Daniel Cioca

Naive T cells in the elderly: are they still there?

Abstract:  One of the most striking changes in the primary lymphoid organs during human aging is the progressive involution of the thymus. As a consequence, the rate of naïve T cell output dramatically declines with age and the peripheral T cell pool shrinks. These changes lead to increased incidence of severe infections and decreased protective effect of vaccinations in the elderly. Little is, however, known of the composition and function of the residual naïve T cell repertoire in elderly persons. To evaluate the impact of aging on the naïve T cell pool, we investigated the quantity, phenotype, function, composition, and senescence status of CD45RA+CD28+ human T cells—a phenotype generally considered as naïve cells—from both young and old healthy donors. We found a significant decrease in the number of CD45RA+CD28+ T cells in the elderly, whereas the proliferative response of these cells is still unimpaired. In addition to their reduced number, CD45RA+CD28+ T cells from old donors display significantly shorter telomeres and have a restricted TCR repertoire in nearly all 24 Vβ families. These findings let us conclude that naïve T cells cannot be classified with conventional markers in old age.

CD25-Expressing CD8+ T Cells Are Potent Memory Cells in Old Age

We have recently described an IL-2/IL-4-producing CD8+CD25+ nonregulatory memory T cell population that occurs in a subgroup of healthy elderly persons who characteristically still have a good humoral response after vaccination. The present study addresses this specific T cell subset and investigates its origin, clonal composition, Ag specificity, and replicative history. We demonstrate that CD8+CD25+ memory T cells frequently exhibit a CD4+CD8+ double-positive phenotype. The expression of the CD8 αβ molecule and the occurrence of signal-joint TCR rearrangement excision circles suggest a thymic origin of these cells. They also have longer telomeres than their CD8+CD25− memory counterparts, thus indicating a shorter replicative history. CD8+CD25+ memory T cells display a polyclonal TCR repertoire and respond to IL-2 as well as to a panel of different Ags, whereas the CD8+CD25− memory T cell population has a more restricted TCR diversity, responds to fewer Ags, and does not proliferate in response to stimulation with IL-2. Molecular tracking of specific clones with clonotypic primers reveals that the same clones occur in CD8+CD25+ and CD8+CD25− memory T cell populations, demonstrating a lineage relationship between CD25+ and CD25− memory CD8+ T cells. Our results suggest that CD25-expressing memory T cells represent an early stage in the differentiation of CD8+ cells. Accumulation of these cells in elderly persons appears to be a prerequisite of intact immune responsiveness in the absence of naive T cells in old age.

Techniques in gerontology: Cell lines as standards for telomere length and telomerase activity assessment

The length of telomeres is believed to critically influence cellular aging processes and disease development. In order to reliably monitor telomere length and the corresponding cellular telomerase activity by optimized procedures, either based on flow cytometry or quantitative PCR technique, we here propose three commonly used cell lines, HEK293, K562 and TCL1301 as standards. In this contribution, efficient methods to determine mean telomere length of eukaryotic chromosomal DNA and determination of the corresponding telomeras activity are outlined. In particular, wide-range standard curves for a precise assessment of telomere length of genomic DNA by quantitative PCR technique are presented, measures, which greatly simplify the evaluation of respective functional roles of telomeres when studying biological processes such as disease progression and aging.

Immunizations in the elderly: do they live up to their promise?

ZusammenfassungZu Beginn des 21sten Jahrhunderts wird unser Gesundheitssystem nicht nur durch neuartige beziehungsweise wiederkehrende Infektionskrankheiten gefordert, sondern auch durch den demographischen Wandel, welcher derzeit in den Industriestaaten stattfindet. Von besonderer Bedeutung ist, dass Infektionserkrankungen bei älteren Menschen häufiger auftreten, einen schwereren Verlauf nehmen und charakteristische Merkmale hinsichtlich klinischer Präsentation und Behandlung aufweisen. Dies ist auf die Abnahme von Immunfunktionen zurückzuführen, welches kollektiv als Immunseneszenz bezeichnet wird. Die bedeutendsten altersassoziierten Veränderungen betreffen das T-Zell-System. Dies hat dementsprechend Auswirkungen auf die Wirksamkeit von Impfungen im Alter. Nichtsdestoweniger spielen Impfungen eine äußerst wichtige Rolle bei der Reduktion der durch Infektionserkrankungen verursachten Morbidität und Mortalität. Der vorliegende Artikel erläutert nun die Auswirkungen von Infektionserkrankungen auf ältere Menschen und vermittelt einen Überblick über den aktuellen Forschungsstand von Impfungen für ältere Menschen. Weiters soll dargestellt werden, welche altersassoziierten Veränderungen des Immunsystems für die verminderte Wirkung von Impfungen verantwortlich sind.SummaryIn the 21st century, public health is not only challenged by newly emerging and re-emerging infectious diseases but also by demographic developments that are taking place in many countries. Importantly, infections in the elderly are more frequent, more severe and have distinct features with respect to clinical presentation and treatment. This is due to a decline in the functions of the immune system referred to as immunosenescence. The most important age-related changes affect the T cell system. Although this derogates the protective effect of some vaccines, vaccinations are still considered the most cost-effective medical procedure for preventing morbidity and mortality caused by infectious diseases. The present article aims at outlining the impact of infectious diseases on the elderly and summarizing the progress made in the field of vaccinations of the elderly and how age-related changes within the immune system contribute to the decreased efficacy of vaccines.

What is the significance of HLA-DR antigen expression in the extraglomerular mesangium in glomerulonephritis?

INTRODUCTION AND AIMS The HLA-DR antigen is a HLA class II molecule involved in the presentation of antigenic peptides to the T cell receptor, thus regulating the immune response. Renal expression of the HLA-DR antigen may indicate specific sites of immunologically-mediated kidney injury in glomerulonephritis (GN). The aim of our study was to assess the presence of the HLA-DR antigen along the nephron including the extraglomerular mesangium in GN. METHODS A cross-sectional study of 22 patients with glomerulonephritis, mean age: 46.59±10.77 years, 14 male and 8 female, was conducted. Conventional stains, as well as immunohistochemistry for the HLA-DR Antigen Alpha-Chain were employed on kidney biopsies. Immunohistochemistry was assessed using a semi-quantitative score: 0-absent, 1-mild, 2-moderate, 3-intense. Statistical analysis was performed using SPSS17. RESULTS Four patients presented Focal and Segmental Glomerulosclerosis (FSGS), 5 patients: membranoproliferative GN, 7 patients: membranous nephropathy, 3 patients: mesangial proliferative GN, 2 patients: minimal change disease (MCD), and 1 patient: crescentic GN. Regarding the percentage of cases with HLA-DR positive cells along the nephron out of 22 patients: glomerular endothelial cells were 100% positive, intraglomerular mesangium cells were 81.8% positive, podocytes were 36.4% positive, extraglomerular mesangium cells were 31.8% positive, proximal tubule cells were 95.5% positive, distal tubule cells were 68.2% positive, interstitial capillaries were 77.3% positive, and cells of interstitial infiltrates were 27.3% positive. The percentage of cases staining positively for the HLA-DR antigen in the extraglomerular mesangium was 25% in FSGS, 60% in membranoproliferative GN, 0% in membranous nephropathy, 33.3% in mesangial proliferative GN, 100% in minimal change disease and 0% in crescentic GN. CONCLUSIONS A prominent HLA-DR antigen distribution was found on glomerular endothelial cells, intraglomerular mesangium cells and proximal and distal tubular cells. Extraglomerular mesangium cells and podocytes stained variably for the HLA-DR antigen, as did the cells of the interstitial infiltrates. The extraglomerular mesangium which serves as a portal of entry into the intraglomerular mesangium is endowed with antigen-presenting capabilities and is a region where induction of immune reactions could take place.

The dynamics of urinary N-acetyl-β-d-glucosaminidase (NAG), a marker of renal tubular dysfunction, in patients with lupus nephritis undergoing oral prednisone therapy

Introduction and aims: N-Acetyl-β-d-glucosaminidase (NAG), a marker of renal tubular dysfunction, is increased in patients with lupus nephritis. In addition to the toxic effects of proteinuria, patients with lupus nephritis may exhibit other factors that contribute to tubular dysfunction, such as pathogenic antitubular basement membrane antibodies. The aim of our study was to assess urinary NAG, proteinuria, and glomerular filtration rate (GFR) before treatment and after 7 and 30 days of oral prednisone therapy in patients with lupus nephritis. Methods: Ten patients with lupus nephritis, all females, mean age: 29.4 ± 10.17 years, were enrolled into the study. All the patients received oral prednisone 1 mg/kg. Twenty healthy subjects served as controls. We measured urinary NAG before treatment and after 7 and 30 days of oral prednisone therapy. Proteinuria, GFR, blood pressure, and side effects of therapy were also followed up. Urinary NAG was measured using the colorimetrical method and expressed as units per gram of creatinine (U/gCr). Statistical analysis (Wilcoxon signed ranks test and Wilcoxon rank sum test) was performed using SPSS 17. Results: In the 10 patients with lupus nephritis, urinary NAG before treatment was 16.9 ± 13.39 U/gCr (P = 0.005 compared with controls). NAG in controls was 1.73 ± 0.51 U/gCr. Proteinuria before treatment was 3.84 ± 1.93 g/24 h. The GFR before treatment was 50.48 ± 11.98 mL/min/1.73 m2. After 7 days of prednisone, urinary NAG was 23.55 ± 25.25 U/gCr (P = 0.878 compared with baseline, and P = 0.02 compared with controls). Proteinuria was 2.94 ± 1.3 g/24 h (P = 0.005 compared with baseline), and the GFR was 58.11 ± 13.64 mL/min/1.73 m2 (P = 0.005 compared with baseline). After 30 days of prednisone, urinary NAG was 11.77 ± 12.18 U/gCr (P = 0.203 compared with baseline, P = 0.022 compared with the value after 7 days of prednisone, and P = 0.01 compared with controls). Proteinuria was 1.73 ± 0.68 g/24 h (P = 0.005 compared with baseline, and P = 0.005 compared with the value after 7 days of prednisone), and the GFR was 67.49 ± 16.42 mL/min/1.73 m2 (P = 0.005 compared with baseline and P = 0.009 compared with the value after 7 days of prednisone). Blood pressure measurements did not show any significant changes. No major side effects of steroid therapy were noticed. Conclusions: Urinary NAG showed a significant reduction between 7 and 30 days of therapy. The reduction in urinary NAG set in later than the decline in proteinuria and the improvement in GFR. Further studies incorporating a longer follow-up are needed to observe whether the reduction in NAG persists upon continuation of prednisone therapy.

The endothelial cell markers von Willebrand factor [vWF], CD31 and CD34 are lost in glomerulonephritis and no longer correlate with the morphological indices of glomerular sclerosis, interstitial fibrosis, activity and chronicity

Endothelial cells (ECs) are active participants of an inflammatory process in glomeruli. EC damage has been shown to play an important role in the progression of glomerulonephritis (GN). The degree of glomerular and peritubular capillary loss in models of progressive renal disease correlates with the severity of glomerulosclerosis and interstitial fibrosis. The aim of our study was to analyze the association of vWF, CD31 and CD34 immunoreactivity with the morphological indices of glomerular sclerosis, interstitial fibrosis, activity and chronicity in GN. A cross-sectional study of 22 patients with GN was conducted. Conventional stains (hematoxylin-eosin, periodic acid Schiff and Trichrome Gömöri stains) and immunohistochemistry (vWF, CD31 and CD34) were employed on kidney biopsies. Activity and chronicity of GN, as well as glomerular segmental sclerosis and interstitial fibrosis, were evaluated according to a scoring system initially used for lupus nephritis and antineutrophil-cytoplasmic-antibody-associated vasculitis. Immunohistochemistry was assessed using a semi-quantitative score. Statistical analysis was performed using EpiInfo 6.04. The mean patient age was 46.68+/-14.09; 14 patients were male, and eight were female. Performing Spearmans rank correlation test, no correlation was found between each marker and glomerular segmental sclerosis, interstitial fibrosis, activity and chronicity, which suggests a loss of these markers and microvasculature involvement.

Does the antiviral therapy of patients with chronic hepatitis exert nephrotoxic effects

Introduction: HBV and HCV chronic hepatitis can be accompanied by secondary renal disease. In addition, these patients receive antiviral drugs with potential nephrotoxicity. It is known that interferon (IFN) therapy in HCV-infected kidney transplant recipients is followed by rejection of the transplant in 50% of the cases. Ribavirin is contraindicated in hemodialyzed patients and in patients with a GFR <50 ml/min/1.73 m2. IFN therapy requires dosage reduction and close monitoring in patients with a GFR <50 ml/min/1.73 m2 and in patients with end stage renal disease. The aim of our study was to assess the nephrotoxicity of antiviral drugs in patients with chronic hepatitis by measuring three renal biomarkers: urinary albumin, N-acetyl-β-d-glucosaminidase (NAG) and α 1-microglobulin, as well as glomerular filtration rate (GFR-MDRD4) before and at 6 months of therapy. Methods: Fifty-five patients (28 male and 27 female, with a mean age of 47.85 ± 12.03 years) with chronic hepatitis (40 patients with HCV, 13 patients with HBV, 1 patient with HBV+HCV, and 1 patient with HBV+HDV) were enrolled into the study. Different antiviral drug associations were used on a case-by-case basis. The 40 patients with HCV chronic hepatitis received either Peg-IFN-α 2a+Ribavirin (37 patients) or Peg-IFN-α 2b+Ribavirin (3 patients). The 13 patients with HBV chronic hepatitis received Peg-IFN-α 2a (9 patients), Lamivudine (2 patients), Entecavir (1 patient), or Adefovir (1 patient). The patient with HBV+HCV chronic hepatitis received Peg-IFN-α 2a+Ribavirin. The patient with HBV+HDV chronic hepatitis received IFN-α 2a. Urinary albumin (ELISA), NAG (colorimetrical method), α 1-microglobulin (ELISA), and serum creatinine were measured before and at 6 months of antiviral therapy. Urinary markers were expressed as either mg/gCr (for albumin and α 1-microglobulin) or U/gCr (for NAG). Statistical analysis (Pearson’s correlation coefficient, paired t-test and χ2-test) was performed. Results: At 6 months of therapy urinary albumin/gCr did not increase significantly: 16.58 ± 23.39 vs. 15.85 ± 24.96 mg/gCr before therapy, p = 0.87. Urinary NAG/gCr did not increase significantly: 4.21 ± 3.37 vs. 3.83 ± 3.2 U/gCr before therapy, p = 0.53. Urinary α 1-microglobulin/gCr was almost unchanged: 4.38 ± 4.47 vs. 4.38 ± 3.57 mg/gCr before therapy, p = 0.99. The GFR did not decline significantly: 92.41 ± 22.21 vs. 94.59 ± 36.1 ml/min/1.73 m2 before therapy, p = 0.7. Ten patients (18.18%) were albuminuric before therapy, and 14 patients (25.45%) were albuminuric at 6 months of therapy, a non-significant increase (p = 0.35). We found a correlation between urinary albumin/gCr and NAG/gCr and between urinary albumin/gCr and α 1-microglobulin/gCr both at baseline and at 6 months of therapy: r = 0.54, p = 0.0005; r = 0.29, p = 0.03; r = 0.51, p = 0.0005; and r = 0.4, p = 0.002, respectively. In the patient receiving Adefovir, a known nephrotoxic drug, two of the three biomarkers (urinary albumin/gCr and NAG/gCr) increased, most notably NAG/gCr. Both HCV and HBV chronic hepatitis therapy were associated with non-significant changes in renal biomarker excretion and GFR. Conclusions: With the exception of Adefovir, all of the drug associations used in this study were safe.

Treatment of systemic lupus erythematosus in two patients with extreme B-cell lymphopenia: importance of immunomonitoring and avoidance of B-cell targeted therapy

Introduction: Because dysfunction of the B-cell compartment is thought to be important in the pathogenesis of systemic lupus erythematosus (SLE), there has been a recent focus on therapies that target humoral immunity via multiple mechanisms. The aim of this paper was to demonstrate the importance of immunomonitoring in two cases with class II lupus nephritis on steroids who presented with a flare-up of disease. After a thorough work-up for infectious triggers of disease activity, conversion to another histopathological class of lupus nephritis was suspected. Deterioration of the patients’ clinical condition made kidney biopsy impossible, and as B-cell targeted therapy was considered, we decided to perform an immunophenotypic analysis and to tailor therapy to the results of the lymphocyte profile. As we incidentally found extremely low B-cell counts, any B-cell–targeted therapy was prohibited, and cyclophosphamide (Cy) was considered a viable therapeutic option. Methods: We performed flow-cytometric lymphocyte (Ly) phenotyping (CD19, CD3, CD3CD4, CD3CD8, CD56/16) on two patients with class II lupus nephritis before and after two intravenous (i.v.) Cy pulse administrations. During all this time, patients were on steroids. Results: Both patients showed extreme B-cell lymphopenia, a marker of active SLE, which was not greatly impacted by the treatment over the follow-up period. Conclusions: As current therapies are aimed at targeting the B cell, an important component of adaptive immunity, caution must be exercised before their use. In addition, monitoring of Ly subsets is essential due to the occurrence of extreme B-cell lymphopenia.