Branko Malenica

Indirect demonstration of the lifetime function of human thymus

The aim of this study was to test the hypothesis that human thymus maintains its function as the site of early T cell development throughout life, but to a progressively diminishing extent. Mononuclear cell suspensions prepared from the samples of 39 human thymuses were analysed for the total number of cells per gram of thymus tissue, percentage of single marker‐positive CD2, CD4 and CD8 cells, percentages of double‐positive CD4 CD8 and CD2 CD8 cells, double‐negative CD4 CD8 cells, absolute numbers of these cells per gram of tissue, and extent of the in vitro proliferation upon stimulation with concanavalin A (Con A), phytohaemagglutinin (PHA) and pokeweed mitogen (PWM) mitogens. The main outcome measures were flow cytometric data on thymus lymphoid cell composition (according to CD classification), expressed as percentages and numbers of cells per gram of thymus tissue. The total number of mononuclear cells expressed per gram of thymus tissue exponentially decreased with age. The slope of none of the analysed cell subpopulations differed from the slope of the line constructed for age‐related decline of the total number of mononuclear cells (−0.024 on a semilogarithmic scale). The thymuses of all ages contained all analysed cell subpopulations in approximately the same proportions: percentages of these cell subpopulations did not change with age, except for all CD4+ (P = 0.017) and double‐positive CD4+ CD8+ (P = 0.016) cells, which tended to decrease with age. The extent of proliferation of thymus cells upon stimulation with T and B cell mitogens was unrelated to age. We conclude that the thymus retains its function as the site of differentiation of T lymphocytes throughout life. With respect to the number of involved lymphoid cells, the function exponentially decreases with age.

Cytokines and growth factors in mostly atherosclerotic patients on hemodialysis determined by biochip array technology.

Abstract Background: The lifespan of patients with chronic renal failure (CRF) is reduced, and coronary artery disease is the leading cause of morbidity and mortality in these patients. The progression of atherosclerosis is accelerated and angiogenesis is impaired in CRF. Risk factors that could contribute to further understanding of vascular pathology include markers of inflammation and growth factors. The purpose of this study was to determine the levels of cytokines (IL-2, IL4, IL-6, IL-8, IL-10, IL-1α, IL-1β), vascular endothelial growth factor (VEGF), epidermal growth factor (EGF), interferon-γ (IFNγ), tumor necrosis factor-α (TNFα) and monocyte chemotactic protein-1 (MCP-1) in patients on chronic hemodialysis (HD; n=75), and to compare values with those of control subjects (n=113). Methods: Evidence® biochip array analyzer was used for quantification of plasma concentrations in samples. Results: Significant differences were found between the control subjects and HD patients. IL-2 (p<0.001), IL-4 (p<0.001) and EGF (p<0.001) levels were higher in controls than in HD patients, while IL-6 (p<0.001), IL-8 (p=0.081), IL-10 (p=0.008), TNFα (p<0.001), IL-1β (p<0.001) and MCP-1 (p<0.001) levels were higher in HD patients. We also found IL-2 (p=0.015) and IL-1α (p=0.035) levels to be significantly higher in males than females, while IL-4 (p=0.025) and IL-1β (p=0.049) levels were significantly higher in females. Among HD patients, IL-2 levels were higher in patients under the age of 50 years (p<0.048). It was also higher in female than in male patients (p<0.035) and in patients on HD for more than 10 years (p<0.009). IL-6 levels were higher in patients over the age of 50 years (p<0.047). Patients with previous glomerulonephritis had the highest level of IL-6 compared to patients with previous pyelonephritis and diabetes mellitus (p<0.063). IL-6 levels were higher in patients with concomitant hepatitis C virus (HCV) infection (p<0.036) and in patients with developed atherosclerosis (p<0.003). IL-8 levels were higher in patients over the age of 50 years (p<0.003) and in the group with previous glomerulonephritis (p<0.031). IL-10 levels were higher in the group with developed atherosclerosis (p<0.045). EGF was the highest in the group of patients with previous diabetes mellitus compared to pyelonephritis and glomerulonephritis groups (p<0.073). TNFα levels were higher in the patient population on HD for more than 10 years (p<0.032) and in the concomitant HCV group (p<0.073). IL-1β levels were higher in the HCV group (p<0.088). Conclusions: Plasma concentrations of some cytokines and growth factors could serve as useful diagnostic and prognostic parameters for patients with CRF on HD. Clin Chem Lab Med 2007;45:1347–52.

Effect of spinal and general anesthesia on serum concentration of pro-inflammatory and anti-inflammatory cytokines.

BACKGROUND Surgery induces release of neuroendocrine hormones, cytokines and acute phase proteins. The aim of this study was to assess the effect of spinal and general anesthesia on serum concentration of pro-inflammatory and anti-inflammatory cytokines, and cytokines which are secreted by Th1 helper lymphocytes. METHODS 30 patients with American Society of Anesthesiologists status I and II who were scheduled for TURP (Transurethral Resection of the Prostata) were anesthetized in regional (spinal) or general anesthesia. Peripheral venous blood samples were collected 2 h before surgery on the first, third and fifth postoperative days. We measured pro-inflammatory cytokines, anti-inflammatory cytokines and cytokines which are secreted by Th1 helper lymphocytes in order to establish differences in patients before and after surgery. RESULTS Statistically significant differences were found in serum levels of interleukin-2 (IL-2) between general and spinal anesthesia (p=0.043). The concentration of IL-2 was continuously elevated in general anesthesia, but not in spinal anesthesia. It is important to note that the preoperative serum IL-2 concentration in general anesthesia group was significantly higher in comparison to spinal anesthesia group (p=0.028). There was also statistically significant increase of interleukin-6 (IL-6) in spinal (p=0.043) and general anesthesia (p=0.03) in comparison to preoperative value. CONCLUSION Surgery-related postoperative release of the pro-inflammatory cytokine IL-6 was increased in patients after spinal and general anesthesia. In our study, increased levels of the typical Th1 cytokine IL-2 were found in patients anesthetized by general anesthesia compared to spinal anesthesia. Serum concentrations of other pro-inflammatory cytokines, anti-inflammatory cytokines and cytokines which are secreted by Th1 helper lymphocytes showed no statistical difference before and after surgery under general and spinal anesthesia.

Monocyte response to LPS after exposure to corticosteroids and chloroquine with implications for systemic lupus erythematosus

Essential part of a response to infection is early pathogen recognition and adequate initiation of innate immunity. One of the hallmarks of systemic lupus erythematosus (SLE) is reduced resistance to infection despite overall hyperactivity of the immune system. Immunosuppressive drugs (high‐dose corticosteroids and cytotoxic agents) are independent risk factors for infection in SLE, with bacteria as predominant cause. To investigate whether less aggressive immunomodulatory treatment may still affect recognition and response to Gram‐negative bacteria, we measured TLR4 expression in monocytes of untreated SLE patients and patients on chloroquine and low‐dose steroid therapy and examined the drugs’ influence on monocyte TLR4 expression in peripheral blood mononuclear cell (PBMC) culture. Additionally, we determined whether induction of monocyte NF‐κB signalling, TNF‐α and IL‐6 production with lipopolysaccharide (LPS), a TLR4 ligand, can be altered with dexamethasone, chloroquine or both. There was no statistically significant difference in TLR4 expression between patients with SLE and controls, even though treated SLE patients tended to have lower frequency of TLR4+ monocytes and TLR4 mean fluorescence intensity than healthy controls. However, neither dexamethasone nor chloroquine had major influence on TLR4 expression in vitro or suppressed LPS‐induced NF‐κB activation in monocytes, although dexamethasone decreased TNF‐α and IL‐6 production. Therefore, even if low‐dose steroids or chloroquine do not seem to affect TLR4 expression and signalling, steroids might decrease cytokine production in response to LPS.

Decrease in circulating DNA, IL-10 and BAFF levels in newly-diagnosed SLE patients after corticosteroid and chloroquine treatment

Arsenal of pattern-recognition receptors alongside antibody production machinery make B cells vulnerable to autoimmune response if an autoantigen elicits both pathways in a self-sustained fashion. Systemic lupus erythematosus is an autoimmune disease characterized by autoantibodies to DNA, RNA and related structures. Murine studies demonstrated autoreactive B cell activation upon TLR9 stimulation with DNA-containing immune complexes. This activation could be abolished with chloroquine, a drug used in SLE treatment that also blocks TLR9 signaling. We investigated whether chloroquine modulates TLR9 expression, circulating DNA levels and B cell-related cytokines in newly discovered, untreated SLE patients. TLR9 was measured in peripheral blood B cells by flow cytometry, serum DNA by real-time PCR, and IL-10 and BAFF by ELISA before treatment, after 3weeks on corticosteroids, and 3months after introduction of chloroquine. We found that circulating DNA is higher in SLE patients than in controls in every time-point and decreases significantly after chloroquine treatment. Untreated patients had higher serum IL-10 than controls or patients on corticosteroids. Also, corticosteroids decreased and chloroquine completely abolished CpG-mediated CD86 upregulation on B cells and IL-10 secretion in PBMC culture. Providing the TLR9 pathway activation demonstrates its importance in pathogenesis of human SLE, this data supports continuation of chloroquine in SLE treatment protocol. In addition, observed modulation of cytokine and DNA levels after immunomodulatory treatment prompts for inclusion of untreated patients in studies of human immune disorders.

Imunological and clinical characteristics in children with polyarteritis nodosa: a retrospective study over the last 20 years

Results PAN was diagnosed in 12 patients (6 girls and 6 boys). The share of PAN amongst all vasculitides was 4%. The mean age at disease onset was (±SD) 11.33±3,08 years. Systemic PAN was diagnosed in 7 children (58%), microscopic polyangiitis in 3 (25%), cutaneus PAN in 2 (17%) and classic PAN in 0 (0%). The most consistent symptoms were skin involvement (90%) and arthritis/ arthralgia (60%). The CNS was affected in 40% of patients. ESR and CRP were elevated in all patients. Antineutrophil cytoplasmic antibodies were elevated in 3 patients (25%). Antistreptolysin O was elevated in 4 patients (25%). The relation between the severity of skin involvement and involvement of other organs was not found. Therapy mode for all patients was corticosteroids. Immunosuppressive drugs and Rituximab (antiCD20) were used as additional therapy for patients with severe symptoms. Two patients with microscopic polyangiitis died due to chronic renal and pulmonary failure during the follow-up. Conclusion In comparison to available studies, we found a difference in distribution of childhood polyarteritis nodosa as well as some clinical characteristics (e.g. higher prevalence of neurological symptoms), while other researched features, laboratory and treatment, were similar.

Binding of Anti-Double Stranded (ds) DNA-Positive Sera to Denatured (d) DNA and Synthetic Poly[dA-dT] x Poly[dA-dT] Double Stranded Copolymer in an ELISA Format

Using an ELISA assay anti-nuclear antibody-positive sera from 300 patients with various immune-related diseases and 64 anti-nuclear antibody-negative sera were analysed for binding to S1-nuclease-treated double stranded (ds) DNA. In addition, the pattern of reactivity of 50 selected anti-dsDNA-positive sera was established using denatured (d) DNA and poly[dA-dT] X poly[dA-dT] double-stranded alternating copolymer (dAT) as additional DNA antigens. None of the 64 anti-nuclear antibody-negative sera and 76 of the 300 anti-nuclear antibody-positive sera (25%) were anti-dsDNA-positive. Of the anti-nuclear antibody-positive and anti-dsDNA-positive sera, 48 (63%) were from systemic lupus erythematosus patients, and 7 (9%) from rheumatoid arthritis patients, whereas 21 patients (27.6%) suffered from various immune and non-immune related diseases. Anti-dsDNA-positive reactivity was highly correlated with dDNA and dAT reactivity (r = 0.906, p < 0.0001 and r = 0.93, p < 0.0001, respectively). Although the majority of the 50 selected (37 systemic lupus erythematosus and 13 non-systemic lupus erythematosus) anti-dsDNA-positive sera concomitantly bound to both additional antigens, 7 of these (14%) did not bind to dAT, and 2 (4%) did not bind to dDNA. Anti-dsDNA-positive sera (n = 37) showed a similar pattern, in which 8.1% and 2.7% of sera did not bind to dAT and to dDNA, respectively. In contrast, anti-dsDNA-negative sera from various immune-related diseases bound either ssDNA (12.5%) or dDNA and dAT (12.5%). These data suggest that dsDNA and dAT-based assays detect similar but not identical specificities in the sera of patients suffering from systemic lupus erythematosus and in a proportion of non-systemic lupus erythematosus patients.