The extracellular matrix in multiple sclerosis: an update

Extracellular matrix (ECM) molecules play important roles in the pathobiology of the major human central nervous system (CNS) inflammatory/demyelinating disease multiple sclerosis (MS). This mini-review highlights some recent work on CNS endothelial cell interactions with vascular basement membrane ECM as part of the cellular immune response, and roles for white matter ECM molecules in demyelination and remyelination in MS lesions. Recent basic and clinical investigations of MS emphasize axonal injury, not only in chronic MS plaques, but also in acute lesions; progressive axonal degeneration in normal-appearing white matter also may contribute to brain and spinal cord atrophy in MS patients. Remodeling of the interstitial white matter ECM molecules that affect axon regeneration, however, is incompletely characterized. Our ongoing immunohistochemical studies demonstrate enhanced ECM versican, a neurite and axon growth-inhibiting white matter ECM proteoglycan, and dermatan sulfate proteoglycans at the edges of inflammatory MS lesions. This suggests that enhanced proteoglycan deposition in the ECM and axonal growth inhibition may occur early and are involved in expansion of active lesions. Decreased ECM proteoglycans and their phagocytosis by macrophages along with myelin in plaque centers imply that there is "injury" to the ECM itself. These results indicate that white matter ECM proteoglycan alterations are integral to MS pathology at all disease stages and that they contribute to a CNS ECM that is inhospitable to axon regrowth/regeneration.

Thymocyte migration: an affair of multiple cellular interactions?

Cell migration is a crucial event in the general process of thymocyte differentiation. The cellular interactions involved in the control of this migration are beginning to be defined. At least chemokines and extracellular matrix proteins appear to be part of the game. Cells of the thymic microenvironment produce these two groups of molecules, whereas developing thymocytes express the corresponding receptors. Moreover, although chemokines and extracellular matrix can drive thymocyte migration per se, a combined role for these molecules appears to contribute to the resulting migration patterns of thymocytes in their various stages of differentiation. The dynamics of chemokine and extracellular matrix production and degradation is not yet well understood. However, matrix metalloproteinases are likely to play a role in the breakdown of intrathymic extracellular matrix contents. Thus, the physiological migration of thymocytes should be envisioned as a resulting vector of multiple, simultaneous and/or sequential stimuli involving chemokines, adhesive and de-adhesive extracellular matrix proteins, as well as matrix metalloproteinases. Accordingly, it is conceivable that any pathological change in any of these loops may result in the alteration of normal thymocyte migration. This seems to be the case in murine infection by the protozoan parasite Trypanosoma cruzi, the causative agent of Chagas' disease. A better knowledge of the physiological mechanisms governing thymocyte migration will provide new clues for designing therapeutic strategies targeting developing T cells.

A method for multiple sequential analyses of macrophage functions using a small single cell sample

Microbial pathogens such as bacillus Calmette-Guérin (BCG) induce the activation of macrophages. Activated macrophages can be characterized by the increased production of reactive oxygen and nitrogen metabolites, generated via NADPH oxidase and inducible nitric oxide synthase, respectively, and by the increased expression of major histocompatibility complex class II molecules (MHC II). Multiple microassays have been developed to measure these parameters. Usually each assay requires 2-5 x 10(5) cells per well. In some experimental conditions the number of cells is the limiting factor for the phenotypic characterization of macrophages. Here we describe a method whereby this limitation can be circumvented. Using a single 96-well microassay and a very small number of peritoneal cells obtained from C3H/HePas mice, containing as little as <=2 x 10(5) macrophages per well, we determined sequentially the oxidative burst (H2O2), nitric oxide production and MHC II (IAk) expression of BCG-activated macrophages. More specifically, with 100 µl of cell suspension it was possible to quantify H2O2 release and nitric oxide production after 1 and 48 h, respectively, and IAk expression after 48 h of cell culture. In addition, this microassay is easy to perform, highly reproducible and more economical.

Plasmablastic multiple myeloma is associated with increased vascular endothelial growth factor immunoexpression

The biologic basis of the negative prognosis of plasmablastic myeloma is not fully understood. To determine whether histologically aggressive multiple myeloma (MM) is associated with a more angiogenic marrow environment, bone marrow samples from 50 recently diagnosed MM patients were evaluated. Twelve percent (6/50) of patients presented plasmablastic MM, and this feature correlated with moderate/strong intensity of vascular endothelial growth factor staining of plasma cells (P = 0.036). Although plasmablastic MM was not associated with increasing of microvessel density, this new evidence of increased expression of vascular endothelial growth factor on plasmablasts suggests that the adverse prognosis conferred by plasmablastic disease may be due, at least in part, to secretion of this angiogenic cytokine, also suggesting that the subset of MM patients with plasmablastic features may derive particular benefit from antiangiogenic therapies.

Pharmacokinetic and parasitological evaluation of the bone marrow of dogs with visceral leishmaniasis submitted to multiple dose treatment with liposome-encapsulated meglumine antimoniate

The aim of the present study was to evaluate the impact of a multiple dose regimen of a liposomal formulation of meglumine antimoniate (LMA) on the pharmacokinetics of antimony in the bone marrow of dogs with visceral leishmaniasis and on the ability of LMA to eliminate parasites from this tissue. Dogs naturally infected with Leishmania chagasi received 4 intravenous doses of either LMA (6.5 mg antimony/kg body weight, N = 9), or empty liposomes (at the same lipid dose as LMA, N = 9) at 4-day intervals. A third group of animals was untreated (N = 8). Before each administration and at different times after treatment, bone marrow was obtained and analyzed for antimony level (LMA group) by electrothermal atomic absorption spectrometry, and for the presence of Leishmania parasites (all groups). There was a significant increase of antimony concentration from 0.76 µg/kg wet organ (4 days after the first dose) to 2.07 µg/kg (4 days after the fourth dose) and a half-life of 4 days for antimony elimination from the bone marrow. Treatment with LMA significantly reduced the number of dogs positive for parasites (with at least one amastigote per 1000 host cells) compared to controls (positive dogs 30 days after treatment: 0 of 9 in the LMA group, 3 of 9 in the group treated with empty liposomes and 3 of 8 in the untreated group). However, complete elimination of parasites was not achieved. In conclusion, the present study showed that multiple dose treatment with LMA was effective in improving antimony levels in the bone marrow of dogs with visceral leishmaniasis and in reducing the number of positive animals, even though it was not sufficient to achieve complete elimination of parasites.

Chemokines in the cerebrospinal fluid of patients with active and stable relapsing-remitting multiple sclerosis

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the human central nervous system. Although its etiology is unknown, the accumulation and activation of mononuclear cells in the central nervous system are crucial to its pathogenesis. Chemokines have been proposed to play a major role in the recruitment and activation of leukocytes in inflammatory sites. They are divided into subfamilies on the basis of the location of conserved cysteine residues. We determined the levels of some CC and CXC chemokines in the cerebrospinal fluid (CSF) of 23 relapsing-remitting MS patients under interferon-ß-1a therapy and 16 control subjects using ELISA. MS patients were categorized as having active or stable disease. CXCL10 was significantly increased in the CSF of active MS patients (mean ± SEM, 369.5 ± 69.3 pg/mL) when compared with controls (178.5 ± 29.1 pg/mL, P < 0.05). CSF levels of CCL2 were significantly lower in active MS (144.7 ± 14.4 pg/mL) than in controls (237.1 ± 16.4 pg/mL, P < 0.01). There was no difference in the concentration of CCL2 and CXCL10 between patients with stable MS and controls. CCL5 was not detectable in the CSF of most patients or controls. The qualitative and quantitative differences of chemokines in CSF during relapses of MS suggest that they may be useful as a marker of disease activity and of the mechanisms involved in the pathogenesis of the disease.

Detection of multiple mycoplasma infection in cell cultures by PCR

A total of 301 cell cultures from 15 laboratories were monitored for mycoplasma (Mollicutes) using PCR and culture methodology. The infection was detected in the cell culture collection of 12 laboratories. PCR for Mollicutes detected these bacteria in 93 (30.9%) samples. Although the infection was confirmed by culture for 69 (22.9%) samples, PCR with generic primers did not detect the infection in five (5.4%). Mycoplasma species were identified with specific primers in 91 (30.2%) of the 98 samples (32.6%) considered to be infected. Mycoplasma hyorhinis was detected in 63.3% of the infected samples, M. arginini in 59.2%, Acholeplasma laidlawii in 20.4%, M. fermentans in 14.3%, M. orale in 11.2%, and M. salivarium in 8.2%. Sixty (61.2%) samples were co-infected with more than one mycoplasma species. M. hyorhinis and M. arginini were the microorganisms most frequently found in combination, having been detected in 30 (30.6%) samples and other associations including up to four species were detected in 30 other samples. Failure of the treatments used to eliminate mycoplasmas from cell cultures might be explained by the occurrence of these multiple infections. The present results indicate that the sharing of non-certified cells among laboratories may disseminate mycoplasma in cell cultures.

Profile of the Brazilian scientific production in multiple sclerosis

This paper analyzes the profile of the Brazilian output in the field of multiple sclerosis from 1981 to 2004. The search was conducted through the MEDLINE and LILACS databases, selecting papers in which the term "multiple sclerosis" was defined as the main topic and "Brazil" or "Brasil" as others. The data were analyzed regarding the themes, the state in Brazil and institution where the papers were produced, the journals where the papers were published, journal's impact factor, and language. The search disclosed 141 documents (91 from MEDLINE and LILACS, and 50 from LILACS only) published in 44 different journals (23 of them MEDLINE-indexed). A total of 111 documents were produced by 17 public universities, 29 by 3 private medical schools and 1 by a non-governmental organization. There were 65 original contributions, 37 case reports, 20 reviews, 6 PhD dissertations, 5 guidelines, 2 validation studies, 2 clinical trials, 2 chapters in textbooks, 1 Master of Science thesis, and 1 patient education handout. The journal impact factor ranged from 0.0217 to 6.039 (median 3.03). Of 91 papers from MEDLINE, 65 were published by Arquivos de Neuro-Psiquiatria. More than 90% of the papers were written in Portuguese. São Paulo was the most productive state in the country, followed by Rio de Janeiro, Minas Gerais and Paraná. Eighty-two percent of the Brazilian output came from the Southeastern region.

Analysis of polymorphism at site -174 G/C of interleukin-6 promoter region in multiple myeloma

It is well established that interleukin-6 (IL-6) is an essential growth factor for multiple myeloma (MM) and patients with increased IL-6 levels have a poor prognosis. In healthy subjects, the presence of the C allele at a polymorphic site (-174 G/C) of the IL-6 gene is related to low IL-6 levels. In view of the potential association of this particular polymorphism with IL-6 concentration, and the relevance of IL-6 in MM pathogenesis, the objective of the present study was to investigate the prevalence of IL-6 (-174 G/C) promoter polymorphism and its association with development of MM in Brazilian individuals. We investigated the prevalence of these alleles in 52 patients and 60 healthy subjects (matched by age, sex, and race) of a Brazilian population. Thirty patients were male (42.4%), 24 (46.2%) were white and the median age at diagnosis was 58.5 years (range: 28 to 84 years). To determine the IL-6 (-174 G/C) polymorphism, molecular analysis was performed by polymerase chain reaction followed by endonuclease restriction digestion. The genotype distributions observed in the group of patients were 4% CC, 42% GC and 54% GG. The C allele frequency was 0.25. These results were similar to the control group, suggesting no impact of this polymorphism on the susceptibility to MM.

Multiple factor analysis of metachronous upper urinary tract transitional cell carcinoma after radical cystectomy

Transitional cell carcinoma (TCC) of the urothelium is often multifocal and subsequent tumors may occur anywhere in the urinary tract after the treatment of a primary carcinoma. Patients initially presenting a bladder cancer are at significant risk of developing metachronous tumors in the upper urinary tract (UUT). We evaluated the prognostic factors of primary invasive bladder cancer that may predict a metachronous UUT TCC after radical cystectomy. The records of 476 patients who underwent radical cystectomy for primary invasive bladder TCC from 1989 to 2001 were reviewed retrospectively. The prognostic factors of UUT TCC were determined by multivariate analysis using the COX proportional hazards regression model. Kaplan-Meier analysis was also used to assess the variable incidence of UUT TCC according to different risk factors. Twenty-two patients (4.6%). developed metachronous UUT TCC. Multiplicity, prostatic urethral involvement by the bladder cancer and the associated carcinoma in situ (CIS) were significant and independent factors affecting the occurrence of metachronous UUT TCC (P = 0.0425, 0.0082, and 0.0006, respectively). These results were supported, to some extent, by analysis of the UUT TCC disease-free rate by the Kaplan-Meier method, whereby patients with prostatic urethral involvement or with associated CIS demonstrated a significantly lower metachronous UUT TCC disease-free rate than patients without prostatic urethral involvement or without associated CIS (log-rank test, P = 0.0116 and 0.0075, respectively). Multiple tumors, prostatic urethral involvement and associated CIS were risk factors for metachronous UUT TCC, a conclusion that may be useful for designing follow-up strategies for primary invasive bladder cancer after radical cystectomy.

Roles of calcium and IP3 in impaired colon contractility of rats following multiple organ dysfunction syndrome

The purpose of the present study was to explore changes in rat colon motility, and determine the roles of calcium and inositol (1,4,5)-triphosphate (IP3) in colon dysmotility induced by multiple organ dysfunction syndrome (MODS) caused by bacteria peritonitis. The number of stools, the contractility of the muscle strips and the length of smooth muscle cells (SMC) in the colon, the concentration of calcium and IP3 in SMC, and serum nitric oxide were measured. Number of stools, fecal weight, IP3 concentration in SMC and serum nitric oxide concentration were 0.77 ± 0.52 pellets, 2.51 ± 0.39 g, 4.14 ± 2.07 pmol/tube, and 113.95 ± 37.89 µmol/L, respectively, for the MODS group (N = 11) vs 1.54 ± 0.64 pellets, 4.32 ± 0.57 g, 8.19 ± 3.11 pmol/tube, and 37.42 ± 19.56 µmol/L for the control group (N = 20; P < 0.05). After treatment with 0.1 mM acetylcholine and 0.1 M potassium chloride, the maximum contraction stress of smooth muscle strips, the length of SMC and the changes of calcium concentration were 593 ± 81 and 458 ± 69 g/cm³, 48.1 ± 11.8 and 69.2 ± 15.7 µM, 250 ± 70 and 167 ± 48%, respectively, for the control group vs 321 ± 53 and 284 ± 56 g/cm³, 65.1 ± 18.5 and 87.2 ± 23.7 µM, 127 ± 35 and 112 ± 35% for the MODS group (P < 0.05). Thus, colon contractility was decreased in MODS, a result possibly related to reduced calcium concentration and IP3 in SMC.

Restless leg syndrome, sleep quality and fatigue in multiple sclerosis patients

We have tested the hypothesis that restless leg syndrome (RLS) is related to quality of sleep, fatigue and clinical disability in multiple sclerosis (MS). The diagnosis of RLS used the four minimum criteria defined by the International Restless Legs Syndrome Study Group. Fatigue was assessed by the Fatigue Severity Scale (FSS >27), quality of sleep by the Pittsburgh Sleep Quality Index (PSQI >6), excessive daytime sleepiness by the Epworth Sleepiness Scale (ESS >10) and clinical disability by the Expanded Disability Status Scale (EDSS). Forty-four patients (32 women) aged 14 to 64 years (43 ± 14) with disease from 0.4 to 23 years (6.7 ± 5.9) were evaluated. Thirty-five were classified as relapsing-remitting, 5 as primary progressive and 4 as secondary progressive. EDSS varied from 0 to 8.0 (3.6 ± 2.0). RLS was detected in 12 cases (27%). Patients with RLS presented greater disability (P = 0.01), poorer sleep (P = 0.02) and greater levels of fatigue (P = 0.03). Impaired sleep was present in 23 (52%) and excessive daytime sleepiness in 3 cases (6.8%). Fatigue was present in 32 subjects (73%) and was associated with clinical disability (P = 0.000) and sleep quality (P = 0.002). Age, gender, disease duration, MS pattern, excessive daytime sleepiness and the presence of upper motor neuron signs were not associated with the presence of RLS. Fatigue was best explained by clinical disability and poor sleep quality. Awareness of RLS among health care professionals may contribute to improvement in MS management.

Phenotypic and genotypic variant of MDR-Mycobacterium tuberculosis multiple isolates in the same tuberculosis episode, Rio de Janeiro, Brazil

Assuming that the IS6110-restriction fragment length polymorphism (RFLP) changes at a constant rate of 3.2 years, this methodology was applied to demonstrate, for the first time, variant patterns of Mycobacterium tuberculosis (MTB) in multiple isolates obtained at short time intervals from sputum and blood of an HIV+ patient with multiple admissions to the Emergency Room and to the multidrug-resistant tuberculosis (MDR-TB) Reference Center of a secondary-care hospital in Rio de Janeiro, Brazil. In sputum, the IS6110-RFLP appeared in isolates with two variant patterns with 10 and 13 IS6110 copies. However, blood presented only the pattern corresponding to 10 copies, suggesting compartmentalization. With regard to the exact match of 10 of 13 bands, this may be a subpopulation with the same clonal origin and this may be related to the IS6110 transposition. A susceptibility test demonstrated an MDR profile (INH R, RIF R, SM R, and EMB R), with the sputum isolate also exhibiting EMB S (R = resistant; S = sensitive). A gene mutation confirmed resistance only to streptomycin. There was agreement between the results of the phenotypic test and the clinical response to MDR-TB treatment, suggesting serious implications with regard to treatment administration based exclusively on molecular methods, and calling attention to the fact that more effective control strategies against the emergence of MDR strains must be implemented by the TB control program to prevent transmission of MDR-MTB strains at health facilities in areas highly endemic for TB.

Brain tissue segmentation using q-entropy in multiple sclerosis magnetic resonance images

The loss of brain volume has been used as a marker of tissue destruction and can be used as an index of the progression of neurodegenerative diseases, such as multiple sclerosis. In the present study, we tested a new method for tissue segmentation based on pixel intensity threshold using generalized Tsallis entropy to determine a statistical segmentation parameter for each single class of brain tissue. We compared the performance of this method using a range of different q parameters and found a different optimal q parameter for white matter, gray matter, and cerebrospinal fluid. Our results support the conclusion that the differences in structural correlations and scale invariant similarities present in each tissue class can be accessed by generalized Tsallis entropy, obtaining the intensity limits for these tissue class separations. In order to test this method, we used it for analysis of brain magnetic resonance images of 43 patients and 10 healthy controls matched for gender and age. The values found for the entropic q index were 0.2 for cerebrospinal fluid, 0.1 for white matter and 1.5 for gray matter. With this algorithm, we could detect an annual loss of 0.98% for the patients, in agreement with literature data. Thus, we can conclude that the entropy of Tsallis adds advantages to the process of automatic target segmentation of tissue classes, which had not been demonstrated previously.

Serum levels of brain-derived neurotrophicfactor correlate with the number of T2 MRI lesions in multiple sclerosis

The objective of the present study was to determine if there is a relationship between serum levels of brain-derived neurotrophic factor (BDNF) and the number of T2/fluid-attenuated inversion recovery (T2/FLAIR) lesions in multiple sclerosis (MS). The use of magnetic resonance imaging (MRI) has revolutionized the study of MS. However, MRI has limitations and the use of other biomarkers such as BDNF may be useful for the clinical assessment and the study of the disease. Serum was obtained from 28 MS patients, 18-50 years old (median 38), 21 women, 0.5-10 years (median 5) of disease duration, EDSS 1-4 (median 1.5) and 28 healthy controls, 19-49 years old (median 33), 19 women. BDNF levels were measured by ELISA. T1, T2/FLAIR and gadolinium-enhanced lesions were measured by a trained radiologist. BDNF was reduced in MS patients (median [range] pg/mL; 1160 [352.6-2640]) compared to healthy controls (1640 [632.4-4268]; P = 0.03, Mann-Whitney test) and was negatively correlated (Spearman correlation test, r = -0.41; P = 0.02) with T2/FLAIR (11-81 lesions, median 42). We found that serum BDNF levels were inversely correlated with the number of T2/FLAIR lesions in patients with MS. BDNF may be a promising biomarker of MS.