Alois Gessl

High-Glucose–Triggered Apoptosis in Cultured Endothelial Cells

High ambient glucose concentration, linked to vascular complications in diabetes in vivo, modulates mRNA expression of fibronectin, collagen, tissue-type plasminogen activator, and plasminogen activator inhibitor and induces delayed replication and excess cell death in cultured vascular endothelial cells. To determine the role of high ambient glucose (30 mmol/1) in apoptosis, paired cultures of individual isolates of human umbilical vein endothelial cells (HUVECs) were exposed to both high (30 mmol/1) and low (5 mmol/1) concentrations of glucose for short-term (24, 48, and 72 h) and long-term (13 ± 1 days) experiments. Incubation of HUVECs with high glucose for >48 h increased DNA fragmentation (13.7 ± 6.5% of total DNA, mean ± SD) versus cultures kept in 5 mmol/1 glucose (10.9 ± 5.6%, P < 0.005), as measured by [3H]thymidine assays. Data were confirmed by apoptosis-specific fluorescence-activated cell sorter analysis of confluent HUVEC cultures, which displayed after long-term exposure to 30 mmol/1 glucose a 1.5-fold higher prevalence of apoptosis than control cultures exposed to 5 mmol/1 glucose (P < 0.005). In contrast, no increase in DNA fragmentation in response to 30 mmol/1 glucose was seen for standardized cell lines (K 562, P 815, YT) and fibroblasts. Expression of clusterin mRNA, originally reported to be a molecular marker of apoptosis, was only slightly affected by short-term (24-h) high-glucose exposure but was significantly reduced after long-term incubation in 30 mmol/1 glucose (82.2 ± 13.8% of control) versus 5 mmol/1 glucose, which questions the role of clusterin gene expression as a marker of apoptosis. The results demonstrate that high ambient glucose can promote apoptosis in HUVECs in vitro and suggest potential endothelial damage by hyperglycemia in diabetic patients.

Modulation by high glucose of adhesion molecule expression in cultured endothelial cells

SummaryWe evaluated the influence of high ambient glucose on cellular expression of adhesion molecules, known to mediate endothelial interaction of leucocytes and monocytes. Paired cultures of individual isolates of human umbilical vein endothelial cells (HUVECs) were studied by fluorescence activated cell sorter analysis after exposure to 30 vs 5 mmol/l glucose. Incubation of HUVECs for 24 h in 30 mmol/l glucose increased ICAM-1 (intercellular adhesion molecule-1; 116.4±16.9% of control, p ≤ 0.05), but not PECAM (platelet endothelial cell adhesion molecule) expression, compared to cultures kept in 5 mmol/l glucose. Long-term exposure (13±1 days) of HUVECs to 30 mmol/l glucose increased expression of ICAM-1 to 122.5±32.2% (p<0.002) and reduced that of PECAM to 86.9±21.3% vs the respective control culture in 5 mmol/l glucose (p<0.02). Stimulation of confluent HUVECs, kept in 30 vs 5 mmol/l glucose for 13±1 days, with 20 U/ ml interleukin-1 for 24 h (ICAM-1) and 4 h (endothelial leukocyte adhesion molecule 1) resulted in reduced ICAM-1 (84.8±27.0%, p<0.05) and endothelial leukocyte adhesion molecule-1 (87.6±22.4%, p<0.05) expression vs control cells, while that of PECAM (t: 24 h) and vascular cell adhesion molecule-1 (t: 16 h) remained unchanged. In conclusion, it appears that differences in expression of adhesion molecules on HUVECs in response to high glucose reflects endothelial glucose toxicity, which may also induce endothelial dysfunction in diabetes.

Thyroid ultrasound versus antithyroid peroxidase antibody determination: a cohort study of four hundred fifty-one subjects.

Autoimmune thyroiditis is mirrored by a hypoechoic ultrasound pattern. We determined diagnostic precision of thyroid sonography compared to that of anti-thyroid peroxidase antibody (TPOAb) concentration. Ambulatory patients with unknown thyroid status (n = 451; 407 female, ages 44 +/- 16 years; 45 male, ages 50 +/- 14 years) excluding those with suspected hyperthyroidism or on drugs known to cause hypothyroidism were recruited consecutively. Subjects were recruited from a specialized thyroid outpatient unit with higher frequencies of thyroid disorders than in the general population. Before determination of thyroid function and TPOAb concentration thyroid volume (normal values: women < 12 mL, men < 14 mL) and echogenicity (grade 1 = normal: similar to submandibular gland, hyperechoic to neck muscles; grade 2: hypoechoic to submandibular gland, hyperechoic to neck muscles, grade 3: iso-/hypoechoic to neck muscles) were determined. Positive predictive value of grade 3 pattern for detection of autoimmune thyroiditis was 94% (with overt hypothyroidism) and 96% (with any degree of hypothyroidism), that of grade 2 or 3 85% and 87%, respectively. Negative predictive value of grade 1 pattern for detection of euthyroid TPOAb negative subjects was 91%. Goiter was present in 31% and 21% of TPOAb postive and negative subjects, respectively, while 11% and 15% had an atrophic thyroid gland (p = not significant [n.s.]). Given a high intraobserver and interobserver agreement abnormal thyroid ultrasound patterns were highly indicative of autoimmune thyroiditis and allowed the detection of thyroid dysfunction with 96% probability.

Phenotype and Function of Peripheral and Prostatic Lymphocytes in Patients with Benign Prostatic Hyperplasia

In a previous report, we demonstrated intense lymphocytic infiltration of all benign prostatic hypertrophy (BPH) tissues analyzed in conjunction with HLA-DR expression on normally MHC-class-II-negative prostate epithelial cells. The composition of these infiltrates (70 to 80% CD3+ T-cells, but no granulocytes) resembles the situation seen in immune responses against altered self or self rather than against foreign antigens (infection). In the present study, phenotypic and functional immunoassays were used in order to investigate whether T-cells in BPH are indeed activated, and whether this activation is systemic or restricted locally to the prostate. Analysis of T-cell activation marker expression and proliferation requirements provided substantial evidence that these infiltrating lymphocytes, in contrast to their peripheral counterparts, are chronically activated. Since local accumulation of activated lymphocytes can cause tissue destruction, high concentrations of cytokines, and consequently tissue rebuilding, this process might contribute to the pathogenesis of BPH.

Differentiation of Rat Bone Marrow Cells Into Macrophages Under the Influence of Mouse L929 Cell Supernatant

Bone marrow cells (BMC) flushed from femora of Lewis rats were cultured in Dulbeccos modification of Eagles medium supplemented with mouse L929 cell supernatant as a source of colony‐stimulating factor (CSF). Differentiation of macrophage progenitor cells into macrophages (Mø) and expression of various markers were kinetically assessed. The proportion of Mø increases from approximately 4% in freshly isolated BMC to 100% after 7‐8 days of cell culture. These cells, termed bone marrow cell‐derived macrophages (BMDMø), adhere to and spread on plastic surface; exhibit Mø morphology; stain intensely for nonspecific esterase; are able to phagocytose latex particles, IgG‐sensitized erythrocytes, and C3‐coated red cells; and express receptors for IgG and C3. A subpopulation of BMDMø expresses MHC class II antigens as demonstrated by immunofluorescence using MRC OX6 and MRC OX17 monoclonal antibodies which recognize antigens coded in the I‐A or I‐E subregion of the MHC, respectively.

Renal Elimination of Sclerostin Increases With Declining Kidney Function

CONTEXT Sclerostin serum levels are increased in patients with chronic kidney disease (CKD). Osteoporosis and CKD often occur simultaneously. Currently antisclerostin antibodies are in clinical development for the treatment of osteoporosis. OBJECTIVE The objective of this study was to study the renal handling of sclerostin. DESIGN This was a cross-sectional study. SETTING The study was conducted at a university hospital and outpatient renal clinic. PATIENTS One hundred twenty men and women with CKD stage 1-5 participated in the study. INTERVENTION Measurements of sclerostin in urine and serum (ELISA), renal function [estimated glomerular filtration rate (eGFR)], electrolytes, α1-microglobulin, PTH, vitamin D, and markers of bone turnover were conducted. Eight human kidney biopsies were stained for sclerostin using immunohistochemistry. MAIN OUTCOME MEASURE Urinary excretion of sclerostin was measured. RESULTS Urinary sclerostin excretion increased with declining eGFR (R=-0.75, P<.001), from 10.4 (±12.7) pmol/L in patients with eGFR greater than 90 mL/min per 1.73 m2 (CKD stage 1) to 117.9 (±65.4) pmol/L in patients with eGFR less than 15 mL/min per 1.73 m2 (CKD stage 5, P<.001). Fractional excretion of sclerostin increased with declining eGFR (R=-0.83, P<.001) from 0.45% (±0.6%) in CKD 1 to 26.3% (±17.6%) in CKD 5 (P<.001). Fractional excretion of sclerostin correlated with fractional excretion of α1-microglobulin (R=0.82, P<.001). No association between serum sclerostin and fractional excretion of phosphorus was found in a multivariate analysis. Sclerostin was detected in proximal tubular cells, showing a diffuse cytoplasmic staining pattern. CONCLUSION Increased sclerostin serum levels in CKD patients are not due to decreased renal elimination. On the contrary, renal elimination increases with declining kidney function. Whether this has consequences on antisclerostin antibody dosing, efficacy, or safety in patients with CKD remains to be determined.

Hyperprolactinaemia in hypothyroidism: clinical significance and impact of TSH normalization

objectives Menstrual irregularities in hypothyroidism have been reported to occur less frequently than previously described. We therefore studied the influence of serum PRL in patients with newly diagnosed subclinical and overt hypothyroidism and in hyperprolactinaemic patients treated with T4 to distinguish the impact of hypothyroidism from that of confounding drugs on hyperprolactinaemia and menstrual irregularities.

Effects of T4 replacement therapy on glucose metabolism in subjects with subclinical (SH) and overt hypothyroidism (OH)

Objective  To evaluate β‐cell function and insulin sensitivity in subjects with overt (OH) and subclinical hypothyroidism (SH) before and after T4 replacement therapy.

Influence of tumour necrosis factor-alpha on the expression of Fc IgG and IgA receptors, and other markers by cultured human blood monocytes and U937 cells.

The expression of Fc receptors for IgG (FcγR) and IgA (FcαR) and of various other antigens on the human monocytic cell line U937 and peripheral blood monocytes, under stimulation with human recombinant tumour necrosis factor‐α (TNF‐α) and other cytokines, was investigated by flow cytomelry. TNF‐α, as well as interferon‐γ (IFN‐γ) or interleukin‐6 (IL‐6) had a significant up‐regulating effect on U937 expression of FcγRI/CD64. Furthermore, the action of TNF‐α was augmented by IL‐6, and more evidently by IFN‐γ. IFN‐α alone had only a marginal effect, but was able to increase the TNF‐α‐driven FcγRI expression. In contrast to U937 cells, TNF‐α did not enhance significantly FcγRI expression on human monocytes. Interestingly, on both U937 cells and monocytes. FcαR was augmented markedly by TNF‐α. Furthermore, TNF‐α induced the expression of HLA‐DR and HLA‐DP antigens on monocytes and U937 cells. The expression of FcγRII/CD32, FcγRIII/CD16. CD14, complement receptor type 1 (CR1/CD35). CR4 (CD11c/CD18), and MHC class‐I antigens, was not influenced significantly by TNF‐α. The results of this study show that TNF‐α may act on human mononuclear phagocytes, alone or in combination with other cytokines, by modulating the expression of various cell‐surface antigens.

Thyrotropin Serum Concentrations in Patients with Papillary Thyroid Microcancers

BACKGROUND Recent studies have shown that elevated serum thyrotropin (thyroid-stimulating hormone [TSH]) concentrations are associated with an increased risk of differentiated thyroid cancers in patients with nodular goiter. Therefore, the measurement of TSH concentrations might support the clinical estimation of cancer risk, especially in patients with thyroid nodules that are too small for fine-needle aspiration biopsies. Thus, the objective of this study was to compare preoperative serum TSH concentrations in patients with papillary thyroid microcarcinoma (PTMC) and individuals in whom the presence of even small differentiated thyroid cancers was excluded by thorough histological examination of the thyroid after total thyroidectomy because of medullary thyroid carcinoma or c-cell hyperplasia. METHODS The study was a retrospective cross-sectional analysis. Thirty-three patients with PTMC who had undergone a hemi- or total thyroidectomy and 54 subjects with medullary thyroid carcinoma or c-cell hyperplasia in whom a total thyroidectomy had been performed between 1994 and 2008 were included. Exclusion criteria were the intake of medication that might affect thyroid function and previous thyroid cancer or thyroid surgery. RESULTS The mean TSH value was comparable between patients with PTMCs (1.40 +/- 0.92 mLU/L, 95% CI = 1.07-1.72) and the control group (1.43 +/- 1.44 mLU/L; 95% CI = 1.04-1.82, p = 0.912). There was a positive trend in correlation between nodule size and TSH levels in patients with PTMC (p = 0.066). CONCLUSIONS TSH is not elevated in subjects with PTMCs, indicating that it is not likely involved in the de novo oncogenesis of these small cancers. However, TSH might rather play a role in the progression of preexisting PTMCs.