HLA-DR15 Molecules Jointly Shape an Autoreactive T Cell Repertoire in Multiple Sclerosis.

The HLA-DR15 haplotype is the strongest genetic risk factor for multiple sclerosis (MS), but our understanding of how it contributes to MS is limited. Because autoreactive CD4+ T cells and B cells as antigen-presenting cells are involved in MS pathogenesis, we characterized the immunopeptidomes of the two HLA-DR15 allomorphs DR2a and DR2b of human primary B cells and monocytes, thymus, and MS brain tissue. Self-peptides from HLA-DR molecules, particularly from DR2a and DR2b themselves, are abundant on B cells and thymic antigen-presenting cells. Furthermore, we identified autoreactive CD4+ T cell clones that can cross-react with HLA-DR-derived self-peptides (HLA-DR-SPs), peptides from MS-associated foreign agents (Epstein-Barr virus and Akkermansia muciniphila), and autoantigens presented by DR2a and DR2b. Thus, both HLA-DR15 allomorphs jointly shape an autoreactive T cell repertoire by serving as antigen-presenting structures and epitope sources and by presenting the same foreign peptides and autoantigens to autoreactive CD4+ T cells in MS.

Long-term Clinical Relevance of De Novo Donor-Specific Antibodies After Pediatric Liver Transplantation.

BACKGROUND: Anti-HLA antibodies and especially donor-specific antibodies (DSA) play a significant role in graft survival after solid organ transplantation. Their impact on long-term survival in adult liver transplantation (LT) is controversial, but they may be a risk factor. The effects of DSA after pediatric LT are still unclear. METHODS: We performed a retrospective evaluation of DSA in sera from 43 children who had received transplants at our tertiary center. Twenty-four patients had good long-term clinical and laboratory graft function (group 1), whereas 19 LT recipients suffered from histologically confirmed and clinically relevant chronic allograft rejection (group 2); 16 of these have already undergone retransplantation due to graft dysfunction. Inclusion criteria were availability of sera before the first LT to identify preformed antibodies in case of DSA positivity after LT and long-term follow-up at our institution. Sera were analyzed for anti-HLA antibodies using Luminex single antigen beads, where a mean fluorescence intensity value of more than 1500 was considered positive. RESULTS: The prevalence of DSA was 33% for group 1 and 68% for group 2. Antibodies were predominantly HLA class II. Values of mean fluorescence intensity were comparable in both groups. Only one of the DSA+ ve patients from group 1 exhibited preformed antibodies. In conclusion, pediatric patients with chronic rejection revealed a higher rate of de novo DSA, especially of HLA-class II DSA. Further studies are necessary to confirm these data with a larger pediatric cohort.

5-azacytidine promotes an inhibitory T-cell phenotype and impairs immune mediated antileukemic activity.

Demethylating agent, 5-Azacytidine (5-Aza), has been shown to be active in treatment of myeloid malignancies. 5-Aza enhances anticancer immunity, by increasing expression of tumor-associated antigens. However, the impact of 5-Aza immune responses remains poorly understood. Here, T-cell mediated tumor immunity effects of 5-Aza, are investigated in vitro and in vivo. T-cells from healthy donors were treated with 5-Aza and analyzed by qRT-PCR and flow cytometry for changes in gene expression and phenotype. Functionality was assessed by a tumor lysis assay. Peripheral blood from patients treated with 5-Aza after alloSCT was monitored for changes in T-cell subpopulations. 5-Aza treatment resulted in a decrease in CD8+ T-cells, whereas CD4+ T-cells increased. Furthermore, numbers of IFN-γ + T-helper 1 cells (Th1) were reduced, while Treg-cells showed substantial increase. Additionally, CD8+ T-cells exhibited limited killing capacity against leukemic target cells. In vivo data confirm the increase of Treg compartment, while CD8+ T-effector cell numbers were reduced. 5-Aza treatment results in a shift from cytotoxic to regulatory T-cells with a functional phenotype and a major reduction in proinflammatory Th1-cells, indicating a strong inhibition of tumor-specific T-cell immunity by 5-Aza.

Reasons for non-uptake and subsequent participation in the NHS Bowel Cancer Screening Programme: a qualitative study.

BACKGROUND: Screening for bowel cancer using the guaiac faecal occult blood test offered by the NHS Bowel Cancer Screening Programme (BCSP) is taken up by 54% of the eligible population. Uptake ranges from 35% in the most to 61% in the least deprived areas. This study explores reasons for non-uptake of bowel cancer screening, and examines reasons for subsequent uptake among participants who had initially not taken part in screening. METHODS: Focus groups with a socio-economically diverse sample of participants were used to explore participants' experience of invitation to and non-uptake of bowel cancer screening. RESULTS: Participants described sampling faeces and storing faecal samples as broaching a cultural taboo, and causing shame. Completion of the test kit within the home rather than a formal health setting was considered unsettling and reduced perceived importance. Not knowing screening results was reported to be preferable to the implications of a positive screening result. Feeling well was associated with low perceived relevance of screening. Talking about bowel cancer screening with family and peers emerged as the key to subsequent participation in screening. CONCLUSIONS: Initiatives to normalise discussion about bowel cancer screening, to link the BCSP to general practice, and to simplify the test itself may lead to increased uptake across all social groups.

Intracellular localization and induction of a dynamic RNA-editing event of macro-algal V-ATPase subunit A (VHA-A) in response to copper.

A V-ATPase subunit A protein (VHA-A) transcript together with a variant (C793 to U), which introduces a stop codon truncating the subunit immediately downstream of its ATP binding site, was identified within a Fucus vesiculosus cDNA from a heavy metal contaminated site. This is intriguing because the VHA-A subunit is the crucial catalytic subunit responsible for the hydrolysis of ATP that drives ion transport underlying heavy metal detoxification pathways. We employed a chemiluminescent hybridization protection assay to quantify the proportion of both variants directly from mRNA while performing quantification of total transcript using Q-PCR. Polyclonal antisera raised against recombinant VHA-A facilitated simultaneous detection of parent and truncated VHA-A and revealed its cellular and subcellular localization. By exploiting laboratory exposures and samples from an environmental copper gradient, we showed that total VHA-A transcript and protein, together with levels of the truncated variant, were induced by copper. The absence of a genomic sequence representing the truncated variant suggests a RNA editing event causing the production of the truncated VHA-A. Based on these observations, we propose RNA editing as a novel molecular process underpinning VHA trafficking and intracellular sequestration of heavy metals under stress.

Evaluation of DNA extraction methods for Bacillus anthracis spores isolated from spiked food samples.

AIMS: Nine commercial DNA extraction kits were evaluated for the isolation of DNA from 10-fold serial dilutions of Bacillus anthracis spores using quantitative real-time PCR (qPCR). The three kits determined by qPCR to yield the most sensitive and consistent detection (Epicenter MasterPure Gram Positive; MoBio PowerFood; ABI PrepSeq) were subsequently tested for their ability to isolate DNA from trace amounts of B. anthracis spores (approx. 6·5 × 10(1) and 1·3 × 10(2)  CFU in 25 ml or 50 g of food sample) spiked into complex food samples including apple juice, ham, whole milk and bagged salad and recovered with immunomagnetic separation (IMS). METHODS AND RESULTS: The MasterPure kit effectively and consistently isolated DNA from low amounts of B. anthracis spores captured from food samples. Detection was achieved from apple juice, ham, whole milk and bagged salad from as few as 65 ± 14, 68 ± 8, 66 ± 4 and 52 ± 16 CFU, respectively, and IMS samples were demonstrated to be free of PCR inhibitors. CONCLUSIONS: Detection of B. anthracis spores isolated from food by IMS differs substantially between commercial DNA extraction kits; however, sensitive results can be obtained with the MasterPure Gram Positive kit. SIGNIFICANCE AND IMPACT OF THE STUDY: The extraction protocol identified herein combined with IMS is novel for B. anthracis and allows detection of low levels of B. anthracis spores from contaminated food samples.

T cell epitope mapping of JC polyoma virus-encoded proteome reveals reduced T cell responses in HLA-DRB1*04:01+ donors.

JC polyomavirus (JCV) infection is highly prevalent and usually kept in a persistent state without clinical signs and symptoms. It is only during immunocompromise and especially impaired CD4(+) T cell function in the brain, as seen in AIDS patients or natalizumab-treated multiple sclerosis patients, that JCV may cause progressive multifocal leukoencephalopathy (PML), an often life-threatening brain disease. Since CD4(+) T cells likely play an important role in controlling JCV infection, we here describe the T cell response to JCV in a group of predominantly HLA-DR-heterozygotic healthy donors (HD) by using a series of overlapping 15-mer peptides spanning all JCV-encoded open reading frames. We identified immunodominant epitopes and compared T cell responses with anti-JCV VP1 antibody production and with the presence of urinary viral shedding. We observed positive JCV-specific T cell responses in 28.6% to 77.6%, humoral immune response in 42.6% to 89.4%, and urinary viral shedding in 36.4% to 45.5% of HD depending on the threshold. Four immunodominant peptides were mapped, and at least one immunogenic peptide per HLA-DRB1 allele was detected in DRB1*01(+), DRB1*07(+), DRB1*11(+), DRB1*13(+), DRB1*15(+), and DRB1*03(+) individuals. We show for the first time that JCV-specific T cell responses may be directed not only against JCV VP1 and large T antigen but also against all other JCV-encoded proteins. Heterozygotic DRB1*04:01(+) individuals showed very low T cell responses to JCV together with normal anti-VP1 antibody levels and no urinary viral shedding, indicating a dominant-negative effect of this allele on global JCV-directed T cell responses. Our data are potentially relevant for the development of vaccines against JCV.

TCR bias and HLA cross-restriction are strategies of human brain-infiltrating JC virus-specific CD4+ T cells during viral infection.

Virus-specific CD4(+) T cells play a central role in control of viral pathogens including JC polyoma virus (JCV) infection. JCV is a ubiquitous small DNA virus that leads to persistent infection of humans with no clinical consequences. However, under circumstances of immunocompromise, it is able to cause an opportunistic and often fatal infection of the brain called progressive multifocal leukoencephalopathy (PML). PML has emerged as a serious adverse event in multiple sclerosis patients treated with the anti-VLA-4 mAb natalizumab, which selectively inhibits cell migration across the blood-brain barrier and the gut's vascular endothelium thus compromising immune surveillance in the CNS and gut. In a multiple sclerosis patient who developed PML under natalizumab treatment and a vigorous immune response against JCV after Ab washout, we had the unique opportunity to characterize in detail JCV-specific CD4(+) T cell clones from the infected tissue during acute viral infection. The in-depth analysis of 14 brain-infiltrating, JCV-specific CD4(+) T cell clones demonstrated that these cells use an unexpectedly broad spectrum of different strategies to mount an efficient JCV-specific immune response including TCR bias, HLA cross-restriction that increases avidity and influences in vivo expansion, and a combination of Th1 and Th1-2 functional phenotypes. The level of combinatorial diversity in TCR- and HLA-peptide interactions used by brain-infiltrating, JCV-specific CD4(+) T cells has not, to our knowledge, been reported before in humans for other viral infections and confirms the exceptional plasticity that characterizes virus-specific immune responses.

Impact of the NK cell receptor LIR-1 (ILT-2/CD85j/LILRB1) on cytotoxicity against multiple myeloma.

The role of different receptors in natural-killer- (NK-) cell-mediated cytotoxicity against multiple myeloma (MM) cells is unknown. We investigated if an enhancement of NK-cell-mediated cytotoxicity against MM could be reached by blocking of the inhibitory leukocyte immunoglobulin-like receptor 1 (LIR-1). Our investigations revealed high levels of LIR-1 expression not only on the NK cell line NK-92, but also on myeloma cells (MOLP-8, RPMI8226) as well as on a lymphoblastoid cell line (LBCL; IM-9). Subsequent cytotoxicity assays were designed to show the isolated effects of LIR-1 blocking on either the effector or the tumor side to rule out receptor-receptor interactions. Although NK-92 was shown to be capable of myeloma cell lysis, inhibition of LIR-1 on NK-92 did not enhance cytotoxicity. Targeting the receptor on MM and LBCL did not also alter NK-92-mediated lysis. We come to the conclusion that LIR-1 alone does not directly influence NK-cell-mediated cytotoxicity against myeloma. To our knowledge, this work provides the first investigation of the inhibitory capability of LIR-1 in NK-92-mediated cytotoxicity against MM and the first functional evaluation of LIR-1 on MM and LBCL.

Angiotensin-Converting Enzyme 2 (ACE2) Is a Key Modulator of the Renin Angiotensin System in Health and Disease.

Angiotensin-converting enzyme 2 (ACE2) shares some homology with angiotensin-converting enzyme (ACE) but is not inhibited by ACE inhibitors. The main role of ACE2 is the degradation of Ang II resulting in the formation of angiotensin 1-7 (Ang 1-7) which opposes the actions of Ang II. Increased Ang II levels are thought to upregulate ACE2 activity, and in ACE2 deficient mice Ang II levels are approximately double that of wild-type mice, whilst Ang 1-7 levels are almost undetectable. Thus, ACE2 plays a crucial role in the RAS because it opposes the actions of Ang II. Consequently, it has a beneficial role in many diseases such as hypertension, diabetes, and cardiovascular disease where its expression is decreased. Not surprisingly, current therapeutic strategies for ACE2 involve augmenting its expression using ACE2 adenoviruses, recombinant ACE2 or compounds in these diseases thereby affording some organ protection.

Hyperfiltration in type 1 diabetes: does it exist and does it matter for nephropathy?

AIMS/HYPOTHESIS: Hyperfiltration is widely regarded as a contributing factor to the development of microalbuminuria and progressive nephropathy in type 1 diabetes. However, recent studies have questioned this conclusion. METHODS: To address this conflicting evidence, we examined the association between hyperfiltration and progression to microalbuminuria in 2,318 adults with type 1 diabetes. We also compared the estimated GFR in our diabetic patients with rates observed in 6,247 adults from the Finnish general population, using age- and sex-specific z scores. RESULTS: The distribution of estimated GFR in adults with type 1 diabetes and normoalbuminuria was not significantly different from that expected in the general population (p = 0.51, Mann-Whitney test). Type 1 diabetic patients with a higher estimated GFR were also no more likely to develop microalbuminuria over a median of 5.2 years of follow-up than those with normal estimated GFR. This was the case regardless of whether hyperfiltration was defined by an absolute threshold, deciles of estimated GFR or a z score, using creatinine- or cystatin-based clearance formulas in men or in women. CONCLUSIONS/INTERPRETATION: Together with other studies, these data suggest that creatinine- or cystatin-based estimates of GFR do not predict the development of microalbuminuria in patients with type 1 diabetes. Moreover, in the absence of incipient or overt nephropathy, conventionally determined renal function in patients with type 1 diabetes appears no different from that in the general population. This is hardly surprising, given that these individuals, by all definitions, do not have kidney disease.

The potential for a two-stage diabetes risk algorithm combining non-laboratory-based scores with subsequent routine non-fasting blood tests: results from prospective studies in older men and women.

AIM: To develop strategies based on simple clinical assessment and blood markers to identify older individuals at high risk for Type 2 diabetes. METHODS: A prospective study of non-diabetic men (n = 3523) and women (n = 3404) aged 60-79 years followed for 7 years, during which there were 297 incident cases of Type 2 diabetes. Logistic regression was used to develop scores to predict incident cases, starting with clinical predictors and adding blood markers that predicted the incidence of diabetes. Receiving operating characteristic analyses were used to assess improvement in prediction. RESULTS: The area under the curve for a simple clinical assessment score, which included age, sex, family history of diabetes, smoking status, BMI, waist circumference, hypertension and recall of doctor diagnosis of coronary heart disease was 0.765 (0.740, 0.791); sensitivity and specificity in the top quintile of the score were 50.3 and 81.4%, respectively. Addition of simple fasting blood markers HDL cholesterol, triglyceride and glucose improved prediction [area under the curve = 0.817 (0.793, 0.840), P < 0.0001; sensitivity 63.8%; specificity 82.0%]. An alternative model adding blood markers not dependent on fasting yielded similar results. Further addition of C-reactive protein made no improvement. Blood measurements made small differences to reclassification of risk in those in the lowest three quintiles of the non-laboratory score. CONCLUSION: In large population settings, simple clinical assessments could be used in the first instance to identify older adults who would benefit from further testing with routine (non-fasting) blood markers to identify those at most likely to be at elevated diabetes risk.

Receptor for AGEs (RAGE) blockade may exert its renoprotective effects in patients with diabetic nephropathy via induction of the angiotensin II type 2 (AT2) receptor.

AIMS/HYPOTHESIS: The receptor for AGEs (RAGE) contributes to the development and progression of diabetic nephropathy. In this study, we examined whether the protective effects of RAGE blockade are exerted via modulation of the renal angiotensin II type 2 (AT2) receptor. METHODS: Control and streptozotocin diabetic mice, wild-type or deficient in the AT2 receptor (At2 knockout [KO]) or RAGE (Rage KO), were studied for 24 weeks. Adenoviral overexpression of full-length Rage in primary rat mesangial cells was also used to determine the effects on AT2 production. RESULTS: With diabetes, Rage-deficient mice had less albuminuria, and an attenuation of hyperfiltration and glomerulosclerosis as compared with diabetic wild-type and At2 KO mice. Renal gene and protein expression of RAGE was elevated with diabetes. Diabetic Rage KO mice had a greater increase in renal AT2 receptor protein than was seen in diabetic wild-type mice. Diabetes-induced increases in renal cytosolic and mitochondrial superoxide generation were prevented in diabetic Rage KO mice, but enhanced in all At2 KO mice. Adenoviral overexpression of RAGE or AGE treatment decreased cell surface AT2 expression, in association with increasing superoxide generation; both were reversed using antioxidants N-acetylcysteine and apocynin, and soluble RAGE in primary mesangial cells. CONCLUSIONS/INTERPRETATION: RAGE appears to be a common and key modulator of AT2 receptor expression, a finding that would implicate a newly defined RAGE-AT2 axis in the development and progression of diabetic nephropathy.

Angiotensin II mediates epithelial-to-mesenchymal transformation in tubular cells by ANG 1-7/MAS-1-dependent pathways.

Epithelial-to-mesenchymal transformation (EMT) of tubular cells into a myofibroblastic phenotype is an important mediator of renal scarring in chronic nephropathy. This study examines the role of the renin-angiotensin system (RAS) in this process. NRK-52E cells were exposed to angiotensin (ANG) II and ANG 1-7 in the presence or absence of inhibitors and agonists of RAS signaling. EMT was assessed at 3 days by expression of alpha-smooth muscle actin (alpha-SMA) and E-cadherin and the induction of a myofibroblastic phenotype. Expression of fibrogenic growth factors and matrix proteins was assessed by RT-PCR and immunofluorescence microscopy. To confirm findings in vivo, rats were also infused with ANG 1-7 (24 microg*kg(-1)*h(-1)) or saline via an osmotic minipump for 10 days, and renal fibrogenesis was then assessed. Treatment of NRK-52E cells with ANG II induced characteristic changes of EMT. Selective blockade of the AT(1) receptor or the AT(2) receptor failed to inhibit ANG II-induced EMT. However, blockade of the ANG 1-7 receptor, Mas-1, was able to prevent ANG II-dependent EMT. To confirm these findings, both ANG 1-7 and the selective Mas receptor agonist, AVE-0991, were able to induce NRK-52E cells in a dose-dependent manner. Exposing cells to recombinant ACE2 was also able to induce EMT. In addition, an infusion of ANG 1-7 induced the tubular expression of alpha-SMA and the expression of matrix proteins in the kidney. ANG II is a potent stimulus for EMT, but not through conventional pathways. This study points to the possible limitations of conventional RAS blockade, which not only fails to antagonize this pathway, but also may enhance it via augmenting the synthesis of ANG 1-7.

Antibody blockade of c-fms suppresses the progression of inflammation and injury in early diabetic nephropathy in obese db/db mice.

AIMS/HYPOTHESIS: Macrophage-mediated renal injury plays an important role in the development of diabetic nephropathy. Colony-stimulating factor (CSF)-1 is a cytokine that is produced in diabetic kidneys and promotes macrophage accumulation, activation and survival. CSF-1 acts exclusively through the c-fms receptor, which is only expressed on cells of the monocyte-macrophage lineage. Therefore, we used c-fms blockade as a strategy to selectively target macrophage-mediated injury during the progression of diabetic nephropathy. METHODS: Obese, type 2 diabetic db/db BL/KS mice with established albuminuria were treated with a neutralising anti-c-fms monoclonal antibody (AFS98) or isotype matched control IgG from 12 to 18 weeks of age and examined for renal injury. RESULTS: Treatment with AFS98 did not affect obesity, hyperglycaemia, circulating monocyte levels or established albuminuria in db/db mice. However, AFS98 did prevent glomerular hyperfiltration and suppressed variables of inflammation in the diabetic kidney, including kidney macrophages (accumulation, activation and proliferation), chemokine CC motif ligand 2 levels (mRNA and urine protein), kidney activation of proinflammatory pathways (c-Jun amino-terminal kinase and activating transcription factor 2) and Tnf-alpha (also known as Tnf) mRNA levels. In addition, AFS98 decreased the tissue damage caused by macrophages including tubular injury (apoptosis and hypertrophy), interstitial damage (cell proliferation and myofibroblast accrual) and renal fibrosis (Tgf-beta1 [also known as Tgfb1] and Col4a1 mRNA). CONCLUSIONS/INTERPRETATION: Blockade of c-fms can suppress the progression of established diabetic nephropathy in db/db mice by targeting macrophage-mediated injury.

Potential reno-protective effects of a gluten-free diet in type 1 diabetes.

AIMS/HYPOTHESIS: Coeliac disease is common in type 1 diabetes. It is managed with a gluten-free diet, characterised by foods low in AGEs. We hypothesised that this diet would lead to lower plasma AGEs and be associated with reduced albuminuria. METHODS: From a single paediatric clinic, we recruited 21 children with type 1 diabetes and biopsy-proven coeliac disease, and 38 individuals with diabetes alone. The groups were matched for age, sex, duration of disease and metabolic control. Participants completed a detailed clinical and dietary history. Blood samples were taken for HbA(1c), coeliac serology, thyroid function, serum IgA levels and plasma AGEs, and urine samples were obtained for estimation of the albumin/creatinine ratio (ACR). RESULTS: All the individuals with coeliac disease were asymptomatic, with negative transglutaminase antibodies. There were no significant differences between the groups in terms of age (14 years), sex (29% male), duration of diabetes (7 years), mean HbA(1c) (8.3%), lipid levels or treatment regimens. However, children with diabetes and coeliac disease had twofold lower levels of urinary ACR than with those diabetes alone (p = 0.04). This was associated with lower levels of circulating AGEs (p = 0.03). These associations were independent of metabolic control, diabetes management and other potentially confounding variables, such as household exposure to cigarette smoke. CONCLUSIONS/INTERPRETATION: Adherence to a gluten-free diet may provide additional benefits for individuals with coeliac disease, and potentially those with type 1 diabetes.

Role of MKK3-p38 MAPK signalling in the development of type 2 diabetes and renal injury in obese db/db mice.

AIMS/HYPOTHESIS: Obesity and diabetes are associated with increased intracellular p38 mitogen-activated protein kinase (MAPK) signalling, which may promote tissue inflammation and injury. Activation of p38 MAPK can be induced by either of the immediate upstream kinases, MAP kinase kinase (MKK)3 or MKK6, and recent evidence suggests that MKK3 has non-redundant roles in the pathology attributed to p38 MAPK activation. Therefore, this study examined whether MKK3 signalling influences the development of obesity, type 2 diabetes and diabetic nephropathy. METHODS: Wild-type and Mkk3 (also known as Map2k3) gene-deficient db/db mice were assessed for the development of obesity, type 2 diabetes and renal injury from 8 to 32 weeks of age. RESULTS: Mkk3 (+/+) db/db and Mkk3 (-/-) db/db mice developed comparable obesity and were similar in terms of incidence and severity of type 2 diabetes. At 32 weeks, diabetic Mkk3 (+/+) db/db mice had increased kidney levels of phospho-p38 and MKK3 protein. In comparison, kidney levels of phospho-p38 in diabetic Mkk3 ( -/- ) db/db mice remained normal, despite a fourfold compensatory increase in MKK6 protein levels. The reduced levels of p38 MAPK signalling in the diabetic kidneys of Mkk3 ( -/- ) db/db mice was associated with protection against the following: declining renal function, increasing albuminuria, renal hypertrophy, podocyte loss, mesangial cell activation and glomerular fibrosis. Diabetic Mkk3 ( -/- ) db/db mice were also significantly protected from tubular injury and interstitial fibrosis, which was associated with reduced Ccl2 mRNA expression and interstitial macrophage accumulation. CONCLUSIONS/INTERPRETATION: MKK3-p38 MAPK signalling is not required for the development of obesity or type 2 diabetes, but plays a distinct pathogenic role in the progression of diabetic nephropathy in db/db mice.

The role of tubular epithelial-mesenchymal transition in progressive kidney disease.

The accumulation of interstitial matrix represents the final common pathway of most forms of kidney disease. Much of this matrix is synthesized by interstitial myofibroblasts, recruited from resident fibroblasts and circulating precursors. In addition, a significant proportion is derived from epithelial-mesenchymal transition (EMT) of tubuloepithelial cells. The importance of EMT has been demonstrated in experimental models, where blockade of EMT attenuates renal fibrosis. Although a number of factors may initiate EMT in the kidney, the most potent is transforming growth factor-beta1 (TGF-beta1). Moreover, many other prosclerotic factors have effects on EMT indirectly, via induction of TGF-beta1. Signaling events in this pathway include activation of Smad/integrin-linked kinase (ILK) and connective tissue growth factor (CTGF). Basement membrane integrity is also a key regulator of EMT. In particular, overexpression of matrix metalloproteinase-2 has a key role in the initiation of EMT, membrane dissolution, and the interstitial transit of transformed mesenchymal cells. Endogenous inhibitors of EMT also play an important counterregulatory role both to prevent EMT and stimulate uncommitted cells to regain their tubular phenotype (mesenchymal-epithelial transition). Such inhibitors represent a potential therapeutic approach, offering a mechanism to slow or even redress established renal fibrosis.

The accuracy of cystatin C and commonly used creatinine-based methods for detecting moderate and mild chronic kidney disease in diabetes.

BACKGROUND: The accuracy of measuring serum cystatin C levels for detecting various stages of chronic kidney disease (CKD) in diabetes is still unclear. METHODS: In a cross-sectional study of 251 subjects, a reference glomerular filtration rate (GFR) was measured using (99c)Tc-DTPA plasma clearance (iGFR). Multivariate analysis was used to identify independent clinical and biochemical associations with serum cystatin C and iGFR levels. The diagnostic accuracy of cystatin C and commonly used creatinine-based methods of measuring renal function (serum creatinine, the MDRD four-variable and Cockcroft-Gault formulae) for detecting mild and moderate CKD was also compared. RESULTS: In the entire study population the same five variables, age, urinary albumin excretion rates, haemoglobin, history of macrovascular disease and triglyceride levels were independently associated with both cystatin C and iGFR levels. A serum cystatin C level cut-off > 82.1 nmol/l (1.10 mg/l) had the best test characteristics as a screening tool for detecting moderate CKD (< 60 ml/min per 1.73 m(2)) when compared with creatinine-based methods. At the upper threshold for mild CKD (< 90 ml/min per 1.73 m(2)), cystatin C also had greater diagnostic accuracy than creatinine, but had similar diagnostic accuracy when compared with creatinine-based formulae for predicting renal function. CONCLUSIONS: This study suggests that the clinical and biochemical parameters associated with serum cystatin C levels are closely linked to those associated with GFR and highlights the potential usefulness of screening for moderate or mild CKD in subjects with diabetes by simply measuring serum cystatin C levels.

L1Tc non-LTR retrotransposons from Trypanosoma cruzi contain a functional viral-like self-cleaving 2A sequence in frame with the active proteins they encode.

A comparative analysis of 40 Trypanosoma cruzi L1Tc elements showed that the 2A self-cleaving sequence described in viruses is present in them. Of these elements, 72% maintain the canonical 2A motif (DxExNPGP). A high percentage has a conserved point mutation within the motif that has not been previously described. In vitro and in vivo expression of reporter polyproteins showed that the L1Tc2A sequence is functional. Mutations within certain L1Tc2A sequences affect the efficiency of the cleavage. The data indicate that the L1Tc2A sequence may be influencing the L1Tc enzymatic machinery determining the composition and level of the translated products. The residues located immediately upstream of the 2A consensus sequence increase the cleaving efficiency and appear to stabilize the relative amount of translated products.