Predictive value of human cytomegalovirus (HCMV) T-cell response in the control of HCMV infection by seropositive solid-organ transplant recipients according to different assays and stimuli.

Human cytomegalovirus (HCMV) is still the most common viral infection in solid-organ transplant recipients (SOTR). Our study aimed to identify the predictive values of the T-cell response able to protect from HCMV disease, according to different assays. Viral DNA was determined by real-time PCR. The T-cell immune response to HCMV infection was investigated in SOTR according to the following assays and stimuli: cytokine flow cytometry (CFC) after peripheral blood mononuclear cell (PBMC) stimulation with autologous HCMV-infected dendritic cells (iDC) vs three ELISPOT assays using PBMCs stimulated with: 1. HCMV-infected cell lysate (iCL); 2. a pool of 34 epitopic peptides (PP) from different HCMV proteins; 3. a commercial pp65 peptide pool (CPM). ELISPOT results were normalized to T-cell counts. Overall, 51 SOTR were enrolled: 29 (57%) had low viral load (LVL) self-resolving infections, 19 (37%) high viral load (HVL) infections treated with antiviral drugs, and 3 (6%) tissue-invasive disease (TID). At DNAemia peak, ROC analysis showed that CFC-iDC CD4+ and the ELISPOT-iCL assays yielded overlapping area under the curve (AUC) results. The time needed to reconstitute protective T-cell immunity in SOTR with HVL infections was significantly longer with each assay compared to LVL infections. Using the CFC-iDC assay as a reference test (requiring 7 days to complete), the 24h ELISPOT-iCL assay provides similar results in terms of protection prediction from HCMV infection.

Genetic Screening of Anderson-Fabry Disease in Probands Referred From Multispecialty Clinics.

BACKGROUND: Anderson-Fabry disease (AFD) is a rare X-linked lysosomal storage disease, caused by defects of the alpha-galactosidase A (GLA) gene. AFD can affect the heart, brain, kidney, eye, skin, peripheral nerves, and gastrointestinal tract. Cardiology (hypertrophic cardiomyopathy), neurology (cryptogenic stroke), and nephrology (end-stage renal failure) screening studies suggest the prevalence of GLA variants is 0.62%, with diagnosis confirmation in 0.12%. OBJECTIVES: This study sought to expand screening from these settings to include ophthalmology, dermatology, gastroenterology, internal medicine, pediatrics, and medical genetics to increase diagnostic yield and comprehensively evaluate organ involvement in AFD patients. METHODS: In a 10-year prospective multidisciplinary, multicenter study, we expanded clinical, genetic, and biochemical screening to consecutive patients enrolled from all aforementioned clinical settings. We tested the GLA gene and α-galactosidase A activity in plasma and leukocytes. Inclusion criteria comprised phenotypical traits and absence of male-to-male transmission. Screening was extended to relatives of probands harboring GLA mutations. RESULTS: Of 2,034 probands fulfilling inclusion criteria, 37 (1.8%) were carriers of GLA mutations. Cascade family screening identified 60 affected relatives; clinical data were available for 4 affected obligate carriers. Activity of α-galactosidase A in plasma and leukocytes was diagnostic in male subjects, but not in female subjects. Of the 101 family members harboring mutations, 86 were affected, 10 were young healthy carriers, and 5 refused clinical evaluation. In the 86 patients, involved organs or organ systems included the heart (69%), peripheral nerves (46%), kidney (45%), eye (37%), brain (34%), skin (32%), gastrointestinal tract (31%), and auditory system (19%). Globotriaosylceramide accumulated in organ-specific and non-organ-specific cells in atypical and classic variants, respectively. CONCLUSIONS: Screening probands with clinically suspected AFD significantly increased diagnostic yield. The heart was the organ most commonly involved, independent of the clinical setting in which the patient was first evaluated.

Normalizing ELISPOT responses to T-cell counts: a novel approach for quantification of HCMV-specific CD4(+) and CD8(+) T-cell responses in kidney transplant recipients.

BACKGROUND: Human cytomegalovirus (HCMV) is the most common opportunistic virus infection in solid organ transplant recipients. The analysis of HCMV-specific T-cell immunity after organ transplant is of relevant clinical interest. OBJECTIVES: To analyze HCMV-specific CD4(+) and CD8(+) T-cell responses in healthy subjects and kidney transplant recipients (KTR). STUDY DESIGN: HCMV-specific T-cell responses were evaluated by interferon-γ (IFN-γ) enzyme-linked immunospot (ELISPOT) using overlapping 15-mer peptide pools of immediate early (IE)-1, IE-2, phosphoprotein 65 (pp65) (for stimulation of both CD4(+) and CD8(+) T-cell responses) and a pool of 34 short peptides (8-12 amino acids in length, for stimulation of CD8(+) T-cell responses). ELISPOT results were normalized to T-cell subset counts and their correlations with a reported dendritic cell (DC)-based assay, which simultaneously quantifies HCMV-specific CD4(+) and CD8(+) T-cell responses, were analyzed. RESULTS: HCMV-seropositive KTR showed higher ELISPOT responses compared to HCMV-seropositive healthy subjects. IE-1 and pp65 ELISPOT responses were mediated mainly by CD8(+) T-cells and, to a lesser extent, CD4(+) T cells; IE-2 peptides appear to stimulate CD56(+) cells (natural killer cells). In HCMV-seropositive healthy subjects, ELISPOT results (expressed either as net spots/million cells or normalized to the corresponding T-cell count) significantly correlated with the DC assay. However, in HMCV-seropositive KTR, only normalized ELISPOT responses to overlapping 15-mer peptide pools significantly correlated with DC-assay responses. CONCLUSIONS: The normalized ELISPOT represents a novel and simple approach for quantifying and monitoring HCMV-specific CD4(+) and CD8(+) T-cell responses in KTR.

Anaemia management in non-dialysis chronic kidney disease (CKD) patients: a multicentre prospective study in renal clinics.

BACKGROUND: Knowledge on anaemia management in non-dialysis chronic kidney disease (ND-CKD) patients regularly followed in renal clinics is scarce although being essential to identifying areas of therapeutic improvement. METHODS: We prospectively evaluated anaemia management in two visits, performed 6 months apart, in 755 prevalent ND-CKD stage 3b-5 patients followed in 19 nephrology clinics from ≥6 months. Anaemia was defined as severe (Hb <11 g/dL) or mild (Hb: 11-13.5 in males and 11-12 g/dL in females); iron deficiency (ID) was defined as transferrin saturation (TSAT) <20% and/or ferritin <100 ng/mL. Primary endpoint was the change of anaemia and ID prevalence between baseline and 6-month visit. Secondary endpoint was the prevalence of clinical inertia to either ESA or iron supplementation, that is, the lack of ESA or iron prescription despite Hb <11 g/dL or ID. RESULTS: Age was 69 ± 13 years and GFR 27.5 ± 10.0 mL/min/1.73 m(2); male gender, diabetes and prior cardiovascular disease were 57.2, 30.1 and 30.1%, respectively. Prevalence of severe and mild anaemia was 18.0 and 44.0% at baseline and remained unchanged at Month 6 (19.3 and 43.2%). ID was prevalent at both visits (60.1 and 60.9%). Clinical inertia to ESA was similar at baseline and at Month 6 (39.6 and 34.2%, respectively, P = 0.487) and it was less frequent than clinical inertia to iron therapy (75.7 and 72.0%, respectively). CONCLUSIONS: This study shows that anaemia prevalence is unexpectedly high in the setting of tertiary nephrology care. This was due to a persistent clinical inertia in the anaemia management, remarkable for iron supplementation and less critical, but still significant, for ESA treatment.

Chronic kidney disease epidemic: myth and reality.

In recent years, an epidemic of chronic kidney disease (CKD) has emerged as one of the major public health problem. The prevalence of CKD is largely sustained by the inclusion of a substantial proportion of the elderly population within stage 3 CKD, according to the Kidney Disease Outcomes Quality Initiative staging system. However, some clarifications are necessary when interpreting these data. In fact, renal function "normally" declines with age, without bearing any unfavourable outcome; in addition, the Modification of Diet in Renal Disease formula used to calculate glomerular filtration rate (GFR) underestimates kidney function in the elderly and in women. Considerable interest in CKD has been generated by the evidence that predialysis CKD is associated with the increased risk of cardiovascular disease (CVD). Again, potential confounding factors must be ruled out. Age is thought to play a major role in this context. The most common causes of CKD, hypertension and diabetes mellitus, are also known to affect cardiovascular outcomes directly, thus preventing the recognition of an independent effect of kidney dysfunction on mortality by CVD. Taken together, these considerations point for a better definition of CKD. Early identification of patients at risk for accelerated decline in renal function is mandatory to plan strategies for screening and preventing CKD and its complications. At present, detection of CKD in the general population requires a multi-dimensional approach that should include the evaluation of clinical risk conditions, evaluation of albuminuria and sequential monitoring of GFR.

Rheumatoid arthritis and renal light-chain deposition disease: long-term effectiveness of TNF-α blockade with etanercept.

A 68-year-old woman diagnosed with erosive rheumatoid arthritis (RA) was treated with intramuscular methotrexate 15 mg weekly and oral prednisone 5 mg daily. A favorable outcome of 6 years was followed by RA flare and nephrotic syndrome (NS). Renal biopsy revealed non-amyloid light-chain deposition disease. Laboratory analysis and bone marrow biopsy excluded monoclonal protein and plasma cell dyscrasia. Addition of subcutaneous etanercept, 25 mg twice weekly allowed rapid control of both arthritis and NS. To date, after over 7-year follow-up, RA is in clinical remission, 24-h albuminuria is consistently below 0.5 g, and serum creatinine is 0.9 mg/dl.

Renal blood flow redistribution during acute kidney injury.

We describe a case of acute kidney injury with decreased kidney perfusion in which contrast-enhanced computed tomography of the abdomen was performed for a nonrenal indication. This imaging procedure showed intrarenal blood flow redistribution from the cortex to the medulla that reversed after recovery of kidney function. Renal blood flow redistribution was described first almost a century ago in experimental models of renal ischemia, but clinical imaging studies are scarce. We provide a clear example of this phenomenon using contrast-enhanced computed tomography.

The antifibrogenic effect of hepatocyte growth factor (HGF) on renal tubular (HK-2) cells is dependent on cell growth.

Although several reports suggest an antifibrogenic effect of hepatocyte growth factor (HGF), an increased deposition of matrix induced by HGF has also been reported. These conflicting effects could result from a diverse proliferative state of the target cells. Aim of the present study was to evaluate HGF effects on growth arrested (quiescent) and actively proliferating renal tubular epithelial (HK-2) cells. HK-2 cells were cultured in RPMI medium either on agarose gel or on plastic surface in order to inhibit or to allow cell proliferation. Cells were incubated with RPMI containing HGF (50 ng/ml) for 24 h at 37 degrees C. Untreated HK-2 were used as control. After 24 h of incubation, cells were counted by Coulter counter. (alpha2)IV collagen, transforming growth factor-beta (TGF-beta), Tissue inhibitor of metalloproteases (TIMP1 and 2) mRNA levels were determined by RT-PCR. The production of type IV collagen, c-met, proliferating cell nuclear antigen (PCNA), and SnoN, a transcriptional Smad corepressor and thus a TGF-beta inhibitor, was evaluated by ELISA or western blotting. MMP-9 and 2 gelatinolytic activity was studied by zymography. Treatment with HGF did not increase HK-2 cell number and PCNA synthesis when the cells were grown on agarose as it did for cells grown on plastic surface. HGF increased (alpha2)IV collagen in proliferating cells whereas it reduced (alpha2)IV collagen and c-met synthesis in growth arrested cells. HGF treatment increased TGF-beta and TIMP-2 in proliferating cells while reduced TIMP-1 mRNA levels of quiescent cells. Furthermore, production of the co repressor SnoN was significantly decreased by HGF in proliferating cells. Quiescent and proliferating HK-2 showed a different pattern of metalloproteases activity with a prevalence of MMP2 in quiescent and MMP9 in proliferating cells. In summary, HGF showed opposite effects on growth arrested and proliferating HK-2 cells favouring matrix deposition in the latter with increasing expression of collagen, TIMP-1 and TGF-beta. Our results demonstrate that the proliferative state of target cells may influence the effects of HGF on extracellular matrix turnover in HK-2 cells.

Renal function and functional reserve in healthy elderly individuals.

BACKGROUND: Aging is characterized by a decline in renal function and by a susceptibility to renal diseases. However it is not clear whether the observed changes are solely hemodynamic, structural or both. We evaluated renal function, functional reserve (RFR) and morphology in healthy elderly individuals. METHODS: Healthy participants (n=19) were divided into young (n=6, age range 25-37 years), middle-aged (n=6, 44-74 years) and elderly (n=7, 81-96 years). Nitric oxide (NO), plasma renin activity (PRA) and aldosterone, renal plasma flow (RPF) by p-aminohippurate clearance (CPAH) and glomerular filtration rate (GFR) by inulin clearance (CIN) were determined before and during maximal vasodilating stimuli, induced with the infusion of dopamine and amino acids. Glomerular sclerosis, lumen area and wall thickness of afferent arterioles were determined by kidney biopsy from 36 healthy kidney donors and from 6 nephrectomies for renal carcinoma. RESULTS: GFR and RPF were slightly reduced in elderly individuals whereas filtration fraction (FF) was increased. GFR and RPF did not increase in the elderly after maximal vasodilating stimuli as in young and middle-aged subjects suggesting a reduction of RFR. NO, increased at baseline, did not increase further after vasodilating stimuli; while on the contrary, PRA, similar in the 3 groups at baseline, was not reduced by vasodilating stimuli in the elderly. Sclerotic glomeruli but not glomerular volume were significantly increased by aging. Afferent arteriole lumens were reduced by aging whereas wall thickness was unchanged. CONCLUSIONS: Renal function is preserved with aging in healthy subjects at the expense of a complete reduction of RFR. RFR may be wasted to compensate for the increased number of sclerotic glomeruli. Vascular changes, suggested by reduced arteriolar lumen, may be so advanced that even in the presence of high levels of vasodilatory molecules, kidneys are not responsive anymore to maximal vasoactive stimuli.

Uremic serum induces proatherogenic changes in human endothelial cells.

BACKGROUND: Cardiovascular complications are the main cause of death in uremic patients. Uremic angiopathy has been regarded as an accelerated form of atherosclerosis. However the mechanism leading to vessel wall injury is still unknown. We hypothesized that uremic serum affects endothelium inducing a proatherogenic state. METHODS: We studied the effects of uremic serum on human endothelial cells (HECs). Cell proliferation and adhesion of mononuclear cells to HEC monolayers were evaluated by cell counting, apoptosis and collagen production by ELISA, and nitric oxide (NO) by measuring the concentration of nitrite/nitrate in the cell supernatant. (alfa2)IV collagen, tissue inhibitor of metalloproteases-1 (TIMP-1) and transforming growth factor-beta (TGF-beta) mRNA levels were measured by reverse transcriptase polymerase chain reaction (RT-PCR). In some experiments cells were preincubated with anti-receptor for advanced glycation end product (anti-RAGE) blocking antibodies. RESULTS: Uremic serum did not modify HEC proliferation but induced apoptosis after 72 hours of incubation. Adhesion of mononuclear cells to HEC monolayers was significantly increased by uremic serum. In addition, uremic serum increased (alfa2)IV collagen, TIMP-1 and TGF-beta mRNA levels. There was no increase in nitric oxide concentration in ure-mic serum-treated endothelial cells, and the expression of TGF-beta was neither modified by L-NAME nor by anti-RAGE antibodies. CONCLUSIONS: Our results indicate that uremic serum affects HEC inducing a proatherogenic state that may be responsible for the accelerated atherosclerosis of uremic patients. Apparently uremic serum effect is not mediated by NO or by AGEs.