Collagen IV of basement membranes: III. Chloride pressure is a primordial innovation that drives and maintains the assembly of scaffolds.

Collagen IV scaffold is a primordial innovation enabling the assembly of a fundamental architectural unit of epithelial tissues-a basement membrane attached to polarized cells. A family of six α-chains (α1 to α6) coassemble into three distinct protomers that form supramolecular scaffolds, noted as collagen IVα121, collagen IVα345, and collagen IVα121-α556. Chloride ions play a pivotal role in scaffold assembly, based on studies of NC1 hexamers from mammalian tissues. First, Cl- activates a molecular switch within trimeric NC1 domains that initiates protomer oligomerization, forming an NC1 hexamer between adjoining protomers. Second, Cl- stabilizes the hexamer structure. Whether this Cl--dependent mechanism is of fundamental importance in animal evolution is unknown. Here, we developed a simple in vitro method of SDS-PAGE to determine the role of solution Cl- in hexamer stability. Hexamers were characterized from 34 animal species across 15 major phyla, including the basal Cnidarian and Ctenophora phyla. We found that solution Cl- stabilized the quaternary hexamer structure across all phyla except Ctenophora, Ecdysozoa, and Rotifera. Further analysis of hexamers from peroxidasin knockout mice, a model for decreasing hexamer crosslinks, showed that solution Cl- also stabilized the hexamer surface conformation. The presence of sufficient chloride concentration in solution or "chloride pressure" dynamically maintains the native form of the hexamer. Collectively, our findings revealed that chloride pressure on the outside of cells is a primordial innovation that drives and maintains the quaternary and conformational structure of NC1 hexamers of collagen IV scaffolds.

Ambulatory intravenous insulin and islet cell transplantation to treat severe type III insulin hypersensitivity in a patient with type 1 diabetes mellitus.

Allogenic pancreatic islet cell transplantation is an appropriate treatment option to consider in the management of refractory cases of severe hypersensitivity to insulin in patients with type 1 diabetes mellitus.

Baseline characteristics of patients with atypical haemolytic uraemic syndrome (aHUS): The Australian cohort in a global aHUS registry.

AIMS: To describe the baseline characteristics and treatment of Australian patients diagnosed with atypical haemolytic uraemic syndrome (aHUS) reported to the Global aHUS Registry. METHODS: Descriptive analysis of the Australian cohort with aHUS (n = 106) was undertaken for demographics, disease characteristics and prior treatment with eculizumab; comparing with the global cohort (n = 1688) for certain pre-specified disease characteristics. RESULTS: In Australia, almost two-thirds of patients diagnosed with aHUS were female and over 80% of patients were Caucasians, with similar proportions reported in the global cohort. Less than 6% of patients in the Australia and global cohorts were reported to have a history of autoimmune disease (4% vs 2%, respectively; P = .21) or cancer (5% vs 5%, respectively; P = .93), conditions that have been associated with secondary HUS. In the Australian cohort, 26% had received a kidney transplant and 68% of patients had received eculizumab. Kidneys were the most common organ involvement, followed by gastrointestinal tract (26%) and cardiovascular system (19%), with 35% of patients reported to have had at least two organs involved within 6 months prior to baseline visit or entry into the registry. Complement factor H was the most common pathogenic complement gene variant in the Australian patients. CONCLUSION: Data from the aHUS registry confirms and defines region-specific disease characteristics among a selected group of Australian children and adults with aHUS reported to the registry. Ongoing and more inclusive data will provide further information about temporal trends and treatment outcomes, representing a unique opportunity for clinicians and researchers to further develop knowledge surrounding this rare disease.

Rapamycin and abundant TCR stimulation are required for the generation of stable human induced regulatory T cells.

OBJECTIVES: Regulatory T cells (Tregs) are a vital sub-population of CD4+ T cells with major roles in immune tolerance and homeostasis. Given such properties, the use of regulatory T cells for immunotherapies has been extensively investigated, with a focus on adoptive transfer of ex vivo expanded natural Tregs (nTregs). For immunotherapies, induced Tregs (iTregs), generated in vitro from naïve CD4+ T cells, provide an attractive alternative, given the ease of generating cell numbers required for clinical dosage. While the combination of TGF-ß, ATRA and rapamycin has been shown to generate highly suppressive iTregs, the challenge for therapeutic iTreg generation has been their instability. Here, we investigate the impact of rapamycin concentrations and α-CD3/CD28 bead ratios on human iTreg stability. METHODS: We assess iTregs generated with various concentrations of rapamycin and differing ratios of α-CD3/CD28 beads for their differentiation, stability, expression of Treg signature molecules and T helper effector cytokines, and Treg-specific demethylation region (TSDR) status. RESULTS: iTregs generated in the presence of TGF-ß, ATRA, rapamycin and a higher ratio of α-CD3/CD28 beads were highly suppressive and stable upon in vitro re-stimulation. These iTregs exhibited a similar expression profile of Treg signature molecules and T helper effector cytokines to nTregs, in the absence of TSDR demethylation. CONCLUSION: This work establishes a method to generate human iTregs which maintain stable phenotype and function upon in vitro re-stimulation. Further validation in pre-clinical models will be needed to ensure its suitability for applications in adoptive transfer.

IL-17A-induced mesenchymal stem cells have promising therapeutic value for clinical translation.

Interferon (IFN) gamma is the prototypic proinflammatory cytokine used to preactivate the immunomodulatory properties of mesenchymal stem cells (MSC). IFN-gamma, however, converts MSC into a cell therapy that can be immunogenic, detrimental, and hence nonfeasible for clinical application. The article by Bai et al. is an in vivo proof-of-concept study that interleukin-17A (IL-17A) enhances the immunosuppressive, tolerance-promoting, and renoprotective properties of MSC. IL-17A is an alternative cytokine to preactivate MSC. IL-17A enhances the therapeutic efficacy of MSC for renal diseases.

Immune profiling and cancer post transplantation.

Half of all long-term (> 10 year) australian kidney transplant recipients (KTR) will develop squamous cell carcinoma (SCC) or solid organ cancer (SOC), making cancer the leading cause of death with a functioning graft. At least 30% of KTR with a history of SCC or SOC will develop a subsequent SCC or SOC lesion. Pharmacological immunosuppression is a major contributor of the increased risk of cancer for KTR, with the cancer lesions themselves further adding to systemic immunosuppression and could explain, in part, these phenomena. Immune profiling includes; measuring immunosuppressive drug levels and pharmacokinetics, enumerating leucocytes and leucocyte subsets as well as testing leucocyte function in either an antigen specific or non-specific manner. Outputs can vary from assay to assay according to methods used. In this review we define the rationale behind post-transplant immune monitoring assays and focus on assays that associate and/or have the ability to predict cancer and rejection in the KTR. We find that immune monitoring can identify those KTR of developing multiple SCC lesions and provide evidence they may benefit from pharmacological immunosuppressive drug dose reductions. In these KTR risk of rejection needs to be assessed to determine if reduction of immunosuppression will not harm the graft.

Spontaneous glomerular mesangial lesions in common marmoset monkeys (Callithrix jacchus): a benign non-progressive glomerulopathy.

BACKGROUND: Common marmosets are known to develop an IgM glomerulopathy, which has been linked with 'wasting marmoset' syndrome. This study investigated renal pathology in a colony of marmosets, with and without weight loss. METHODS: Renal histology, immunofluorescence, and electron microscopy were performed on marmosets euthanized for research or for weight loss. Serum and urine biochemistry were measured during life and at euthanasia. RESULTS: Histology from 25 adult marmosets (19 research and 6 weight loss) showed mesangial expansion in the majority of glomeruli. Mesangial changes correlated with electron-dense deposits and IgM deposition by immunofluorescence; negligible other pathology was seen. Glomerular basement membrane thickness appeared increased compared to reported human measurements. Low-grade proteinuria was present in all animals, but did not progress. Renal function was normal in all animals. CONCLUSIONS: Marmosets develop a glomerulopathy characterized by mesangial expansion, IgM deposition, and proteinuria. This is a benign occurrence and not specifically associated with weight loss.

Propagation and characterisation of dendritic cells from G-CSF mobilised peripheral blood monocytes and stem cells in common marmoset monkeys.

The common marmoset is a small New World Primate that has been used as an immunological model for a number of human diseases. Dendritic cells (DC) have not been extensively characterised in this species and in particular protocols to derive DC from living donors without the need for animal sacrifice are presently lacking. This study establishes new protocols to generate substantial numbers of marmoset DC for use in cell therapy studies. Recombinant human G-CSF was used to mobilise peripheral blood monocytes and CD34(+) stem cells in sufficient numbers for large scale in-vitro DC propagation using cytokine conditioning including IL-4, GM-CSF, FLT3-L, stem cell factor and thrombopoietin. Marmoset DC exhibited morphology similar to human DC, were capable of antigen uptake and presentation and had moderate allo-stimulatory ability. Monocyte-derived DC had a maturation-resistant immature phenotype, whereas haematopoietic precursor-derived DC were semi-mature in phenotype and function. This study confirms the feasibility of the marmoset as a unique small primate model in which to pursue DC-based immunotherapy strategies.

Hyperparathyroidism and vitamin D deficiency predispose to bone loss in renal transplant recipients.

BACKGROUND: Bone disease is common postrenal transplantation resulting in increased fracture rates and morbidity. The cause is multifactorial including hyperparathyroidism, corticosteroids, and possibly calcium and vitamin D deficiencies. The aim of this study was to identify modifiable factors contributing to bone disease in long-term renal transplant (RT) recipients. METHODS: Ninety-seven RT recipients were prospectively recruited over a 6-month period from a single center. Bone-related parameters were collected including bone mineral density at lumbar spine and total hip sites, serum and urinary markers of bone-turnover and calcium metabolism, and intact parathyroid hormone levels. RESULTS: The mean time posttransplant of RT recipients was 9.5 years and mean estimated glomerular filtration rate was 70.3 mL/min. Up to 50% of recipients had biochemical evidence of calcium and vitamin D deficiencies. In the multiple regression models, elevated intact parathyroid hormone levels and calcium deficiency, which are affected by estimated glomerular filtration rate and vitamin D levels, are significantly associated with reduction in bone mineral density measurements. CONCLUSIONS: Hyperparathyroidism and vitamin D deficiency are common and are likely to contribute to bone loss postrenal transplantation. Measures aim to correct these problems pre- and posttransplant may improve bone health in RT recipients.

Calcific uraemic arteriolopathy: an update.

PURPOSE OF REVIEW: Calcific uraemic arteriolopathy (CUA) or calciphylaxis is a rare but important cause of morbidity and mortality in patients with chronic kidney disease. The prevalence of CUA is increasing in patients with renal failure, and the condition is also being recognized in nonuraemic patients. RECENT FINDINGS: There has been increasing understanding of the molecular basis of vascular calcification, in particular on the important role of the uraemic microenvironment in the factors implicated in the differentiation of vascular smooth muscle cells into osteoblasts. New options for treatment of hyperphosphataemia and secondary hyperparathyroidism in patients with chronic kidney disease have become available in the last few years and these have begun to be used in patients with CUA. These include bisphosphonates, newer noncalcium/nonaluminium-containing phosphate binders and case reports of use of cinacalcet. Other treatments for CUA that are not targeted directly at calcium/phosphate homeostasis include hyperbaric oxygen and the antioxidant cation chelator sodium thiosulphate. SUMMARY: Clinicians managing patients with CUA should consider a combination approach of treating deranged calcium/phosphate with newer therapeutic agents and promoting wound healing with other older modalities such as hyperbaric oxygen and sodium thiosulphate infusions. Randomized controlled trials for treatments in CUA are still lacking.

Human plasmacytoid dendritic cells regulate immune responses to Epstein-Barr virus (EBV) infection and delay EBV-related mortality in humanized NOD-SCID mice.

Epstein-Barr virus (EBV) is associated with posttransplant lymphoproliferative disease (PTLD), which is a leading cause of cancer death in recipients of transplants. We investigated the role of plasmacytoid dendritic cells (PDCs) in the development of EBV infection and the onset of lymphoproliferative disease (LPD) in humanized NOD-SCID mice and studied the effect of EBV on PDC function. NOD-SCID mice reconstituted with PDC-depleted peripheral blood mononuclear cells (PBMCs) from EBV IgG+ human donors had significantly enhanced mortality from disseminated EBV infection (median survival, 43 days) compared to PBMC-only mice (median survival, 72 days; log-rank P<.05). Mice reconstituted with PDC-enriched PBMCs challenged with EBV exhibited delayed mortality from EBV-LPD (median survival, 80 days) compared to PBMC-only mice challenged with EBV (median survival, 50 days; log-rank P<.05). EBV-stimulated pDCs produced interferon alpha (IFN-alpha) and promoted the activation of natural killer cells and IFN-gamma-producing CD3+T cells. PDC activation of CD3+T cells in response to EBV stimulation was dependent on cell-to-cell contact, in part mediated by toll-like receptor 9 (TLR-9) signaling that was inhibited by chloroquine and TLR-9 inhibitory CpG. Thus, PDCs play an important role in anti-EBV cellular immune responses that may be targets for manipulation in novel strategies for the treatment of PTLD.

MHC Class II DRB genotyping is highly predictive of in-vitro alloreactivity in the common marmoset.

The common marmoset (Callithrix jacchus) is emerging as a promising alternative pre-clinical model for transplantation and immunological research. It is therefore important to establish a rapid and reliable method of confirming alloreactivity between donor-recipient pairs. In this study of a large marmoset colony (n=49), we firstly characterised MHC Class II genes (Caja-DRB*W1201, Caja-DRB1*03, Caja-DRB*W16) using, for the first time in this species, sequence-based allelic typing techniques. Exon 2 was amplified using M13-tailed PCR primers specific for known marmoset alleles, and sequenced using universal M13 sequencing primers and dye terminator cycle sequencing. Twenty-six genotypes involving monomorphic Caja-DRB*W1201, 8 Caja-DRB*W16 and 5 Caja-DRB1*03 alleles were observed. Two new DRB*W16 alleles were identified. Subsequently we investigated whether matching at MHC-DRB loci alone could accurately predict in-vitro alloreactivity as assessed by mixed lymphocyte reactions. Peripheral blood mononuclear cells (PBMC) isolated from fully and partially DRB-matched and fully mismatched animal pairs were mixed and co-cultured for T-cell proliferation. PBMC co-cultured from fully or partially mismatched pairs exhibited significant T cell proliferation above single cell controls (p<0.01). Mixed PBMC from fully DRB-matched pairs exhibited no proliferation over controls (p=0.3). Thus using Caja-DRB genotyping, suitably alloreactive donor-recipient pairs can be rapidly and accurately identified for use in further studies of cellular and solid organ transplantation.