but sparsely distributed. Marker amplification in the former case not only improved the visibility of existing positive sites, but also revealed additional sites that previously were undetectable. In the latter case, positive sites were rendered more intense and therefore more easily seen during low magnification examination of large areas of tissue.

Heads you win, tails you lose: filtration processing for the microscopical examination of sperm heads.

The sperm head plays a key role in many fertilisation events and determining the precise location of molecules within the head region is important in mechanistically dissecting the fertilisation process. Such molecules may be present in low copy number and many sperm head profiles must be examined to localise them to particular subcellular structures with confidence. Filtration has traditionally been used for the purpose of concentrating biological material, such as free-living cells, spores, and subcellular fractions, and little attempt has been made to extend the procedure to encompass the entire processing schedule, mainly due to the incompatibility of intermediate dehydrating solvents with membrane filters. The novel and simple technique of filtration processing that we describe produced a dense mat of cells, with several sperm heads being visible in coronal orientation in a high-power field at the light microscopic level, and allowed positive immunocytochemical staining to be identified with confidence. This new technique exploits the low viscosity of LR White acrylic resin to allow the entire processing procedure to be undertaken in the filtration apparatus. In contrast, conventional techniques for preparing free-living cells, namely pre-embedding in a supportive matrix prior to processing, and centrifugation at each stage of the processing procedure, proved suboptimal, partly due to the final concentration that could be achieved, but mainly due to the random orientation of cells that these techniques afforded.

Metallosis following implantation of magnetically controlled growing rods in the treatment of scoliosis: a case series.

AIMS: We present a case series of five patients who had revision surgery following magnetic controlled growing rods (MGCR) for early onset scoliosis. Metallosis was found during revision in four out of five patients and we postulated a mechanism for rod failure based on retrieval analysis. PATIENTS AND METHODS: Retrieval analysis was performed on the seven explanted rods. The mean duration of MCGR from implantation to revision was 35 months (17 to 46). The mean age at revision was 12 years (7 to 15; four boys, one girl). RESULTS: A total of six out of seven rods had tissue metallosis and pseudo-capsule surrounding the actuator. A total of four out of seven rods were pistoning. There were two rods which were broken. All rods had abrasive circumferential markings. A significant amount of metal debris was found when the actuators were carefully cut open. Analytical electron microscopy demonstrated metal fragments of predominantly titanium with a mean particle size of 3.36 microns (1.31 to 6.61). CONCLUSION: This study highlights concerns with tissue metallosis in MCGR. We recommend careful follow-up of patients who have received this implant. Cite this article: Bone Joint J 2016;98-B:1662-7.

LR White sections as slot grid support films for transmission electron microscopy.

The utility of LR White sections as slot grid support films for the examination of thin resin-embedded tissue sections by transmission electron microscopy was investigated and compared with traditional formvar-carbon films. Throughout a variety of staining procedures, which involved the use of organic solvent, oxidizing agents, strong acid and prolonged incubation, LR White support films remained intact and the attached tissue sections remained adherent. By contrast, complete loss of formvar-carbon support films occurred in 25% of preparations during routine staining with aqueous reagents. This loss increased to 62% following staining with either alcoholic or oxidizing and acidic stains, and to 66% following prolonged (immunohistochemical) staining. Tissue contrast, ultrastructural detail and immunohistochemical staining intensity were comparable between sections on the two types of support film. The use of LR White sections as support films for slot grids represents a quick, cheap, simple and robust alternative to traditional support films and, furthermore, requires no carbon coating.

The amplification of polymerized diaminobenzidine with physical developers: sensitizing effects of transition metal salts and sulphide.

Amplification of metal-complexed polymerized diaminobenzidine by two light-insensitive physical developers was systematically examined in a dot blot model system following either polymerizing diaminobenzidine in the presence of transition metal salts or applying the metal salts post-diaminobenzidine polymerization. The effect of sodium sulphide treatment on subsequent amplification was also investigated. Those metal-diaminobenzidine complexes that facilitated the most powerful amplification were subsequently tested in an immunohistochemical setting. The most dramatic amplification of polymerized diaminobenzidine was observed following its post-polymerization treatment with salts of platinum alone, or gold or vanadium with subsequent sulphide treatment, and allowed previously invisible quantities of polymerized diaminobenzidine to be clearly seen. Three other transition metal salts also improved the amplification of polymerized diaminobenzidine but to a lesser degree, namely nickel alone, and silver or rhodium with subsequent sulphide treatment. Sensitivity was comparable with the colloidal gold-silver amplification system.

Conditionally immortalized human glomerular endothelial cells expressing fenestrations in response to VEGF.

Glomerular endothelial cells (GEnC) are specialized cells with important roles in physiological filtration and glomerular disease. Despite their unique features, GEnC have been little studied because of difficulty in maintaining them in cell culture. We have addressed this problem by generation of conditionally immortalized (ci) human GEnC using technology with which we have previously produced ci podocytes. Primary culture GEnC were transduced with temperature-sensitive simian virus 40 large tumour antigen and telomerase using retroviral vectors. Cells were selected, cloned, and then characterized by light and electron microscopy (EM), response to vascular endothelial growth factor (VEGF), and tumour necrosis factor (TNF)alpha, expression of endothelial markers by focused gene array, immunofluorescence and Western blotting, and formation and behaviour of monolayers. CiGEnC proliferated at the permissive temperature (33 degrees C) and became growth arrested at the non-permissive temperature (37 degrees C). CiGEnC retained morphological features of early-passage primary culture GEnC up to at least p41, confirming successful immortalization. EM demonstrated fenestrations, increased in number by VEGF. mRNA analysis confirmed expression of the endothelial markers platelet endothelial cell adhesion molecule 1, intercellular adhesion molecule 2, VEGF receptor 2, and von Willebrand factor, validated by immunofluorescence and Western blotting. CiGEnC also expressed Tie2, and TNFalpha upregulated E-selectin. CiGEnC formed monolayers with barrier properties responsive to cyclic adenosine 3',5' monophosphate (cAMP) and thrombin. CiGEnC retain the markers and behaviour of primary culture GEnC. They express fenestrations which are upregulated in response to VEGF. These cells are a unique resource for further study of GEnC and their roles in glomerular filtration, glomerular disease, and response to glomerular injury.

A cytochemical method for visualising blood vessels selectively in semithin LR white sections.

Traditional cytochemical techniques for illustrating blood vessels are usually unsuitable for use in high resolution, 0.35 microm-thick LR White sections due to the thinness of the tissue and, if collagenous, high background staining. We report that a modification of the periodic acid-thiocarbohydrazide-silver proteinate-silver enhancement method (PATCH-SP-SE) selectively visualises, with low background, even the smallest blood vessels in semithin LR White sections of highly collagenous tissues such as human parietal peritoneum.

Caveolin-1 expression and caveolae biogenesis during cell transdifferentiation in lung alveolar epithelial primary cultures.

Caveolae are omega-shaped invaginations of the plasmalemma possessing a cytoplasmic membrane protein coat of caveolin. Caveolae are present in the in vivo alveolar epithelial type I (ATI) lung cell, but absent in its progenitor, the alveolar epithelial type II (ATII) cell. In primary culture ATII cells grown on a plastic substratum acquire with time an ATI-"like" phenotype. We demonstrate that freshly isolated rat ATII cells lack caveolae and expression of caveolin-1 (a critical caveolae structural protein). As the ATII cells acquire an ATI-like phenotype in primary culture caveolin-1 expression increases, with caveolin-1 signal at 192 h postseeding up to 50-fold greater than at 60 h; caveolae were morphologically evident only after 132 h. When maintaining the differentiated ATII phenotype with time, i.e., culture upon collagen with an apical interface of air, a temporal increase in caveolin-1 expression was not observed, with only very faint signals evident even at 192 h postseeding; at no time did these cultures display caveolae. In late primary ATII cultures caveolin-1 expression and caveolae biogenesis occur as a function of in vitro transformation from the ATII to the ATI-like phenotype. The results have broad implications for the in vitro study of the role of caveolae and caveolin in alveolar epithelial cell biology.

Caveolin and its cellular and subcellular immunolocalisation in lung alveolar epithelium: implications for alveolar epithelial type I cell function.

Caveolae are flask-shaped invaginations of the plasmalemma which pinch off to form discrete vesicles within the cell cytoplasm. Biochemically, caveolae may be distinguished by the presence of a protein, caveolin, that is the principal component of filaments constituting their striated cytoplasmic coat. Squamous alveolar epithelial type I (ATI) cells, comprising approximately 95% of the surface area of lung alveolar epithelium, possess numerous plasmalemmal invaginations and cytoplasmic vesicles ultrastructurally indicative of caveolae. However, an ultrastructural appearance does not universally imply the biochemical presence of caveolin. This immunocytochemical study has utilised a novel application of confocal laser scanning and electron microscopy unequivocally to localise caveolin-1 to ATI cells. Further, cytoplasmic vesicles and flask-shaped membrane invaginations in the ATI cell were morphologically identified whose membranes were decorated with anti-caveolin-1 immunogold label. Coexistent with this, however, in both ATI and capillary endothelial cells could be seen membrane invaginations morphologically characteristic of caveolae, but which lacked associated caveolin immunogold label. This could reflect a true biochemical heterogeneity in populations of morphologically similar plasmalemmal invaginations or an antigen threshold requirement for labelling. The cuboidal alveolar epithelial type II cell (ATII) also displayed specific label for caveolin-1 but with no ultrastructural evidence for the formation of caveolae. The biochemical association of caveolin with ATI cell vesicles has broad implications for the assignment and further study of ATI cell function.

Isolation of diabetes-associated kidney genes using differential display.

Differential Display was used to isolate genes that show transcriptional changes in the kidney during the development of diabetes in the GK rat. Eight candidate diabetes-associated cDNA fragments, CDK1-8, were isolated and characterised. cDNA sequencing and subsequent database analysis revealed that CDK2, 4, 5 and 6 showed no significant sequence similarity to previously reported genes, suggesting that they represent novel genes, whereas CDK 1, 3, 7 and 8 showed significant similarity with rat lactate dehydrogenase, rat amiloride sensitive sodium channel, EST109013 and mouse ubiquitin-like protein respectively. The differential mRNA expression of CDK1-8 was confirmed using differential screening of slot blots. CDK1, 2, 4 and 8 mRNAs appeared to increase whereas CDK3, 5, 6 and 7 mRNAs decreased in the kidneys of GK rats with increasing hyperglycaemia. The altered renal mRNA expression of these genes in association with increased hyperglycemia in the GK rat suggest that they are candidates for a role in the development of diabetic nephropathy.

Effects of a selective adenosine A1 receptor antagonist on the development of cyclosporin nephrotoxicity.

1. The clinical application of cyclosporin as an immunosuppressive agent is limited by its nephrotoxicity. 2. The effect of FK453, a selective A1-receptor antagonist, administered twice daily to rats at a dose of 100 mg kg-1 was assessed on the development of nephrotoxicity induced by cyclosporin (10 mg kg-1 i.p. daily) administered for 14 days. The effects of nifedipine administered twice daily (0.3 mg kg-1 s.c.) for 14 days, on cyclosporin nephrotoxicity were also studied. 3. Cyclosporin induced a 46.58% and 35.78% decline in glomerular filtration rate (GFR) and effective renal plasma flow (ERPF) respectively and a reduction of 16.69% in filtration fraction (FF). Co-administration of FK453 resulted in falls of 30.5%, 18.59% and 14.7% in GFR, ERPF and FF respectively, the former two significantly less than the falls seen with cyclosporin (CyA) alone (P < 0.05 vs CyA, ANOVA). 4. Nifedipine appeared to have a more pronounced protective effect resulting in a decline of only 20.91% in GFR, with no significant change in ERPF (increase of 0.93%) when co-administered with CyA. 5. These observations indicate adenosine plays a minor role in the pathophysiology of CyA nephrotoxicity.

Low-dose cyclosporin nephrotoxicity in the rat.

The nephrotoxicity of cyclosporin (CsA) continues to be a clinical problem that detracts from its obvious benefits as an immunosuppressive agent. Animal models designed to study the problem have generally relied either on chronic administration of high doses of the drug or acute administration of single i.v. doses. The present study establishes a model of CsA nephrotoxicity using doses of the drug comparable to those used in man administered over a time period sufficient for haemodynamic and structural changes to become evident. The technique used measures glomerular filtration rate (GFR) and effective renal plasma flow (ERPF) by the plasma clearance of chromium EDTA and iodohippuran respectively. This has the advantage of allowing sequential measurements in individual animals. Significant impairment of GFR was seen in animals treated intraperitoneally with doses of CsA as low as 5 mg/kg/day. CsA 7.5 mg/kg/day caused a significant reduction in ERPF, and at 10 mg/kg/day and greater filtration fraction also declined significantly. Detailed histological examination of the kidneys from these animals also revealed significant tubular dilatation at 10 mg/kg/day and above. This model of CsA toxicity circumvents many of the problems associated with other models. The animals can be studied longitudinally and the period of administration has relevance to clinic practice. This work provides the basis for further studies which can closely mimic the clinical situation using doses similar to those used for human maintenance immunosuppression.

The effects of nifedipine on cyclosporine nephrotoxicity in rats.

We have investigated the effect of nifedipine on cyclosporine nephrotoxicity in the Sprague-Dawley rat employing a repeatable, single-shot, isotopic technique of measuring the glomerular filtration rate (GFR) and effective renal plasma flow (ERPF). Groups of 10 rats received either cyclosporine 5 mg/kg daily or cremaphor with either nifedipine 0.5 mg/kg daily or its vehicle for 14 days. In the cyclosporine group the GFR (P < 0.001, paired t-test), ERPF and filtration fraction (FF) (P < 0.01) all fell significantly. The cyclosporine plus nifedipine group underwent an increase in the ERPF (P < 0.01), the GFR remained unchanged, and the FF fell significantly (P < 0.0001). In this model, nifedipine completely abolished the renal arteriolar vasospasm produced by cyclosporine. That the FF fell in the cyclosporine plus nifedipine-treated animals indicates that cyclosporine has an effect which is not mediated by arteriolar vasoconstriction. This action may be at the glomerular level and is resistant to calcium channel blockade.