Assessing multiple sclerosis activity: is the in vitro production of tumor necrosis factor-alpha, interleukins 2, 6, 4, and 10, and immunoglobulin G of value?

Tumor necrosis factor (TNF) alpha, interleukins (IL) 2, 4, 6, and 10, and IgG oligoclonal bands (IgG OB) in vitro production was assessed, after whole-blood stimulation with lipopolysaccharide or concanavalin A, in 61 patients presenting with relapsing-remitting, relapsing-progressive, or chronic progressive multiple sclerosis. Multiple sclerosis patients were receiving no treatment or azathioprine (AZA), cyclosporin, cyclophosphamide, subcutaneous interferon (IFN) beta 1 a, or corticosteroids (CST). Statistical correlations significantly showed that: (a) AZA lowers TNF-alpha (P = 0.002) and increases IL-4 production (P = 0.0024), and IFN-beta 1 a increases TNF-alpha and decreases IL-4 levels; (b) CST has a negative effect on TNF-alpha, IL-6, and IL-4 synthesis; and (c) AZA, IFN-beta 1 a, and CST diminish IgG OB synthesis (P = 0.001). Although our study of the dynamics of TNF-alpha, IL-2, IL-4, IL-6, and IL-10 in vitro production generally found no statistically significant correlations (partly explained by the limited number of values in the various groups), IL-6 was shown to drop during the periods surrounding relapse (P = 0.05) in the absence of treatment, while TNF-alpha (P = 0.04) and IL-6 (P < 0.05) dropped before exacerbation in the presence of AZA. In vitro production of TNF-alpha was closely and positively correlated with that of IL-6, independently of clinical features. The enhanced production of IL-10 detected before or at relapse with AZA and IFN-beta 1 a (trends) may interfere with initiation of the immune reaction and with the development of new CNS lesions. Some discrepancies with previously published results stress the difficulties in studying the state of stimulation of different populations of leukocytes by using a variety of in vitro stimuli and in establishing a correlation between mRNA studies and the amount of final or active protein produced.

Effects of ciliary muscle plasmid electrotransfer of TNF-alpha soluble receptor variants in experimental uveitis.

Intraocular inflammation has been recognized as a major factor leading to blindness. Because tumor necrosis factor-alpha (TNF-alpha) enhances intraocular cytotoxic events, systemic anti-TNF therapies have been introduced in the treatment of severe intraocular inflammation, but frequent re-injections are needed and are associated with severe side effects. We have devised a local intraocular nonviral gene therapy to deliver effective and sustained anti-TNF therapy in inflamed eyes. In this study, we show that transfection of the ciliary muscle by plasmids encoding for three different variants of the p55 TNF-alpha soluble receptor, using electrotransfer, resulted in sustained intraocular secretion of the encoded proteins, without any detection in the serum. In the eye, even the shorter monomeric variant resulted in efficient neutralization of TNF-alpha in a rat experimental model of endotoxin-induced uveitis, as long as 3 months after transfection. A subsequent downregulation of interleukin (IL)-6 and iNOS and upregulation of IL-10 expression was observed together with a decreased rolling of inflammatory cells in anterior segment vessels and reduced infiltration within the ocular tissues. Our results indicate that using a nonviral gene therapy strategy, the local self-production of monomeric TNF-alpha soluble receptors induces a local immunomodulation enabling the control of intraocular inflammation.

Mycobacterium bovis BCG but not Mycobacterium leprae induces TNF-alpha secretion in human monocytic THP-1 cells

In this study, we compared the level of TNF-alpha secretion induced in monocytic THP-1 cells after phagocytosis of Mycobacterium leprae, the causative agent of leprosy, and M. bovis BCG, an attenuated strain used as a vaccine against leprosy and tuberculosis. The presence of M. leprae and BCG was observed in more than 80% of the cells after 24 h of exposure. However, BCG but not M. leprae was able to induce TNF-alpha secretion in these cells. Moreover, THP-1 cells treated simultaneously with BCG and M. leprae secreted lower levels of TNF-alpha compared to cells incubated with BCG alone. M. leprae was able, however, to induce TNF-alpha secretion both in blood-derived monocytes as well as in THP-1 cells pretreated with phorbol myristate acetate. The inclusion of streptomycin in our cultures, together with the fact that the use of both gamma-irradiated M. leprae and heat-killed BCG gave similar results, indicate that the differences observed were not due to differences in viability but in intrinsic properties between M. leprae and BCG. These data suggest that the capacity of M. leprae to induce TNF-alpha is dependent on the stage of cell maturation and emphasize the potential of this model to explore differences in the effects triggered by vaccine strain versus pathogenic species of mycobacteria on the host cell physiology and metabolism.

Peroxisome proliferator-activated receptors beta/delta: emerging roles for a previously neglected third family member.

PURPOSE OF REVIEW: Peroxisome proliferator-activated receptors alpha, beta/delta and gamma are members of the nuclear receptor superfamily. They mediate the effects of fatty acids and their derivatives at the transcriptional level, and are considered to be lipid sensors that participate in the regulation of energy homeostasis. Compared with the alpha and gamma peroxisome proliferator-activated receptor isotypes, peroxisome proliferator-activated receptor beta functions have long remained an enigma. In this review, we focus on emerging knowledge about peroxisome proliferator-activated receptor beta activation and roles. RECENT FINDINGS: We review recent data that suggest key roles in basic cell functions, such as proliferation, differentiation and survival, and in embryonic development and lipid metabolism in peripheral tissues. SUMMARY: The newly unveiled roles of peroxisome proliferator-activated receptor beta in important basic cell functions certainly justify a further exploration of its potential as a therapeutic target in pathologies such as metabolic syndrome X or skin diseases.

FXYD7 is a brain-specific regulator of Na,K-ATPase alpha 1-beta isozymes.

Recently, corticosteroid hormone-induced factor (CHIF) and the gamma-subunit, two members of the FXYD family of small proteins, have been identified as regulators of renal Na,K-ATPase. In this study, we have investigated the tissue distribution and the structural and functional properties of FXYD7, another family member which has not yet been characterized. Expressed exclusively in the brain, FXYD7 is a type I membrane protein bearing N-terminal, post-translationally added modifications on threonine residues, most probably O-glycosylations that are important for protein stabilization. Expressed in Xenopus oocytes, FXYD7 can interact with Na,K-ATPase alpha 1-beta 1, alpha 2-beta 1 and alpha 3-beta 1 but not with alpha-beta 2 isozymes, whereas, in brain, it is only associated with alpha 1-beta isozymes. FXYD7 decreases the apparent K(+) affinity of alpha 1-beta 1 and alpha 2-beta 1, but not of alpha 3-beta1 isozymes. These data suggest that FXYD7 is a novel, tissue- and isoform-specific Na,K-ATPase regulator which could play an important role in neuronal excitability.

The peroxisomal Acyl-CoA thioesterase Pte1p from Saccharomyces cerevisiae is required for efficient degradation of short straight chain and branched chain fatty acids.

The role of the Saccharomyces cerevisae peroxisomal acyl-coenzyme A (acyl-CoA) thioesterase (Pte1p) in fatty acid beta-oxidation was studied by analyzing the in vitro kinetic activity of the purified protein as well as by measuring the carbon flux through the beta-oxidation cycle in vivo using the synthesis of peroxisomal polyhydroxyalkanoate (PHA) from the polymerization of the 3-hydroxyacyl-CoAs as a marker. The amount of PHA synthesized from the degradation of 10-cis-heptadecenoic, tridecanoic, undecanoic, or nonanoic acids was equivalent or slightly reduced in the pte1Delta strain compared with wild type. In contrast, a strong reduction in PHA synthesized from heptanoic acid and 8-methyl-nonanoic acid was observed for the pte1Delta strain compared with wild type. The poor catabolism of 8-methyl-nonanoic acid via beta-oxidation in pte1Delta negatively impacted the degradation of 10-cis-heptadecenoic acid and reduced the ability of the cells to efficiently grow in medium containing such fatty acids. An increase in the proportion of the short chain 3-hydroxyacid monomers was observed in PHA synthesized in pte1Delta cells grown on a variety of fatty acids, indicating a reduction in the metabolism of short chain acyl-CoAs in these cells. A purified histidine-tagged Pte1p showed high activity toward short and medium chain length acyl-CoAs, including butyryl-CoA, decanoyl-CoA and 8-methyl-nonanoyl-CoA. The kinetic parameters measured for the purified Pte1p fit well with the implication of this enzyme in the efficient metabolism of short straight and branched chain fatty acyl-CoAs by the beta-oxidation cycle.

Human invariant V alpha 24-J alpha Q TCR supports the development of CD1d-dependent NK1.1+ and NK1.1- T cells in transgenic mice.

A sizable fraction of T cells expressing the NK cell marker NK1.1 (NKT cells) bear a very conserved TCR, characterized by homologous invariant (inv.) TCR V alpha 24-J alpha Q and V alpha 14-J alpha 18 rearrangements in humans and mice, respectively, and are thus defined as inv. NKT cells. Because human inv. NKT cells recognize mouse CD1d in vitro, we wondered whether a human inv. V alpha 24 TCR could be selected in vivo by mouse ligands presented by CD1d, thereby supporting the development of inv. NKT cells in mice. Therefore, we generated transgenic (Tg) mice expressing the human inv. V alpha 24-J alpha Q TCR chain in all T cells. The expression of the human inv. V alpha 24 TCR in TCR C alpha(-/-) mice indeed rescues the development of inv. NKT cells, which home preferentially to the liver and respond to the CD1d-restricted ligand alpha-galactosylceramide (alpha-GalCer). However, unlike inv. NKT cells from non-Tg mice, the majority of NKT cells in V alpha 24 Tg mice display a double-negative phenotype, as well as a significant increase in TCR V beta 7 and a corresponding decrease in TCR V beta 8.2 use. Despite the forced expression of the human CD1d-restricted TCR in C alpha(-/-) mice, staining with mCD1d-alpha-GalCer tetramers reveals that the absolute numbers of peripheral CD1d-dependent T lymphocytes increase at most by 2-fold. This increase is accounted for mainly by an increased fraction of NK1.1(-) T cells that bind CD1d-alpha-GalCer tetramers. These findings indicate that human inv. V alpha 24 TCR supports the development of CD1d-dependent lymphocytes in mice, and argue for a tight homeostatic control on the total number of inv. NKT cells. Thus, human inv. V alpha 24 TCR-expressing mice are a valuable model to study different aspects of the inv. NKT cell subset.

Different epitopes of the LACK protein are recognized by V beta 4 V alpha 8 CD4+ T cells in H-2b and H-2d mice susceptible to Leishmania major.

After inoculation of Leishmania major, a rapid production of IL-4 by LACK-specific CD4+ T cells has been shown to drive Th2 cell development in susceptible mice i.e. BALB/c and C57BL/6 mice rendered susceptible by neutralization of IFN-gamma at the onset of infection. Here, we showed that peptide AA 156-173 induced an early IL-4 mRNA expression not only in BALB/c mice but also in resistant B10.D2 mice when IFN-gamma is neutralized. Epitope mapping of LACK protein demonstrated that peptide containing AA 293-305 induced early IL-4 mRNA transcripts in susceptible H-2b mice i.e. BALB/b and resistant C57BL/6 mice when IFN-gamma is neutralized. Stringently, the early IL-4 response to the H-2d (AA 156-173) or the H-2b (AA 293-305) epitopes occurred in V beta 4 V alpha 8 CD4+ T cells from either H-2d or H-2b susceptible mice, respectively.

Cutting edge: influence of the TCR V beta domain on the avidity of CD1d:alpha-galactosylceramide binding by invariant V alpha 14 NKT cells.

CD1d tetramers loaded with alpha-galactosylceramide (alpha-GalCer) bind selectively to mouse invariant Valpha14 (Valpha14i) NKT cells and their human counterparts. Whereas tetramer binding strictly depends on the expression of a Valpha14-Jalpha18 chain in murine NKT cells, the associated beta-chain (typically expressing Vbeta8.2 or Vbeta7) appears not to influence tetramer binding. In this study, we describe novel alpha-GalCer-loaded mouse and human CD1d-IgG1 dimers, which revealed an unexpected influence of the TCR-beta chain on the avidity of CD1d:alpha-GalCer binding. A subset of Valpha14i NKT cells clearly discriminated alpha-GalCer bound to mouse or human CD1d on the basis of avidity differences conferred by the Vbeta domain of the TCR-beta chain, with Vbeta8.2 conferring higher avidity binding than Vbeta7.

Malignant transformation of DMBA/TPA-induced papillomas and nevi in the skin of mice selectively lacking retinoid-X-receptor alpha in epidermal keratinocytes.

Retinoid-X-receptor alpha (RXRalpha), a member of the nuclear receptor (NR) superfamily, is a ligand-dependent transcriptional regulatory factor. It plays a crucial role in NR signalling through heterodimerization with some 15 NRs. We investigated the role of RXRalpha and its partners on mouse skin tumor formation and malignant progression upon topical DMBA/TPA treatment. In mutants selectively ablated for RXRalpha in keratinocytes, epidermal tumors increased in size and number, and frequently progressed to carcinomas. As keratinocyte-selective peroxisome proliferator-activated receptor gamma (PPARgamma) ablation had similar effects, RXRalpha/PPARgamma heterodimers most probably mediate epidermal tumor suppression. Keratinocyte-selective RXRalpha-null and vitamin-D-receptor null mice also exhibited more numerous dermal melanocytic growths (nevi) than control mice, but only nevi from RXRalpha mutant mice progressed to invasive human-melanoma-like tumors. Distinct RXRalpha-mediated molecular events appear therefore to be involved, in keratinocytes, in cell-autonomous suppression of epidermal tumorigenesis and malignant progression, and in non-cell-autonomous suppression of nevi formation and progression. Our study emphasizes the crucial role of keratinocytes in chemically induced epidermal and melanocytic tumorigenesis, and raises the possibility that they could play a similar role in UV-induced tumorigenesis, notably in nevi formation and progression to melanoma.

Mtp-40 and alpha antigen gene fragment amplification for the detection of Mycobacterium tuberculosis in Colombian clinical specimens

In this study, the use of Mtp-40 and alpha antigen polymerase chain reaction (PCR) amplification fragments for the precise tuberculosis (TB) diagnosis was evaluated. One hundred and ninety two different samples were obtained from 113 patients with suspected TB. Mtp-40 and alpha antigen protein genes were amplified by the PCR technique and compared to both the "gold standard" (culture) test, as well as the clinical parameters (including a clinical record and X-ray film exam in 113 patients). Thirty-eight of the 113 patients had a presumptive clinical diagnosis of TB; 74% being detected by PCR technique, 58% by culture and 44% by direct microscopic visualization. Weconclude that it is possible to use PCR as a suitable technique for the detection of any mycobacteria by means of the alpha antigen product, or the specific infection of Mycobacterium tuberculosis by means of the mtp-40 gene. This might be a good supporting tool in difficult clinical TB diagnosis and pauci-bacillary cases.

Increased flow of fatty acids toward beta-oxidation in developing seeds of Arabidopsis deficient in diacylglycerol acyltransferase activity or synthesizing medium-chain-length fatty acids.

Synthesis of polyhydroxyalkanoates (PHAs) from intermediates of fatty acid beta-oxidation was used as a tool to study fatty acid degradation in developing seeds of Arabidopsis. Transgenic plants expressing a peroxisomal PHA synthase under the control of a napin promoter accumulated PHA in developing seeds to a final level of 0. 06 mg g(-1) dry weight. In plants co-expressing a plastidial acyl-acyl carrier protein thioesterase from Cuphea lanceolata and a peroxisomal PHA synthase, approximately 18-fold more PHA accumulated in developing seeds. The proportion of 3-hydroxydecanoic acid monomer in the PHA was strongly increased, indicating a large flow of capric acid toward beta-oxidation. Furthermore, expression of the peroxisomal PHA synthase in an Arabidopsis mutant deficient in the enzyme diacylglycerol acyltransferase resulted in a 10-fold increase in PHA accumulation in developing seeds. These data indicate that plants can respond to the inadequate incorporation of fatty acids into triacylglycerides by recycling the fatty acids via beta-oxidation and that a considerable flow toward beta-oxidation can occur even in a plant tissue primarily devoted to the accumulation of storage lipids.

Roles of mGluR5 in synaptic function and plasticity of the mouse thalamocortical pathway.

The group I metabotropic glutamate receptor 5 (mGluR5) has been implicated in the development of cortical sensory maps. However, its precise roles in the synaptic function and plasticity of thalamocortical (TC) connections remain unknown. Here we first show that in mGluR5 knockout (KO) mice bred onto a C57BL6 background cytoarchitectonic differentiation into barrels is missing, but the representations for large whiskers are identifiable as clusters of TC afferents. The altered dendritic morphology of cortical layer IV spiny stellate neurons in mGluR5 KO mice implicates a role for mGluR5 in the dendritic morphogenesis of excitatory neurons. Next, in vivo single-unit recordings of whisker-evoked activity in mGluR5 KO adults demonstrated a preserved topographical organization of the whisker representation, but a significantly diminished temporal discrimination of center to surround whiskers in the responses of individual neurons. To evaluate synaptic function at TC synapses in mGluR5 KO mice, whole-cell voltage-clamp recording was conducted in acute TC brain slices prepared from postnatal day 4-11 mice. At mGluR5 KO TC synapses, N-methyl-D-aspartate (NMDA) currents decayed faster and synaptic strength was more easily reduced, but more difficult to strengthen by Hebbian-type pairing protocols, despite a normal developmental increase in alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR)-mediated currents and presynaptic function. We have therefore demonstrated that mGluR5 is required for synaptic function/plasticity at TC synapses as barrels are forming, and we propose that these functional alterations at the TC synapse are the basis of the abnormal anatomical and functional development of the somatosensory cortex in the mGluR5 KO mouse.

Isoelectric focusing of alpha-1 acid glycoprotein (orosomucoid) in immobilized pH-gradients with 8M urea: detection of its desialylated variants using an alkaline phosphatase-linked secondary antibody system.

A method allowing a clear separation of the different variants of desialylated alpha 1-acid glycoprotein (orosomucoid) has been developed using isoelectric focusing in immobilized pH gradients, supplemented with 8 M urea and 2% v/v 2-mercaptoethanol. Immunoblotting with two antibody-steps afforded high sensitivity and permitted the detection of about 700 pg of alpha 1-acid glycoprotein in a 20 microL plasma sample diluted 1:28 672. A one year old bloodstrain, kept at room temperature, could easily be phenotyped.