Comparison of the ImmuView and the BinaxNOW antigen tests in detection of Streptococcus pneumoniae and Legionella pneumophila in urine.

The use of urinary antigen tests (UATs) may provide early etiology in pneumonia, and facilitates rapid and directed antibiotic treatment. In this study, we evaluated the novel lateral flow ImmuView Streptococcus pneumoniae and Legionella pneumophila UAT, which detects pneumococcal and L. pneumophila serogroup 1 antigens in a combined test. We compared the ImmuView UAT with the BinaxNOW S. pneumoniae UAT and the BinaxNOW L. pneumophila UAT in 147 patients with pneumococcal bacteremia (n = 48), non-pneumococcal non-Legionella bacteremia (n = 93) and Legionella infections in the lower airways (L. pneumophila, n = 5; L. bozemanii, n = 1). In three cases, the ImmuView test was invalid before and after boiling while the BinaxNOW tests were valid in all cases. In 144 cases, the three UATs demonstrated a very good inter-assay agreement for detection of pneumococcal antigen (κ = 0.86) and L. pneumophila antigen (κ = 1.00). The ImmuView and BinaxNOW S. pneumoniae tests had similar sensitivities (62% vs 60%; p = ns) in 48 cases with pneumococcal bacteremia and both tests had specificities of 97% in 96 cases with non-pneumococcal infections. Furthermore, the ImmuView and BinaxNOW L. pneumophila tests were positive for Legionella antigen in five patients with confirmed L. pneumophila serogroup 1 infections, and negative in all non-L. pneumophila cases. The ImmuView and BinaxNOW tests performed similarly when evaluated on urine samples from bacteremic and non-bacteremic patients with identified etiology.

Genomic analysis reveals the presence of a class D beta-lactamase with broad substrate specificity in animal bite associated Capnocytophaga species.

Capnocytophga canimorsus and Capnocytophga cynodegmi can be transmitted from cats and dogs to humans, and can cause a wide range of infections including wound infections, sepsis, or endocarditis. We and others recently discovered two new Capnocytophaga species, C. canis and C. stomatis, mainly associated with wound infections. The first-line treatment of animal bite related infections is penicillin, and in case of allergy, doxycycline and trimethoprim/sulfamethoxazole. However, there is a lack of antibiotic susceptibility patterns for animal bite associated Capnocytophaga species. Thus, we set out to study the antibiotic profiles against animal bite associated Capnocytophaga species isolated from wound and blood cultures after cat and dog bites and coupled the findings to whole genome sequencing data. A total of 24 strains were included in the study. Phenotypic analysis of antibiotic resistance was performed with E-tests. The web-based tool 'Resfinder' was used to identify resistance genes in the whole genome dataset. Two strains of C. cynodegmi and two strains of the recently discovered C. stomatis were resistant to penicillin (MIC > 24 mg/L) and cephalosporins (MIC > 24 mg/L), and three out of these strains also exhibited resistance to imipenem (MIC = 32 mg/L). Genomic analysis revealed that these strains carried a class D beta-lactamase gene, which has not previously been found in Capnocytophaga spp. A class D beta lactamase with broad substrate specificity was found in animal bite associated Capnocytophaga species, which could have important implications when treating wound infections after cat and dog bites. It also suggests that pet animal bacteria can harbour resistance genes with relevance for human infections.

Rapid identification of Streptococcus pneumoniae in blood cultures by using the ImmuLex, Slidex and Wellcogen latex agglutination tests and the BinaxNOW antigen test.

Rapid identification of Streptococcus pneumoniae in blood culture (BC) bottles is important for early directed antimicrobial therapy in pneumococcal bacteraemia. We evaluated a new latex agglutination (LA) test on BC bottles, the ImmuLex™ S. pneumoniae Omni (Statens Serum Institut, Denmark), and compared the performance with the Slidex® pneumo-Kit (bioMérieux, France) and the Wellcogen™ S. pneumoniae (Remel, UK) LA tests, as well as the BinaxNOW® S. pneumoniae (Alere, USA) antigen test. The four tests were directly applied on 358 positive BC bottles with Gram-positive cocci in pairs or chains and on 15 negative bottles. Valid test results were recorded in all cases for ImmuLex and BinaxNOW and in 88.5 % (330/373) and 94.1 % (351/373) of cases for Slidex and Wellcogen, respectively. Based on bottles positive for S. pneumoniae by conventional methods, the sensitivity of ImmuLex was 99.6 %, similar to the other tests (range, 99.6-100 %). Based on bottles positive for non-pneumococcal pathogens, the specificity of ImmuLex was 82.6 %, in comparison to 97.6 % for Slidex (p < 0.01) and 85.4 % for Wellcogen (p = ns). The BinaxNOW test had a lower specificity (64.1 %) than any LA test (p < 0.01). On BC bottles positive for α-haemolytic streptococci, ImmuLex was positive in 12/67 (17.9 %) cases, Slidex in 2/59 (3.4 %) cases, Wellcogen in 11/64 (17.2 %) cases and BinaxNOW in 25/67 (37.3 %) cases. In conclusion, the ImmuLex test provides a valid and sensitive technique for the rapid detection of S. pneumoniae in BC bottles, similar to the other compared methods. However, the specificity was sub-optimal, since the test may cross-react with other Gram-positive bacteria.

The origin of endodontic Enterococcus faecalis explored by comparison of virulence factor patterns and antibiotic resistance to that of isolates from stool samples, blood cultures and food.

AIM: To elucidate the origin of Enterococcus faecalis isolated from secondary root canal infections and the possibility for a foodborne transmission by comparing them to strains recovered from food, blood and stool regarding putative virulence factors and antibiotic susceptibility profiles, where strains from common origin were hypothesized to harbour similar characteristics. METHODOLOGY: A total of 108 E. faecalis strains recovered in the county of Stockholm, Sweden, were screened using PCR for putative virulence factors esp, cylA, gelE/gelatinase-negative phenotype (ef1841/fsrC), efaA, ace and asa1. The minimum inhibitory concentration (MIC) for ampicillin, piperacillin-tazobactam, imipenem, gentamicin, vancomycin, ciprofloxacin and linezolid was determined using the agar dilution method. RESULTS: Next to strains from blood, the food isolates presented the highest average number of virulence determinants and were frequently enriched with asa1 coding for aggregation substance. None of the endodontic strains carried cylA, and the gelatinase-negative phenotype caused by a deletion dominated the group. Altogether, the most prevalent genes were gelE, efaA and ace, and a combination of them was equally present in approximately 80% of the strains from food, stool and root canals in comparison with 43.3% of the blood isolates. High-level resistance to ciprofloxacin and gentamicin was observed in 30% of the blood isolates, whereas the isolates from other origins, with single exceptions, were susceptible to all tested antibiotics. CONCLUSIONS: Evidence for a foodborne transmission, explaining the high reported prevalence of E. faecalis in root filled teeth, could not be determined based on the similarities in virulence factor patterns and antibiotic susceptibility. The only linkage between isolates from food and root canals consisted of a shared common combination of the genes gelE, efaA and ace. The high occurrence of putative virulence traits in food isolates questions the safety of E. faecalis in food products.

The Uni-Gold™ Streptococcus pneumoniae urinary antigen test: an interassay comparison with the BinaxNOW® Streptococcus pneumoniae test on consecutive urine samples and evaluation on patients with bacteremia.

The performance of the recently commercialized Uni-Gold™ Streptococcus pneumoniae test for the detection of pneumococcal antigen in urine was studied in a multicenter study. First, we studied the interassay agreement between Uni-Gold™ and the BinaxNOW® S. pneumoniae urinary antigen test on 337 consecutive urine samples sent to the laboratory for the detection of pneumococcal antigen. The two tests performed similarly (κ = 0.82): both tests positive in 27 cases, both tests negative in 299 cases, and with divergent test results in 11 cases. Secondly, the tests were run on urine samples from 203 patients with bacteremia, including 51 patients with pneumococcal bacteremia. The sensitivities and specificities were 67 and 86 % for Uni-Gold™, and 57 % and 94 % for BinaxNOW®, respectively. The false-positivity rate was significantly higher for Uni-Gold™ compared with BinaxNOW® in patients with Escherichia coli bacteremia (15 vs. 2.1 %, p = 0.04), and tended to be higher in patients with bacteremia with alpha-hemolytic streptococci (32 vs. 11 %, p = 0.13). When cases with E. coli and alpha-hemolytic streptococci were excluded from the analysis, the overall false-positivity rate was 9/85 (11 %) for Uni-Gold™ and 6/85 (7.1 %) for BinaxNOW®. In conclusion, the study showed that Uni-Gold™ was not inferior to BinaxNOW® for the detection of pneumococcal urinary antigen in patients with pneumococcal bacteremia. The specificity of Uni-Gold™ was suboptimal due to false-positive results in cases with E. coli and alpha-hemolytic streptococci bacteremia. However, in patient populations usually subjected to testing for pneumococcal urinary antigen, such as pneumonia and meningitis patients, bacteremia with these pathogens is uncommon. The diagnostic usefulness of the Uni-Gold™ test should be further evaluated.

Identification of blood and wound isolates of C. canimorsus and C. cynodegmi using VITEK2 and MALDI-TOF.

Capnocytophaga canimorsus and C. cynodegmi are gram negative bacteria that can be transmitted to humans from dogs or cats and cause serious infections. Routine bacteriological methods, including fermentation and phenotypic tests are insufficient to correctly identify C. canimorsus or C. cynodegmi. The aim of this study was to evaluate the performance of VITEK2 and MALDI-TOF in identification of these bacteria. Twenty two isolates that were identified as C. canimorsus / C. cynodegmi by 16S rRNA sequencing were included in the study and were further investigated with VITEK2 and MALDI-TOF. A Capnocytophaga species-specific PCR was used as the reference method. Out of 22 included isolates, the species-specific PCR identified six blood isolates as C. canimorsus and 14 wound isolates as C. cynodegmi. Two isolates could not be identified with the reference method. VITEK2 identified 10/20 isolates correctly to Capnocytophaga spp. MALDI-TOF analysis correctly identified 6/6 C. canimorsus and 13/14 C. cynodegmi isolates. The mean time to identification with VITEK2 was 6 hours whereas MALDI-TOF required approximately 10 minutes per sample. Here we show that MALDI-TOF rapidly identified C. canimorsus and C. cynodegmi and thus constitutes a valuable diagnostic tool in the clinical laboratory.

Comparison of the two blood culture systems, Bactec 9240 and BacT/Alert 3D, in the detection of Candida spp. and bacteria with polymicrobial sepsis.

The purpose of this investigation was to evaluate the performance of the Bactec 9240 and BacT/Alert 3D blood culture systems in the detection of Candida spp. and bacteria in simulated polymicrobial sepsis models. A total of 28 clinical isolates of Escherichia coli, Staphylococcus aureus, Candida albicans, and Candida glabrata were studied. Five polymicrobial models of C. albicans + S. aureus, C. albicans + E. coli, C. glabrata + S. aureus, C. glabrata + E. coli, and C. albicans + C. glabrata were prepared. Each combination was inoculated in five different blood culture vials. The two systems were compared for culture positivity and time to detection (TTD). Twenty-four mixed cultures with a yeast and a bacteria were tested. Bactec Mycosis vials could detect yeasts in all 24 cultures. The aerobic vials from both Bactec and BacT/Alert could detect both yeasts and bacteria in 22/24 (91.66 %) cultures. Bactec Plus Anaerobic/F and BacT/Alert FN vials could detect both microorganisms in 19/24 (79.16 %) and 4/24 (16.67 %) vials, respectively. Seven polymicrobial sepsis models with C. albicans + C. glabrata were also tested. Mycosis vials could detect both yeasts in 7/7 mixed cultures. The aerobic vials from Bactec and BacT/Alert could detect both yeasts in 3/7 and 2/7 mixed cultures, respectively. Bactec Plus Aerobic/F had a shorter TTD compared to BacT/Alert FA and Bactec Plus Anaerobic/F vials (p < 0.0001 and p < 0.01, respectively). The present study shows that the Bactec and BacT/Alert systems have different characteristics in the detection of yeasts and bacteria with polymicrobial sepsis.

Evaluation of species-specific PCR, Bruker MS, VITEK MS and the VITEK 2 system for the identification of clinical Enterococcus isolates.

The purpose of this investigation was to compare the performance of species-specific polymerase chain reaction (PCR), matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) and phenotypic identification systems for the identification of Enterococcus species. A total of 132 clinical isolates were investigated by the following: (1) a multiplex real-time PCR assay targeting ddl Enterococcus faecium, ddl Enterococcus faecalis, vanC1 and vanC2/C3 genes, and a high-resolution melting (HRM) analysis of the groESL gene for the differentiation of Enterococcus casseliflavus and Enterococcus gallinarum; (2) Bruker MS; (3) VITEK MS; and (4) the VITEK 2 system. 16S rRNA gene sequencing was used as a reference method in the study. The 132 isolates were identified as 32 E. faecalis, 63 E. faecium, 16 E. casseliflavus and 21 E. gallinarum. The multiplex PCR, Bruker MS and VITEK MS were able to identify all the isolates correctly at the species level. The VITEK 2 system could identify 131/132 (99.2 %) and 121/132 (91.7 %) of the isolates at the genus and species levels, respectively. The HRM-groESL assay identified all (21/21) E. gallinarum isolates and 81.3 % (13/16) of the E. casseliflavus isolates. The PCR methods described in the present study are effective in identifying the enterococcal species. MALDI-TOF MS is a rapid, reliable and cost-effective identification technique for enterococci. The VITEK 2 system is less efficient at detecting non-faecalis and non-faecium Enterococcus species.

Rapid culture and identification: a practical method for early preliminary laboratory diagnosis of sepsis.

This study describes the development of a method for rapid preliminary species identification of bacteria from positive blood culture vials. The method yielded preliminary identification results for 496 (92%) of 541 positive blood cultures within 5 h. The method was capable of identifying the most frequently isolated bacteria (i.e., Staphylococcus aureus, coagulase-negative staphylococci, Escherichia coli, Streptococcus pneumoniae and Enterococcus spp.) to the species level. The method can be established easily, with a materials cost of 2-5 Euros per sample.

Non-T(H)1 cytokines are augmented systematically early in Guillain-Barré syndrome.

The T(H)1 vs non-T(H)1 cytokine balance in Guillain-Barré syndrome (GBS) is unknown. Using enzyme-linked immunospot (ELISPOT) assays, we observed elevated numbers of interleukin (IL)-6 and IL-10-secreting blood mononuclear cells (BMNC) during the acute phase in untreated patients, and low levels of tumor necrosis factor alpha-secreting BMNC in the recovery phase of GBS. Numbers of IL-12p70-secreting BMNC were not affected over the course of GBS. The non-T(H)1 cytokine profile observed early in GBS may explain the self-limited clinical course associated with GBS.

Monocytes in multiple sclerosis: phenotype and cytokine profile.

Multiple sclerosis (MS) is an inflammatory demyelinating disease characterised by immune abnormalities in the central nervous system (CNS) as well as systemically. Activated, blood-borne monocytes are abundant in MS lesions, the properties of circulating monocytes are incompletely known. To delineate phenotype and levels of cytokine secreting monocytes in MS patients' blood, ELISPOT assays were used for detection and enumeration of monocytes secreting the cytokines IL-6, IL-12, TNF-alpha and IL-10. In parallel, the expression by monocytes of co-stimulatory molecules (CD40, CD80, CD86), major histocompatibility complex molecules (HLA-ABC, HLA-DR) and Fcgamma receptors (CD16, CD64) was examined by flow cytometry. Levels of blood monocytes secreting IL-6 and IL-12 were higher in patients with untreated MS and other neurological diseases (OND) compared to healthy controls, while levels of monocytes secreting TNF-alpha and IL-10 did not differ between groups. MS patients' blood monocytes also displayed elevated mean fluorescence intensity for the co-stimulatory molecule CD86, and MS patients with longer disease duration (>10 years) and higher disease severity (EDSS >3) had higher percentages of CD80 expressing monocytes compared to patients with short duration or lower severity. In conclusion, monocyte aberrations occur in MS and may change over the disease course.

Multiple sclerosis is associated with high levels of circulating dendritic cells secreting pro-inflammatory cytokines.

Recent evidence emphasises a pivotal role for dendritic cells (DC) in the control of immunity by priming and tolerising T cells. DC capture and process antigens, express co-stimulatory molecules, migrate to lymphoid organs and secrete cytokines to initiate immune responses. In multiple sclerosis (MS), autoreactive T cells are proposed to play a pathogenic role by secreting pro-inflammatory cytokines, but studies on DC are lacking. To evaluate the involvement of DC in patients with MS, a modified procedure was used to prepare DC from blood of patients with MS and healthy subjects. DC were found to be potent stimulators of T cells in allogeneic and, to a lesser extent, in autologous mixed leukocyte reaction (MLR). Enzyme-linked immunospot (ELISPOT) assays were adopted to determine levels of IFN-gamma, TNF-alpha, IL-6 and IL-10 secreting DC vs. mononuclear cells (MNC). Proportionally more DC than MNC secreted IFN-gamma and IL-10 in both MS and healthy subjects. Patients with MS had higher levels of IFN-gamma, TNF-alpha and IL-6 secreting DC than healthy subjects. The differences for IFN-gamma and TNF-alpha secreting cells were confined to the subgroup of untreated MS patients and not observed in the subgroup examined during ongoing treatment with IFN-beta. Circulating DC secreting pro-inflammatory cytokines may represent another focus for the study of both immuno-pathogenesis and therapeutic interventions in MS.

No evidence for elevated numbers of mononuclear cells expressing MCP-1 and RANTES mRNA in blood and CSF in multiple sclerosis.

The perivascular accumulation of mononuclear cells (MNC) in brain white matter is critical in the development of active lesions in multiple sclerosis (MS). Chemokines contribute to leukocyte recruitment by increasing the adhesiveness of integrins expressed on leukocytes and by promoting migration through endothelium and extracellular matrix. By using an in situ hybridization technique, it was possible to enumerate blood and CSF MNC expressing mRNA for the two CC chemokines monocyte chemoattractant protein-1 (MCP-1) and RANTES (regulated upon activation, normal T cells, expressed and secreted) in MS patients and controls. No differences in numbers of blood MNC expressing MCP-1 or RANTES could be found in MS patients compared to healthy individuals or patients with acute aseptic meningoencephalitis (AM). High numbers of CSF MNC expressing MCP-1 and RANTES were found in some MS patients, but also in patients with AM. This shows that elevated numbers of MCP-1 and RANTES mRNA expressing CSF MNC are not specific for the inflammatory process in MS. We conclude that there is no evidence for a systemic dysregulation of the CC chemokines MCP-1 and RANTES in MS.

IL-12/IL-12R system in multiple sclerosis.

IL-12/IL-12 receptor (IL-12R) system orchestrates the Th1 pathway of the immune system by maintaining one of the major bridges between innate and adaptive immune responses. Here, we studied both sides of this system in patients with multiple sclerosis (MS) and in controls. MS patients displayed elevated IL-12Rbeta1 and IL-12Rbeta2 expression on PHA-activated T cells compared to healthy subjects. Higher percentages of IL-12Rbeta1 and IL-12Rbeta2 positive T cells in cerebrospinal fluid (CSF) compared to blood were observed both in MS and other neurological diseases (OND). In contrast, numbers of IL-12 secreting blood mononuclear cells (MNC) were similar in MS and controls. The functional importance of high IL-12Rbeta2 in MS was underlined by the finding that IL-12 stimulated IFN-gamma production and proliferation of PHA-activated T cells correlated with levels of IL-12Rbeta2 expression. Our data indicates a dysregulation of the IL-12/IL-12R system in MS. It is suggested that even in the absence of increased IL-12 levels, the net effect of IL-12 might be augmented in MS by elevated expression of its receptor.

Multiple sclerosis: pro- and anti-inflammatory cytokines and metalloproteinases are affected differentially by treatment with IFN-beta.

Interferon-beta (IFN-beta) has a beneficial influence on the course of multiple sclerosis (MS) and has become standard treatment of this disease, though its mechanisms of action are incompletely understood. This study examines the effect of IFN-beta treatment on the cytokines IL-6, TNF-alpha, IFN-gamma and IL-10; the metalloproteinases MMP-3, -7 and -9 and the tissue inhibitor of metalloproteinase-1 (TIMP-1). IFN-beta treatment resulted in decreased numbers of mononuclear cells (MNC) secreting IL-6 and TNF-alpha and expressing mRNA of MMP-3 and MMP-9 compared to pretreatment levels. On the contrary, numbers of IL-10 secreting MNC and TIMP-1 mRNA expressing were augmented during IFN-beta therapy. Whether the down-regulatory effects on pro-inflammatory and upregulatory effects on anti-inflammatory molecules are a direct result of IFN-beta on the immune system or secondary to clinical stabilization of MS pathology induced by IFN-beta remains to be evaluated.

Phenotypes and cytokine profiles of enriched blood dendritic cells in healthy individuals.

Dendritic cells (DC) are highly specialized for initiating adaptive immune responses and are capable of producing a wide variety of cytokines. However, cytokine profiles of the DC naturally present in human blood have received relatively little attention. The objective of this study was to investigate expression of surface markers and cytokines by blood DC not subjected to prolonged culture and/or polyclonal activation, to identify surface phenotypes of cytokine-expressing DC and to evaluate sex and age differences in cytokine profiles of DC. For this purpose, DC were enriched from blood of healthy donors by the use of the adherence method, and expression of surface molecules and intracellular IFN-g, IL-10, IL-12 and IL-15 was studied by flow cytometry. Enriched blood DC expressed higher levels of IFN-g, IL-12 and IL-15, compared to whole mononuclear cells (MNC) incubated for the same time. Expression of IFN-g and IL-12 was confined to the mature CD83+CD11c+ DC subset. Enriched DC from females' blood displayed higher levels of CD80, IL-10 and IL-15. Taken together, enriched blood DC spontaneously express larger amounts of IFN-g, IL-12 and IL-15 than MNC. Sex differences in expression of CD80, IL-10 and IL-15 may have a modulatory influence on immune responses in males and females.

Modulation of immune responses and suppression of experimental autoimmune myasthenia gravis by surgical denervation of the spleen.

Critical interactions between the nervous system and the immune system during experimental autoimmune myasthenia gravis (EAMG) were examined in an animal model for human MG after immunization of adult female Lewis rats with Torpedo acetylcholine receptor (AChR) and complete Freund's adjuvant. Immunized rats depicted marked clinical severity of the disease. Using enzyme-linked immunospot (ELISPOT) assay and in situ hybridization techniques, immune responses in these animals were examined and showed elevated numbers of anti-AChR IgG secreting B cells and AChR reactive interferon (IFN)-gamma-secreting cells, enhanced mRNA expression of the proinflammatory cytokines IFN-gamma and tumour necrosis factor (TNF)-alpha as Th1 subset and the anti-inflammatory cytokines interleukin (IL)-4 and IL-10 as a Th2 subset, and transforming growth factor (TGF)-beta as a Th3 cytokine. Corticosterone and prostaglandin E(2) (PGE(2)) levels were measured by radioimmunoassay and illustrated increased production after immunization. Surgical denervation of the spleen reduced significantly the clinical severity of the disease, suppressed the numbers of IgG and IFN-gamma-secreting cells, down-regulated the mRNA expression for cytokines and reduced corticosterone and PGE(2) production. As controls, sham-operated rats were used and showed results as the EAMG non-denervated control rats. The data present herein, and for the first time, substantial effects of the nervous system on immune responses that may influence the outcome of EAMG. These effects were not dependent on cytokine inhibitory mediators such as prostaglandins or stress hormones. IL-10 and TGF-beta, the two potent immunosuppressive cytokines, were also suppressed, indicating a general suppression by splenic denervation. More investigations are initiated at our laboratories to understand the evident neural control over the immune system during challenges leading to the break of tolerance and development of autoimmunity, which may assist in innovative therapeutic approaches.

Cytokines in multiple sclerosis: methodological aspects and pathogenic implications.

Multiple sclerosis (MS) is one of the leading causes of disability among young adults of Caucasian origin. One hundred and fifty years after the first description of the disease, the cause of MS remains unknown. Ironically, the few hypotheses concerning MS pathogenesis that are valid today were first proposed over a hundred years ago. However, equipped with the advanced technology of molecular biology and imaging systems, we are at present progressively uncovering dues to understanding the pathogenesis of the disease. It is dearly evident that aberrant immune responses occur in MS, and it is likely that the spectrum of cytokines produced decisively influences disease outcome. The detrimental consequences of IFN-gamma and the beneficial effects of IFN-beta treatment in MS support this hypothesis. However, there are still major gaps in our knowledge of the involvement of cytokines in MS. Numerous studies have addressed the question of cytokine levels in MS, often with conflicting results; elevated, normal and decreased levels of almost all cytokines have been reported. This scenario most probably reflects methodological dilemmas as well as the complex biology of cytokines. Here we focus on possible reasons for the discrepancies of results reported on cytokines in MS and summarize findings obtained in particular by the application of enzyme-linked immunospot (ELISPOT) assays to cytokine studies in MS.

Matrix metalloproteinase and cytokine profiles in monocytes over the course of stroke.

Stroke is a common cause of death and disability in our society. Stroke is associated with changes in immune responses within the central nervous system as well as systemically. The cells contributing to such changes as well as the factors contributing to formation of the inflammatory infiltrate observed in stroke remain to be clarified. In this study, blood monocytes and corresponding mononuclear cells (MNC) were separated and examined in parallel within 4 days and 1-3 months after onset of ischemic stroke. Numbers of TNF-alpha-, IL-12-, IL-6-, and IL-10-secreting cells and of cells expressing mRNA for matrix metalloproteinase (MMP)-1, -2, -7, -9 and tissue inhibitor of MMP (TIMP)-1 were studied. The TNF-alpha-, IL-12-, and IL-6-secreting monocytes and MNC were elevated during the acute phase compared to healthy controls. Such differences were not observed when stroke patients were examined during convalescence. The IL-10-secreting monocytes did not change over the course of stroke. Levels of monocytes expressing MMP-1, MMP-7 and TIMP-1 mRNA were elevated in the acute phase of stroke patients compared to convalescence and healthy controls, as were levels of MMP-1, -2, -7, -9 and TIMP-1 mRNA expressing blood MNC. The MMP-2 and -9 activity as measured by zymography also was higher in MNC supernatants in the acute phase of stroke compared to convalescence. The high levels of proinflammatory cytokines and MMPs in blood monocytes and MNC further demonstrate the presence of systemic aberrations in the acute phase of stroke. Such changes may contribute to the influx of blood-borne cells into the ischemic lesions during the acute phase of stroke.

Elevated CD40 ligand expressing blood T-cell levels in multiple sclerosis are reversed by interferon-beta treatment.

Myelin protein reactive CD4+ T cells are considered to be involved in the proposed immunopathogenesis of multiple sclerosis (MS). One particularly important molecule for T-cell activation is the CD40L (gp39) that is expressed on the surface of T cells. This study focuses on the CD40 and the CD40L expression on mononuclear cells prepared from blood from patients with MS, other neurological diseases (OND) and healthy subjects. Immunostaining followed by a three channel flow cytometry was adopted. Patients with MS had higher levels of CD3+CD40L+, CD4+CD40L+ and CD8+CD40L+ T cells compared to patients with OND and healthy subjects. Cross-sectional comparisons revealed that the elevation of CD40L+ T cell subtypes was confined to the patients with untreated MS and not observed in the patients with MS treated with interferon-beta (IFN-beta). Follow up studies showed that levels of CD3+CD40L+ and CD4+CD40L+ T cells decreased in individual patients after the initiation of the IFN-beta treatment. The enhanced expression of CD40L on CD3+, CD4+ and CD8+ T cells in patients with MS may implicate a role for this molecule in disease immunopathogenesis.