Cytokine profile in patients with acute bacterial meningitis.

BACKGROUND: Bacterial meningitis is a life-threatening disease with high mortality and common long-term sequelae. The inflammatory response in the subarachnoid space, modulated by different cytokines, plays a major role in the pathogenesis of acute central nervous system infections. We aimed to examine correlations of interleukin (IL)-6, IL-8, IL-10, IL-12(p40), and tumor necrosis factor (TNF)-α levels with disease severity, complications, and outcome in patients with acute bacterial meningitis. METHODS: The study involved 30 patients with bacterial meningitis/meningoencephalitis admitted to the University Hospital St. George, Plovdiv over a period of 4 years. Patients were selected based on clinical presentation and laboratory abnormalities, consistent with a neuroinfection. Enzyme-linked immunosorbent assay was used to measure the studied cytokines in both cerebrospinal fluid (CSF) and serum in parallel. For microbiological diagnosis multiplex, polymerase chain reaction, and CSF culture were used. RESULTS: In patients with acute bacterial meningitis CSF levels of IL-6, IL-8, IL-10, and TNF-α are significantly increased than in serum. CSF TNF-α, CSF IL-8, and CSF IL-10 had a moderate negative correlation to CSF glucose. It was found that serum IL-8 is significantly elevated in patients who experienced neurological complications, have severe clinical course, and in deceased patients. CSF IL-10 is increased only in patients with severe acute bacterial meningitis. CONCLUSION: Among patients with acute bacterial meningitis serum IL-8 could delineate these with increased risk of neurological complications, severe clinical course, and fatal outcome. Serum IL-8 and CSF IL-10 could be used as indicators of disease severity.

Quantitative system for diagnosis of vulvovaginal candidiasis.

INTRODUCTION: Vaginal infections are one of the most common reason for gynecological consultations. Many of them are the result of overgrowth of resident microorganisms. The clinical symptoms of vulvovaginal candidiasis are nonspecific and an accurate diagnosis is a problem that often leads to inadequate treatment or delays in treatment. The lack of an exact and practical diagnostic method is a common cause of misdiagnosis. AIM: To create a complex, quantitative method for the diagnosis of vulvovaginal candidiasis which to enables differentiation from vaginal fungal colonization. MATERIAL AND METHODS: A total of 2306 vaginal samples were examined. Clinical, microbiological, epidemiological methods and statistical models are used. RESULTS AND DISCUSSION: The proposed score system is a specific, sensitive and inexpensive method to routinely diagnose vulvovaginal candidiasis. Statistical processing of the obtained data shows the impact of the individual components on which the method is based: the presence of vaginal discharge, pruritus, direct microscopy and assessment of the fungal growth. The data analysis reveals good sensitivity (71%) and high specificity (98%) of the method. This allows accurate interpretation of the result of the clinical and microbiological examination of each patient. CONCLUSION: The system for diagnosing vulvovaginal candidiasis is complex and based on quantitative indicators. The method can be used to differentiate vulvovaginal candidiasis from vaginal fungal colonization (the cut-off value is 5.5 points) and to more accurately interpret a Candida positive result from quantitative real-time PCR in asymptomatic patients or in women with mixed vaginal infection.

Combined testing of cerebrospinal fluid IL-12 (p40) and serum C-reactive protein as a possible discriminator of acute bacterial neuroinfections.

INTRODUCTION: Central nervous system infections (CNS) are life-threatening diseases, with meningitis being the most common. Viral infections are usually self-limiting diseases but bacterial pathogens are associated with higher mortality rates and persistent neurological sequelae. We aimed to study the role of IL-6, IL-8, IL-10, IL-12(p40), TNF-α cytokines, classical cerebrospinal fluid (CSF) parameters, and serum C-reactive protein levels (CRP) for discriminating bacterial from viral central nervous system infections. MATERIAL AND METHODS: This prospective study included 80 patients with clinical signs and abnormal cerebrospinal fluid laboratory findings typical for neuroinfection admitted to St. George University Hospital-Plovdiv. Routine methods such as direct microscopy, culturing and identification were used for microbiological analysis as well as latex-agglutination test and multiplex PCR. Cytokines' concentrations were measured by ELISA. CRP and CSF parameters were collected from the patients' medical records. RESULTS: We observed the highest discriminatory power among cytokines for cerebrospinal IL-12(p40) (AUC = 0.925; p = 0.000). CSF protein levels were the best predictor for bacterial neuroinfection (AUC = 0.973; p = 0.000). The AUC for the serum CRP as a stand-alone biomarker was estimated to be 0.943. The discriminatory power can be increased up to 0.995 (p = 0.000) when combining cerebrospinal fluid IL-12(p40) and serum CRP, with an optimal cut-off value of 144 (Sensitivity 100%; Specificity 90.9%). CONCLUSION: The combined testing of CSF IL-12(p40) and serum CRP is associated with the highest diagnostic accuracy.

Diagnosis of spontaneous and secondary bacterial peritonitis in patients with hepatic cirrhosis and ascites.

Spontaneous bacterial peritonitis (SBP) is an infection that is not caused by intra-abdominal source requiring surgery. Nowadays SBP is the main cause of death in patients with cirrhosis. Treatment is carried out with third generation cephalosporins and albumin infusions. The aim of the study is to identify patients with SBP and to be distinguished from the cases with secondary bacterial peritonitis (SecBP) in patients with cirrhosis and ascites. We studied 167 patients with cirrhosis and ascites and SBP was observed in 25 of them, while SecBP--in 22. The diagnosis of SBP is set in neutrophilic leukocytes in ascites > or = 250 cells/mm3 as bacterial cultures are positive in only 16% of them. Completely asymptomatic course had 16% of patients with SBP. Diagnosis of SecBP (according to Runyon's criteria) is based on increased total protein in ascitic fluid > 10 g/l (in 63.7% of patients > 30 g/l), elevated lactate dehydrogenase in ascites (LDH is > 240 U/l in all patients) and glucose < 2,7 mmol/l (only 4.5% of cases with secondary bacterial peritonitis). In support of SecBP are the polymicrobial flora, the isolation of anaerobes, enterococci, fungi, and the very high number of neutrophilic leukocytes in the peritoneal effusion and the refractoriness from conservative treatment. The examination of ascites with Multistix is more informative in secondary than in spontaneous bacterial peritonitis. In suspected secondary bacterial peritonitis CT is indicated.

Changes in plasma levels of acute phase proteins in pancreatitis.

UNLABELLED: The objective of our study was to determine whether the changes in the plasma levels of C-reactive protein, haptoglobin, transferrin, and alpha 1-acid glycoprotein (orosomucoid) in acute pancreatitis patients examined on days 1, 3, 5, 7, 9, and 14 after their admission to hospital can be used to assess the course and determine the severity of the disease, and to discriminate edematous from necrotizing pancreatitis. The study included 18 patients with mild pancreatitis and 20 patients with severe pancreatitis. Pancreatitis was classified as mild or severe according to Ranson and the Acute Physiology and Chronic Health Evaluation (APACHE) II scores. Acute phase proteins were determined using the immunoturbidometric assay. The C-reactive protein in the mild pancreatitis patients was six-fold higher at admission, then gradually increased for 5 days and started to drop. In the severe clinical forms of pancreatitis the C-reactive protein had significantly high values throughout the whole time of study retaining this high level to day 14; these elevated levels correlated with the persistent severe general state of the patients and the extent of necrosis in the pancreas. The changes in the haptoglobin and transferrin levels were not significant. Orosomucoid level in mild pancreatitis cases remained constant whereas it was elevated and continuously above the reference values in severe pancreatitis. IN CONCLUSION: C-reactive protein level changes significantly in patients with acute pancreatitis; it is in the range of 100-120 mg/l in the mild forms of the disease and between 120 and 360 mg/l in the severe pancreatitis. This renders it a valuable indicator for the discrimination of edematous from necrotizing acute pancreatitis.

In vitro study on cytotoxic effect of some chemicals on serum McCoy and serum-free McCoy-Plovdiv cell culture systems.

The cytotoxicity of test agents on serum-free McCoy cultures has not been studied at all. The cytotoxic effect of EDTA, methanol, DMSO, and cycloheximide on serum-free McCoy-Plovdiv cell culture (SF) was detected visually on inverted microscope and quantitatively by tests for viability (NR) and total protein (KBP). The IC50 values for the tested chemicals were calculated. SF showed the lowest IC50 values for cycloheximide, DMSO and EDTA and the highest for methanol according to both tests. EDTA, methanol, DMSO and cycloheximide had dose-effect relationship in the cell test systems after treatment. The data indicate that McCoy-Plovdiv cell line is a suitable serum-free cell system for in vitro cytotoxic studies.

Study on proinflammatory cytokines (IL-1 beta, IL-6, TNF-alpha) and IL-2 in patients with acute hepatitis B.

Cytokine production in patients with hepatitis B may be related either to multiple immune abnormalities or to favourable outcome of the disease. Serum levels of IL-1 beta, IL-2, IL-6 and TNF-alpha were determined dynamically by ELISA kits in patients with self-limited form of acute hepatitis B infection (A-HBV) during the first, second and third decade after icterus appearance. IL-1 beta, IL-6 and TNF-alpha had characteristically high levels in patients measured in all decades, but these were the highest during the first. Then they gradually decreased, reaching normal values at the stage of convalescence. Serum IL-2 levels were found to be most significantly elevated during the second decade and also dropped to normal values in the course of the disease. Patients who cleared HBsAg on the third month after dehospitalisation had higher mean values of IL-2, IL-6 and TNF-alpha in comparison with those who were still HBsAg carriers, thus indicating that proinflammatory cytokine production in self-limited HBV may be important for viral clearance.

Susceptibility and resistance to antigen-induced apoptosis in the thymus of transgenic mice.

Injection of TCR transgenic mice with antigenic peptide results in the deletion of immature thymocytes expressing the transgenic TCR. We have analyzed this process in mice transgenic for a TCR (F5) that recognizes a peptide from the influenza nucleoprotein (NP68). To determine whether deletion of immature thymocytes is the result of specific recognition of the antigenic peptide by the thymocytes or mature T cell activation, bone marrow chimeric mice were generated using a mixture of cells from F5 transgenic and nontransgenic mice. Injection of these mice with antigenic peptide leads to the preferential depletion of F5 transgenic thymocytes, whereas nontransgenic thymocytes remain largely unaffected. Furthermore, exposure of F5 fetal thymic lobes to peptide leads to thymocyte deletion even though no mature single positive T cells are present at this stage. These data suggest that Ag-induced death of immature thymocytes is due to peptide-specific recognition, although activated mature T cells appear to potentiate such deletion. Further administration of antigenic peptide to F5 mice results in the appearance of double-positive thymocytes that are resistant to Ag or anti-CD3-induced apoptosis. These data suggest a change in the ability of the cells to signal through the TCR-CD3 complex, resembling the state of anergy induced in peripheral T cells following chronic exposure to Ag.

Inhibition of thymocyte negative selection by T cell receptor antagonist peptides.

The T cell receptor (TCR) recognizes antigenic peptide presented by major histocompatibility complex (MHC) molecules. Analogs of antigenic peptides have been shown to inhibit antigen-specific T cell responses, a phenomenon described as TCR antagonism. We have examined the effect of a natural variant of an antigenic peptide and a synthetic peptide analog, on the responses of mature T cells and immature thymocytes from an alpha-beta TCR-transgenic mouse (F5), the TCR of which recognizes a nonamer peptide from the nucleoprotein (NP) of influenza virus in the context of the H-2Db MHC molecule. Both peptides were shown to antagonize specifically the T cells cytolytic response without being able directly to stimulate mature T cells from these transgenic mice. Furthermore, a negative selection assay in vitro was used to demonstrate for the first time that antagonistic peptides are capable of antagonizing thymocyte deletion induced by antigenic peptides. These data suggest that the final selection of a T cell could be the result of a balance between the positive and negative influences of endogenous peptide ligands.

Inhibition of I-Ad-, but not Db-restricted peptide-induced thymic apoptosis by glucocorticoid receptor antagonist RU486 in T cell receptor transgenic mice.

Thymocytes differentiate by positive and negative selection of immature CD4+ CD8+ T cells. Negative selection occurs by default or by high-affinity recognition of peptides bound to proteins encoded by the major histocompatibility complex (MHC). MHC class I molecules are expressed on many different cell types, although at different levels, whereas MHC class II molecules are selectively expressed on thymic epithelial cells (TEC) and dendritic cells (DC). We investigated the role of the glucocorticoid receptor (GR) in thymic negative selection using the receptor antagonist RU486. Glucocorticoids (GC) are known to be potent inducers of apoptosis in CD4+ CD8+ thymocytes, and we have earlier shown that anti-CD3-induced thymic apoptosis can be blocked by RU486 in vivo. We now show that anti-CD3 induces thymic apoptosis in mice that have been adrenalectomized (ADX), and that RU486 inhibits anti-CD3 antibody-mediated thymocyte killing in newborn thymic organ cultures. Thymocyte apoptosis induced by ovalbumin peptide OVA323-339 treatment of mice transgenic for the DO11.10T cell receptor (TCR), which recognizes this peptide in the context of I-Ad, was found to be inhibited by RU486. These mice responded to peptide treatment by an extensive activation of the peripheral immune system, which became lethal in 60% of the mice when accompanied by simultaneous RU486 treatment. In contrast, RU486 had no effect on thymic apoptosis induced by the influenza A nucleoprotein NP366-374 peptide, recognized in context of Db, in F5 TCR transgenic mice. We interpret the results to demonstrate that different deletion systems operate in the thymus. We propose that endogenous GC may be important for negative selection by default and by high-affinity recognition of endogenous MHC-presented peptides on TEC.

The programmed cell death of an immature thymocyte cell line transgenic for an alpha beta TCR and the c-myc proto-oncogene.

The c-myc proto-oncogene linked to the mouse Thy-1 gene transcriptional unit predisposes mice to development of thymic tumors consisting predominantly of immature CD4+ CD8+ cells. In an attempt to immortalize immature T cells expressing a known T-cell antigen receptor (TCR), Thy-1/c-myc transgenic mice were bred to alpha beta TCR transgenic mice (F5), and CD4+ CD8+ cell lines were established from thymic tumors in double-transgenic mice. These cells expressed high-level heat-stable antigen (HSA) and were able to undergo programmed cell death upon induction with steroids and CD3 cross-linking, but not with cognate peptide. In addition, one line had rearranged and transcribed endogenous TCR alpha and beta genes, in spite of the fact that transgenic alpha and beta genes were also expressed. Furthermore, we show that Thy-1/myc transgenic mice deficient in recombination activating gene-1 (RAG-1) do not develop tumors, in contrast to RAG-1-/- mice, which are also transgenic for both Thy-1/myc and the F5 TCR. This indicates that in order for thymocytes to be transformed by the Thy-myc transgene, they need to proceed to the double-positive stage.

Tolerance in TCR/cognate antigen double-transgenic mice mediated by incomplete thymic deletion and peripheral receptor downregulation.

Influenza nucleoprotein (NP)-specific T-cell receptor transgenic mice (F5) were crossed with transgenic mice expressing the cognate antigenic protein under the control of the H-2Kb promoter. Double-transgenic mice show negative selection of thymocytes at the CD4+8+TCRlo to CD4+8+TCRhi transition stage. A few CD8+ T cells, however, escape clonal deletion, and in the peripheral lymphoid organs of these mice, they exhibit low levels of the transgenic receptor and upregulated levels of the CD44 memory marker. Such cells do not proliferate upon exposure to antigen stimulation in vivo or ex vivo, however, they can develop low but detectable levels of antigen-specific cytotoxic function after stimulation in vitro in the presence of IL-2.