A zero dead-time multi-particle time and position sensitive detector based on correlation between brightness and amplitude.

A new multi-particle time and position sensitive detector using only a set of microchannel plates, a waveform digitizer, a phosphor screen, and a CMOS camera is described. The assignment of the timing information, as taken from the microchannel plates by fast digitizing, to the positions, as recorded by the camera, is based on the COrrelation between the BRightness of the phosphor screen spots, defined as their integrated intensity and the Amplitude of the electrical signals (COBRA). Tests performed by observing the dissociation of HeH, the fragmentation of H3 into two or three fragments, and the photo-double-ionization of Xenon atoms are presented, which illustrate the performances of the COBRA detection scheme.

[Frontal syndrome resulting from an intracerebral amyloidoma]. / Frontaal syndroom door een intracerebraal amyloïdoom.

A 51-year-old woman suffering an epileptic seizure came to the emergency unit. A ct scan showed a mass lesion in the right frontal lobe. The psychiatric examination indicated a frontal syndrome with severe cognitive impairment. A stereotactical biopsy was carried out. Histopathology produced an unusual diagnosis, namely an intracerebral amyloidoma. By reviewing the literature on intracerebral amyloidoma we were able to compare the psychiatric symptoms of published cases with those of this case.

Molecular characterization of the first two enzymes of the pentose-phosphate pathway of Trypanosoma brucei. Glucose-6-phosphate dehydrogenase and 6-phosphogluconolactonase.

Trypanosomatids are parasitic protists that have part of their glycolytic pathway sequestered inside peroxisome-like organelles: the glycosomes. So far, at least one enzyme of the pentose-phosphate pathway has been found to be associated partially with glycosomes. Here, we describe how two genes from Trypanosoma brucei, coding for the first two enzymes of the pentose-phosphate pathway, i.e. glucose-6-phosphate dehydrogenase and 6-phosphogluconolactonase, were identified by in silico screening of trypanosome genome project data bases. These genes were cloned and sequenced. Analysis of the lactonase sequence revealed that it contained a C-terminal peroxisome targeting signal in agreement with its subcellular localization in the bloodstream form trypanosome (15% glycosomal and 85% cytosolic). However, the dehydrogenase sequence did not reveal any targeting signal, despite its localization inside glycosomes. The corresponding enzymes have been overexpressed in Escherichia coli and purified, and their biochemical characteristics have been determined.

Trypanosoma brucei contains a 2,3-bisphosphoglycerate independent phosphoglycerate mutase.

Assays of phosphoglycerate mutase (PGAM) activity in lysates of bloodstream form Trypanosoma brucei appeared not to require exogenous 2,3-bisphosphoglycerate, thus suggesting that this protist contains an enzyme belonging to the class of cofactor-independent PGAMs. A gene encoding a polypeptide with motifs characteristic for this class of enzymes was cloned. The predicted T. brucei PGAM polypeptide contains 549 amino acids, with Mr 60 557 and pI 5.5. Comparison with 15 cofactor-independent PGAM sequences available in databases showed that the amino-acid sequence of the trypanosome enzyme has 59-62% identity with plant PGAMs and 29-35% with eubacterial enzymes. A low 28% identity was observed with the only available invertebrate sequence. The trypanosome enzyme has been expressed in Escherichia coli, purified to homogeneity and subjected to preliminary kinetic analysis. Previous studies have shown that cofactor-dependent and -independent PGAMs are not homologous. It has been inferred that the cofactor-independent PGAMs are in fact homologous to a family of metalloenzymes containing alkaline phosphatases and sulphatases. Prediction of the secondary structure of T. brucei PGAM and threading the sequence into the known crystal structure of E. coli alkaline phosphatase (AP) confirmed this homology, despite the very low sequence identity. Generally, a good match between predicted (PGAM) and actual (AP) secondary structure elements was observed. In contrast to trypanosomes, glycolysis in all vertebrates involves a cofactor-dependent PGAM. The presence of distinct nonhomologous PGAMs in the parasite and its human host offers great potential for the design of selective inhibitors which could form leads for new trypanocidal drugs.

Apocynin, a plant-derived, cartilage-saving drug, might be useful in the treatment of rheumatoid arthritis.

OBJECTIVE: To investigate whether apocynin, 1-(4-hydroxy-3-methoxyphenyl)ethanone, is able to diminish inflammation-induced cartilage destruction in rheumatoid arthritis (RA), studied in a human in vitro model. METHODS: Apocynin was added to cultures of RA peripheral blood mononuclear cells (PBMNC). Cartilage-destructive activity was determined by addition of culture supernatant to tissue samples of human articular cartilage. In addition, the proliferation of PBMNC, their production of tumour necrosis factor alpha (TN-Falpha), interleukin (IL)-1 and IL-10, and T-cell production of interferon gamma (IFN-gamma) and IL-4, as measures for T1 and T2 cell activity, were determined. RESULTS: Apocynin was able to counteract RA PBMNC-induced inhibition of cartilage matrix proteoglycan synthesis, while no effect on inflammation-enhanced proteoglycan release was found. The effect was accompanied by a decrease in IL-1 and TNF-alpha production by the MNC. No effect on T-cell proliferation was found, but the production of IFN-gamma, IL-4 and T-cell-derived IL-10 was strongly diminished. Most important, apocynin did not show any direct adverse effects on chondrocyte metabolism; on the contrary, it diminished the release of proteoglycans from the cartilage matrix. CONCLUSION: Apocynin in vitro inhibits inflammation-mediated cartilage destruction without having adverse effects on cartilage. The latter may be an advantage of apocynin over many other non-steroidal anti-inflammatory drugs. Therefore, apocynin might have an added beneficial effect in protecting RA patients from joint destruction.

Stimulation of suppressive T cell responses by human but not bacterial 60-kD heat-shock protein in synovial fluid of patients with rheumatoid arthritis.

In several animal models of rheumatoid arthritis (RA), T cell responses to self 60-kD heat-shock protein 60 (hsp60) protect against the induction of arthritis. The nature of this suppressive T cell activity induced by self hsp60 is not clear. In the present study, T cell responses to human (self) hsp60 in RA in terms of type 1 (T1) and type 2 (T2) T cell activity were assessed. The results show that human and not bacterial hsp60-reactive synovial fluid (SF) T cells of patients with RA proliferate in the presence of the T2 cell growth factor IL-4. SF T cells stimulated with human hsp60 produced significantly lower amounts of IFN-gamma and higher amounts of IL-4 than SF T cells stimulated with bacterial hsp60 (P

Decrease in peripheral type 1 over type 2 T cell cytokine production in patients with rheumatoid arthritis correlates with an increase in severity of disease.

OBJECTIVES: To compare peripheral type 1 (T1) and type 2 (T2) T cell activities in rheumatoid arthritis (RA) patients with that found for osteoarthritic (OA) patients and healthy controls and to correlate peripheral T1/T2 cell activity in RA with parameters of the disease. METHODS: Peripheral blood mononuclear cells were isolated from patients with RA (n = 66), OA (n = 19), and healthy controls (n = 15). Primary T cell activity in these mononuclear cells was enhanced by means of anti-CD3/anti-CD28, which mimicks stimulation of T cells by activation of the T cell receptor and a major co-stimulatory signal. Interferon gamma (IFN gamma) production and interleukin 4 (IL4) production in the three groups were quantified as measures of T1 and T2 cell activity, respectively, and compared. Serum tumour necrosis factor alpha (TNF alpha), erythrocyte sedimentation rate (ESR), C reactive protein (CRP), and joint destruction assessed radiographically of RA patients were determined as parameters of disease activity and correlated with T1/T2 cell activity. RESULTS: Peripheral T cells from RA patients produced significantly less IFN gamma and more IL4 than T cells from both age and sex matched OA patients and healthy controls. Moreover, in RA patients both a decrease in IFN gamma and an increase in IL4 production correlated with an increase in serum TNF alpha, ESR, CRP, and joint destruction. CONCLUSIONS: These results suggest a role for differential T cell activity in RA. In view of the intra-articular T1 cell predominance the results might be explained by selective T1 cell migration into the joint or peripheral suppression of T1 cell activity.

Prevention and reversal of cartilage degradation in rheumatoid arthritis by interleukin-10 and interleukin-4.

OBJECTIVE: Inflammation-induced articular cartilage degradation is a major problem in rheumatoid arthritis (RA). Type 1 T cell activity (characterized by interferon-gamma/interleukin-2 [IL-2] production), and consequently, the production of the proinflammatory cytokines IL-1 and tumor necrosis factor alpha (TNF alpha), have been reported to play a major role in cartilage damage. IL-10 and IL-4, both produced by type 2 T cells, are cytokines with the capacity to down-regulate proinflammatory responses. The present study was undertaken to investigate the way in which these cytokines affect activated mononuclear cells (MNC) of RA patients in relation to human articular cartilage degradation in vitro. METHODS: MNC from synovial fluid and peripheral blood of RA patients were stimulated with bacterial antigen and treated with IL-10 and/or IL-4. Bacterial antigen is known to activate type 1 T cells and to induce proinflammatory IL-1/TNF alpha-dependent cartilage damage. Cytokine production and effects of conditioned media, as well as effects of IL-10 and IL-4 on proteoglycan (PG) turnover (as a measure for cartilage damage), were determined. RESULTS: IL-10 and IL-4 inhibited proinflammatory cytokine production of stimulated RA MNC and completely reversed inhibition of cartilage PG synthesis induced by these stimulated RA MNC. IL-10 was more potent than IL-4 in this respect, and the combination of IL-10 and IL-4 had an additive effect. In addition, IL-10 directly stimulated cartilage PG synthesis. CONCLUSION: IL-10 reverses the cartilage degradation induced by antigen-stimulated MNC, and IL-4 has an additive effect on this process. Furthermore, IL-10 has a direct stimulatory effect on PG synthesis, and IL-4, as a growth factor for type 2 T cells, can reduce the ratio of type 1 to type 2 T cell activity. These results provide evidence in favor of the use of a combination of the two cytokines in the treatment of RA.

In vitro effects of methotrexate on human articular cartilage and bone-derived osteoblasts.

Conflicting data have been published on whether low-dose methotrexate (MTX) treatment of rheumatoid arthritis (RA) is able to slow down radiological joint damage, i.e. retard the destruction of articular cartilage and (subchondral) bone. We studied the effects of MTX on proteoglycan (PG) turnover and interleukin-1 (IL-1)- and RA mononuclear cell (RA-MNC)-induced cartilage damage in human articular cartilage tissue cultures, and the effects of MTX on basal and RA-MNC-influenced proliferation and differentiation of osteoblasts in cultures of human bone-derived osteoblasts. MTX exerted no direct effect on cartilage nor did MTX influence IL-1- or RA-MNC-induced cartilage damage, despite strong suppression of basal as well as mitogen- and antigen-induced RA-MNC proliferation. MTX induced strong inhibition of osteoblast proliferation, but did not significantly interfere with osteoblast differentiation (i.e. alkaline phosphatase activity). RA-MNC-enhanced proliferation and differentiation of osteoblasts were abolished by MTX. These results suggest that if MTX is able to induce retardation of radiological progression in RA, this is not based on an initial direct effect of MTX on cartilage as measured by PG turnover, nor on an initial inhibition of IL-1- or RA-MNC-induced cartilage damage. However, longstanding MTX-induced inhibition of RA-MNC proliferation may lead to reduction of the catabolic activity involved in cartilage destruction. On the other hand, long-term inhibition of osteoblast proliferation may eventually lead to decreased bone formation and osteopenia. Whether this will turn out to be a problem of clinical importance in the treatment of RA has to be established.

The stimulation of mononuclear cells from patients with rheumatoid arthritis to degrade articular cartilage is not modulated by cartilage itself.

OBJECTIVE: To study the modulation of mononuclear cell (MNC) activity in patients with rheumatoid arthritis (RA) by constituents released from human articular cartilage, which may be present in vivo during early events of the disease, when articular cartilage is not only mildly damaged. METHODS: In an attempt to stimulate RA MNC, cells were co-cultured with healthy or mildly damaged articular cartilage tissue. In addition, because of the reported cross-reactivity between cartilage constituents and mycobacterial heat-shock protein (hsp60), RA MNC sensitized with hsp60 were also co-cultured with cartilage tissue. Activation of the RA MNC was assessed by analysing the production of catabolic factors involved in joint damage. For this purpose culture supernatants of the treated RA MNC, comprising the catabolic factors, were added to freshly isolated articular cartilage explants. As a read out for catabolic activity, proteoglycan (PG) turnover by the explants was determined. RESULTS: Spontaneous activity of untreated RA MNC caused inhibition of PG synthesis and increased PG release upon addition of their culture supernatants to the cartilage explants. This MNC activity was not enhanced by the constituents released from healthy or mildly damaged cartilage tissue, whereas sensitization of RA MNC with hsp60 resulted in a 40% enhanced inhibition of PG synthesis. However, even under these pre-activated conditions no reactivity towards the cartilage constituents could be observed. CONCLUSION: Cartilage constituents released from mildly damaged cartilage tissue, as may be present during the early events of RA. do not modulate the catabolic activity of RA MNC.

Proinflammatory cytokine production and cartilage damage due to rheumatoid synovial T helper-1 activation is inhibited by interleukin-4.

OBJECTIVES: To investigate the role of T helper-1 cell (Th1) activation in the induction of proinflammatory cytokine production and cartilage damage by rheumatoid arthritis (RA) synovial fluid mononuclear cells (SFMNC) and the subsequent possible beneficial role of the T helper-2 cell (Th2) cytokine interleukin-4 (IL-4) in the inhibition of this process. METHODS: SFMNC were stimulated with bacterial antigen (hsp60) to activate Th1 cells. Th1 and Th2 specific cytokine profiles (interferon gamma (IFN gamma) and IL-4) and proinflammatory cytokines interleukin-1 (IL-1) and tumour necrosis factor alpha (TNF alpha) in the conditioned media were analysed. In addition, the conditioned media were tested for their ability to induce cartilage damage. The same parameters were measured in the presence of IL-4. RESULTS: Stimulation of SFMNC with bacterial antigen resulted in an increase in IFN gamma, IL-1, and TNF alpha production which was accompanied by the induction of cartilage damage. Th1 activation could be inhibited by IL-4 as shown by a reduction of IFN gamma. This was accompanied by a decrease in IL-1 and TNF alpha production and inhibition of cartilage damage. CONCLUSIONS: Th1 activation is a possible mechanism by which inflammation in RA joints is enhanced. The Th2 cytokine IL-4 inhibits this Th1 activity and may diminish inflammation and induction of cartilage damage in RA joints.

Molecular analysis of glyceraldehyde-3-phosphate dehydrogenase in Trypanoplasma borelli: an evolutionary scenario of subcellular compartmentation in kinetoplastida.

In Trypanoplasma borelli, a representative of the Bodonina within the Kinetoplastida, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity was detected in both the cytosol and glycosomes. This situation is similar to that previously found in Trypanosomatidae, belonging to a different Kinetoplastida suborder. In Trypanosomatidae different isoenzymes, only distantly related, are responsible for the activity in the two cell compartments. In contrast, immunoblot analysis indicated that the GAPDH activity in cytosol and glycosomes of T. borelli should be attributed to identical or at least very similar proteins related to the glycosomal GAPDH of Trypanosomatidae. Moreover, only genes related to the glycosomal GAPDH genes of Trypanosomatidae could be detected. All attempts to identify a gene related to the one coding for the trypanosomatid cytosolic GAPDH remained unsuccessful. Two tandemly arranged genes were found which are 95% identical. The two encoded polypeptides differ in 17 residues. Their sequences are 72-77% identical to the glycosomal GAPDH of the other Kinetoplastida and share with them some characteristic features: an excess of positively charged residues, specific insertions, and a small carboxy-terminal extension containing the sequence -AKL. This tripeptide conforms to the consensus signal for targeting of proteins to glycosomes. One of the two gene copies has undergone some mutations at positions coding for highly conserved residues of the active site and the NAD(+)-binding domain of GAPDH. Modeling of the protein's three-dimensional structure suggested that several of the substitutions compensate each other, retaining the functional coenzyme-binding capacity, although this binding may be less tight. The presented analysis of GAPDH in T. borelli gives further support to the assertion that one isoenzyme, the cytosolic one, was acquired by horizontal gene transfer during the evolution of the Kinetoplastida, in the lineage leading to the suborder Trypanosomatina (Trypanosoma, Leishmania), after the divergence from the Bodonina (Trypanoplasma). Furthermore, the data clearly suggest that the original GAPDH of the Kinetoplastida has been compartmentalized during evolution.

Selective interaction of glycosomal enzymes from Trypanosoma brucei with hydrophobic cyclic hexapeptides.

Hydrophobic cyclic hexapeptides have been reported to selectively inhibit glycosomal triosephosphate isomerase from Trypanosoma brucei (Kuntz et al, 1992, Eur. J. Biochem., 207, 441-447). Here it is shown that this inhibition is not due to a specific interaction between the enzyme and soluble hydrophobic cyclic hexapeptides, but that it is the result of a coprecipitation of trypanosome triosephosphate isomerase with cyclic hexapeptides when the solubilities of the latter are exceeded. A study of the interaction of these hexapeptides with other glycosomal enzymes revealed that several of them, such as phosphoglycerate kinase and hexokinase, also coprecipitated with these peptides, whereas most of the homologous enzymes from other organisms did not coprecipitate, nor were they inactivated.

Suppression of human cartilage proteoglycan synthesis by rheumatoid synovial fluid mononuclear cells activated with mycobacterial 60-kd heat-shock protein.

OBJECTIVE: To examine whether T cell reactivity toward heat-shock proteins (HSP) contributes to cartilage destruction in rheumatoid arthritis (RA). METHODS: An in vitro system was used, in which human cartilage explants were cocultured with hsp60-activated synovial fluid mononuclear cells (SFMC) from patients with RA, and proteoglycan (PG) synthesis was measured. RESULTS: The hsp60-activated SFMC suppressed cartilage PG synthesis. This effect was dependent on the production of interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF alpha). CONCLUSION: Mycobacterial 60-kd heat-shock protein can activate rheumatoid SFMC to suppress human cartilage PG synthesis. This suppression is mediated by IL-1 and TNF alpha.

Articular cartilage explant culture; an appropriate in vitro system to compare osteoarthritic and normal human cartilage.

Proteoglycan metabolism of normal and histologically mild to moderate osteoarthritic cartilage explants were studied. Explants were obtained from the human knee of donors aged over 40 years. Proteoglycan content, synthesis and release were very similar in normal cartilage obtained from donors with focal osteoarthritis and cartilage obtained from donors without any sign of osteoarthritis. This suggests that cartilage obtained from donors with focal osteoarthritis indeed can be considered as "normal". The relatively large surface area-compared to their natural setting in the joint- of cartilage explants in culture did not affect the parameters measured, as there was a strong linear correlation between these parameters and the weight of the explants. From our results, we conclude that the use of full depth cartilage tissue explants is a reliable way to assess and compare proteoglycan content, synthesis and release in normal and osteoarthritic cartilage from the same donor.

Antigen-activated T cells inhibit cartilage proteoglycan synthesis independently of T-cell proliferation.

Previously we have shown that blood mononuclear cells (MNC) obtained from patients with rheumatoid arthritis (RA) have the capacity to induce depletion of proteoglycans (PG) in human cartilage explants. This was observed especially after stimulating MNC with mycobacterial antigens, rather than with the mitogen Concanavalin A (Con A). We have now co-cultured cartilage explants in the presence of T-cell clone A2b obtained from the rat model of adjuvant arthritis (AA). We show that inhibition of the cartilage PG synthesis is a consequence of antigen-specific T-cell activation and that it is mediated by a humoral factor. This seems to be a cytokine rather than an enzyme. Moreover, at the level of polyclonally responding T cells, inhibition of PG synthesis due to T-cell activation by mycobacterial antigens was shown to depend on prior mycobacterial immunization. Arthritogenic T-cell clone A2b also showed PG synthesis inhibitory effects when co-cultured with cartilage alone. The inhibitory activity was shown to be unrelated to the degree of T-cell proliferation. We conclude that antigen-specific T-cell activation may be one of the initiating events leading to cartilage damage in arthritic processes. The measurement of T-cell-mediated PG synthesis inhibition may be a more sensitive and relevant assay for the detection of pathogenic T cells than T-cell proliferation.

Influence of rheumatoid synovial fluid and cells on proteoglycans in human cartilage explants. Modulation by piroxicam.

We have studied the effects of cell-free rheumatoid synovial fluid (RASF) and the conditioned medium (CM) from these cells on the proteoglycans (PGs) of normal human cartilage and the influence which piroxicam might have on these processes. Both RASF and the CM from RASF cells enhanced the PG release from the cartilage explants. The effects of the above mentioned fluids on the cartilage PG content depended on the metabolic state of the cartilage i.e. correlated inversely with the PG synthesis. Whether this was due to the presence of anabolic and catabolic factors in these fluids is discussed. Piroxicam had no adverse effect on the PGs of human cartilage in vitro. Piroxicam prevented the cartilage PG depletion when it was induced by the CM from RASF cells.

Role of TNF alpha, in relation to IL-1 and IL-6 in the proteoglycan turnover of human articular cartilage.

In both young and old human articular cartilage explants, TNF alpha induced a concentration-dependent, reversible suppression of the proteoglycan (PG) synthesis. Young cartilage was more sensitive to TNF alpha than old cartilage: 50% suppression of PG synthesis was reached at a TNF alpha concentration of 5 U/ml for young and 30 U/ml for old cartilage, whereas at 10(3) U/ml the PG synthesis of young cartilage was blocked and that of old cartilage suppressed by 80%. These inhibition levels of PG synthesis resulted in 25% PG depletion of the explants after 8 days of culture. The release of cartilage PG was not enhanced. TNF alpha induced no detectable amounts of IL-1 (less than 0.01 U) in young or old cartilage but did induce IL-6 production. The induced amounts of IL-6 were higher in young than in old cartilage but no dose-dependency was evident. Antibodies to neither IL-1 nor IL-6 had any influence on the TNF alpha-induced suppression of PG synthesis. The combination of TNF alpha and IL-1 led to an additive inhibition of PG synthesis which had no relationship to induced IL-6. TNF alpha was about 100-fold less active than IL-1.

In vitro influence of ketoprofen on the proteoglycan metabolism of human normal and osteoarthritis cartilage.

We investigated the influences of ketoprofen on the proteoglycan (PG) turnover of human articular cartilage explants in three groups: normal young with a high basal PG synthesis, normal adult and osteoarthritis cartilage, both with a low basal PG synthesis. Ketoprofen had no influence on the mean PG synthesis rate of normal adult and OA cartilage after 4 days of culture nor on the cartilage PG content after 8 days of culture. There was no relation between the histological grade of OA and effects of ketoprofen. In normal young cartilage ketoprofen induced an increase of the PG synthesis rate when added to the culture in a concentration of 10(-4) M. No correlation existed between the effect of ketoprofen and the basal PG synthesis of normal cartilage.