Urinary Biomarkers for Kidney Disease in ATTR Amyloidosis

Aim: The detection and prognosis of nephropathy in transthyretin amyloidosis depends on albuminuria and renal function. Knowing that urinary levels of alpha-1 microglobulin and beta-2 microglobulin reflect tubular dysfunction while urinary alpha-2 macroglobulin implies glomerular damage, we decide investigate the diagnostic value of these markers in the patients with transthyretin amyloidosis. Methods: Serum and urinary samples collected from 30 patients and 11 asymptomatic carriers were tested for alpha-1 microglobulin, beta-2 microglobulin, alpha-2 macroglobulin, albumin, creatinine and cystatin C. Results: Pathological urinary alpha-1 microglobulin was detected in 17 patients, beta-2 microglobulin in 6 and alpha-2 macroglobulin in 5; 5 patients had albuminuria (mg/g creatinine) 30-300 and in 20 patients values >300 were present. Asymptomatic carriers did not present pathological excretion of these biomarkers and albuminuria was >30 in 1 individual. The excretion rates of alpha-1 microglobulin and beta-2 microglobulin were positively correlated with albuminuria (P<0.001), serum creatinine (P<0.05) and cystatin C (P<0.001). Urinary alpha-2 macroglobulin was almost exclusively found in the presence of albuminuria, although their levels do not correlate. Conclusion: Urinary biomarkers emerge as a potential approach to detect renal disease but unexpectedly, urinary alpha-2 macroglobulin was not a marker of the severity of albuminuria

Layer guided-acoustic plate mode biosensors for monitoring MHC-peptide interactions

The transduction signals from the immobilisation of a class I heavy chain, HLA-A2, on a layer guided acoustic plate mode device, followed by binding of beta(2)-microglobulin and subsequent selective binding of a target peptide are reported

Age-related increase of kynurenic acid in human cerebrospinal fluid-IgG and beta(2)-microglobulin changes

Kynurenic acid (KYNA) is an endogenous metabolite in the kynurenine pathway of tryptophan degradation and is an antagonist at the glycine site of the N-methyl-D-aspartate as well as at the alpha 7 nicotinic cholinergic receptors. In the brain tissue KYNA is synthesised from L-kynurenine by kynurenine aminotransferases (KAT) I and II. A host of immune mediators influence tryptophan degradation. In the present study, the levels of KYNA in cerebrospinal fluid (CSF) and serum in a group of human subjects aged between 25 and 74 years were determined by using a high performance liquid chromatography method. In CSF and serum KAT I and II activities were investigated by radioenzymatic assay, and the levels of β2-microglobulin, a marker for cellular immune activation, were determined by ELISA. The correlations between neurochemical and biological parameters were evaluated. Two subject groups with significantly different ages, i.e. 50 years, p < 0.001, showed statistically significantly different CSF KYNA levels, i.e. 2.84 ± 0.16 fmol/μl vs. 4.09 ± 0.14 fmol/μl, p < 0.001, respectively; but this difference was not seen in serum samples. Interestingly, KYNA is synthesised in CSF principally by KAT I and not KAT II, however no relationship was found between enzyme activity and ageing. A positive relationship between CSF KYNA levels and age of subjects indicates a 95% probability of elevated CSF KYNA with ageing (R = 0.6639, p = 0.0001). KYNA levels significantly correlated with IgG and β2-microglobulin levels (R = 0.5244, p = 0.0049; R = 0.4253, p = 0.043, respectively). No correlation was found between other biological parameters in CSF or serum. In summary, a positive relationship between the CSF KYNA level and ageing was found, and the data would suggest age-dependent increase of kynurenine metabolism in the CNS. An enhancement of CSF IgG and β2-microglobulin levels would suggest an activation of the immune system during ageing. Increased KYNA metabolism may be involved in the hypofunction of the glutamatergic and/or nicotinic cholinergic neurotransmission in the ageing CNS

Limited proteolysis in the investigation of beta2-microglobulin amyloidogenic and fibrillar states.

Amyloid fibrils of patients treated with regular haemodialysis essentially consists of β2-microglobulin (β2-m) and its truncated species ΔN6β2-m lacking six residues at the amino terminus. The truncated fragment shows a higher propensity to self-aggregate and constitutes an excellent candidate for the analysis of a protein in the amyloidogenic conformation. The surface topology and the conformational analysis of native β2-m and the truncated ΔN6β2-m species both in the soluble and in the fibrillar forms were investigated by the limited proteolysis/mass spectrometry strategy. The conformation in solution of a further truncated mutant ΔN3β2-m lacking three residues at the N-terminus was also examined. This approach appeared particularly suited to investigate the regions that are solvent-exposed, or flexible enough to be accessible to protein-protein interactions and to describe the conformation of transient intermediates. Moreover, proteolysis experiments can also be tailored to investigate amyloid fibrils by discriminating the protein regions constituting the unaccessible core of the fibrils and those still flexible and exposed to the solvent. Although native β2-m and ΔN3β2-m shared essentially the same conformation, significative structural differences exist between the native and the ΔN6β2-m proteins in solution with major differences located at the end moiety of strand V and subsequent loop with strand VI and at both the N- and C-termini of the proteins. On the contrary, an identical distribution of preferential proteolytic sites was observed in both proteins in the fibrillar state, which was nearly superimposible to that observed for the soluble form of ΔN6β2-m. These data revealed that synthetic fibrils essentially consists of an unaccessible core comprising residues 20-87 of the β2-m protein with exposed and flexible N- and C-terminal ends. Moreover, proteolytic cleavages observed in vitro at Lys 6 and Lys 19 reproduce specific cleavages that have to take place in vivo to generate the truncated forms of β2-m occurring in natural fibrils. On the basis of these results, a molecular mechanism for fibril formation has been propose

Renal tubular epithelial cells add value in the diagnosis of upper urinary tract pathology

Background: Diagnosis of upper urinary tract infections (UTI) is challenging. We evaluated the analytical and diagnostic performance characteristics of renal tubular epithelial cells (RTECs) and transitional epithelial cells (TECs) on the Sysmex UF-5000 urine sediment analyzer. Methods: Urinary samples from 506 patients presenting with symptoms of a UTI were collected. Only samples for which a urinary culture was available were included. Analytical (imprecision, accuracy, stability and correlation with manual microscopy) and diagnostic performance (sensitivity and specificity) were evaluated. Results: The Sysmex UF-5000 demonstrated a good analytical performance. Depending on the storage time, storage conditions (2-8 degrees C or 20-25 degrees C) and urinary pH, RTECs and TECs were stable in urine for at least 4 h. Using Passing-Bablok and Bland-Altman analysis, an acceptable agreement was observed between the manual and automated methods. Compared to TECs, RTECs demonstrated an acceptable diagnostic performance for the diagnosis of upper UTI. Conclusions: While TECs do not seem to serve as a helpful marker, increased urinary levels of RTECs add value in the diagnosis of upper UTI and may be helpful in the discrimination between upper and lower UTIs

altered expression of cD300a inhibitory receptor on cD4+ T cells From human immunodeficiency Virus-1-infected Patients: association With Disease Progression Markers.

The ability of the CD300a inhibitory receptor to modulate immune cell functions and its involvement in the pathogenesis of many diseases has aroused a great interest in this molecule. Within human CD4+ T lymphocytes from healthy donors, the inhibitory receptor CD300a is differentially expressed among different T helper subsets. However, there are no data about the expression and regulation of CD300a receptor on CD4+ T cells from human immunodeficiency virus (HIV)-1-infected patients. The objective of this study was to investigate the expression of CD300a on CD4+ T cells from HIV-infected patients on suppressive combined antiretroviral therapy (cART) and cART naïve patients. Our results have demonstrated that the expression levels of this inhibitory receptor were higher on CD4+ T cells from HIV-1 infected subjects compared with healthy donors, and that cART did not reverse the altered expression of CD300a receptor in these patients. We have observed an increase of CD300a expression on both PD1+CD4+ and CD38+CD4+ T cells from HIV-1 infected people. Interestingly, a triple positive (CD300a+PD1+CD38+) subset was expanded in naïve HIV-1 infected patients, while it was very rare in healthy donors and patients on cART. Finally, we found a negative correlation of CD300a expression on CD4+ T lymphocytes and some markers associated with HIV-1 disease progression. Thus, our results show that HIV-1 infection has an impact in the regulation of CD300a inhibitory receptor expression levels, and further studies will shed light into the role of this cell surface receptor in the pathogenesis of HIV infection

Testing the theory of immune selection in cancers that break the rules of transplantation

Modification of cancer cells likely to reduce their immunogenicity, including loss or down-regulation of MHC molecules, is now well documented and has become the main support for the concept of immune surveillance. The evidence that these modifications, in fact, result from selection by the immune system is less clear, since the possibility that they may result from reorganized metabolism associated with proliferation or from cell de-differentiation remains. Here, we (a) survey old and new transplantation experiments that test the possibility of selection and (b) survey how transmissible tumours of dogs and Tasmanian devils provide naturally evolved tests of immune surveillance