Impact of genetic background on placental glycogen storage in mice.

129 and C57BL/6 are two of the most widely used laboratory mouse strains. While it is well known that genetic modifiers between the two strains can directly influence embryonic and adult phenotypes, less is known regarding morphological differences in placental development. Here we identify differences in the junctional zone, glycogen storage and the maternal-fetal interface between these two strains and provide examples where these differences impact the phenotypic characterisation of placental mutations.

[Multiple sclerosis management system 3D. Moving from documentation towards management of patients]. / Multiple-Sklerose-Dokumentationssystem 3D. Von der Dokumentation zum Patientenmanagement.

BACKGROUND: The increasing therapeutic options for relapsing-remitting multiple sclerosis require a specific treatment and risk management to recognize the individual response as well as potential side effects. To switch from pure MS documentation to MS management by implementing a new multiple sclerosis management and documentation tool may be of importance. METHOD: This article presents the novel computer-based patient management system "multiple sclerosis management system 3D" (MSDS 3D). RESULTS: MSDS 3D allows documentation and visualization of visit schedules and mandatory examinations via defined study modules by integration of data input from patients, attending physicians and MS nurses. It provides forms for the documentation of patient visits as well as clinical and diagnostic findings. Information is collected via interactive touch screens. A specific module which is part of MSDS 3D's current version allows the monthly monitoring of patients under treatment with natalizumab. A checklist covering clinical signs of progressive multifocal leukoencephalopathy (PML) and a detailed questionnaire about the handling of natalizumab in practice have additionally been added. DISCUSSION: The MS patient management system MSDS 3D has successfully been implemented and is currently being evaluated in a multi-centre setting. Advanced assessment of patient data may allow improvements in clinical practice and research work. The addition of a checklist and a questionnaire into the natalizumab module may support the recognition of PML during its early, treatable course.

BACs as tools for the study of genomic imprinting.

Genomic imprinting in mammals results in the expression of genes from only one parental allele. Imprinting occurs as a consequence of epigenetic marks set down either in the father's or the mother's germ line and affects a very specific category of mammalian gene. A greater understanding of this distinctive phenomenon can be gained from studies using large genomic clones, called bacterial artificial chromosomes (BACs). Here, we review the important applications of BACs to imprinting research, covering physical mapping studies and the use of BACs as transgenes in mice to study gene expression patterns, to identify imprinting centres, and to isolate the consequences of altered gene dosage. We also highlight the significant and unique advantages that rapid BAC engineering brings to genomic imprinting research.

[MS registry in Germany--design and first results of the pilot phase]. / MS-Register in Deutschland -- Design und erste Ergebnisse der Pilotphase.

In the summer of 2001, a nationwide epidemiological multiple sclerosis (MS) register was initiated under the auspices of the German MS Society (DMSG). This project aimed at collecting epidemiological data on the number of patients with MS, course of the disease, and their social situation in Germany. During the 2-year pilot phase, five MS centers with various regional differences and treatment methods participated, leading to a representative selection of patients. In December 2003, standardised data sets of 3,458 MS patients were available for evaluation. After examining the quality of the data, 3,223 sets remained for further analysis. The demographics were similar to those obtained from other epidemiological studies: 72% of the patients were female, mean age was 42.9+/-11.2 years, mean disease duration 12.6+/-8.7 years, and 64% suffered from the relapsing-remitting form of the disease. The median EDSS was 3.0, and 69% of patients had an EDSS

The use of multiple sclerosis databases at neurological university hospitals in Germany.

The development of easy-to-use, clinically oriented multiple sclerosis (MS) database programs has been started, thus paving the way for MS centers to computerize their patient records and to improve quality management To evaluate the prevalence of such programs at German neurological hospitals, a questionnaire was designed and sent to all clinic directors. With a return of more than 92%, it became evident that MS databases are still being used only by a minority of 22% on a regular basis. We did not recognize the predominance of a single program. A new MS database system that is being presently implemented in Germany is described.

[The Multiple Sclerosis Documentation System MSDS. Discussion of a documentation standard for multiple sclerosis]. / Das Multiple-Sklerose-Dokumentationssystem MSDS. Diskussionsgrundlage für einen Dokumentationsstandard Multiple Sklerose?

The MSDS (multiple sclerosis documentation system) has been developed at the Department of Neurology, Technical University of Dresden, Germany, during the last 4 years. The first version of this database application has been in use since October 2000. The MSDS manages information on MS patients, their treating physicians, patient history (symptoms, other diseases, biographical history, family history, habits, medication), clinical signs, results of laboratory examinations (blood chemistry, autoantibodies, borrelia serology, evoked potentials, cranial and spinal cord magnetic resonance imaging), clinical scores relevant for MS, and biosamples. In principle, MSDS allows online data input and semiautomatically generates reports to all general practitioners and neurologists treating the respective patient. Patient information sheets and internal treatment guidelines are part of the system. During a 3-month evaluation, the first version of MSDS was tested at eight university multiple sclerosis ambulatory care units and one general neurology hospital. The overall judgement was favorable. Suggestions for changes and improvements, as well as practical experiences, were considered when developing MSDS 2.0, which will be available by the end of 2001.

Human autoreactive CD4+ T cells from naive CD45RA+ and memory CD45RO+ subsets differ with respect to epitope specificity and functional antigen avidity.

T cells with specificity for self-Ags are normally present in the peripheral blood, and, upon activation, may target tissue Ags and become involved in the pathogenesis of autoimmune processes. In multiple sclerosis, a demyelinating disease of the CNS, it is postulated that inflammatory damage is initiated by CD4+ T cells reactive to myelin Ags. To investigate the potential naive vs memory origin of circulating myelin-reactive cells, we have generated myelin basic protein (MBP)- and tetanus toxoid-specific T cell clones from CD45RA+/RO- and CD45RO+/RA- CD4+ T cell subsets from the peripheral blood of multiple sclerosis patients and controls. Our results show that 1) the response to MBP, different from that to TT, predominantly emerges from the CD45RA+ subset; 2) the reactivity to immunodominant MBP epitopes mostly resides in the CD45RA+ subset; 3) in each individual, the recognition of single MBP epitopes is skewed to either subset, with no overlap in the Ag fine specificity; and 4) in spite of a lower expression of costimulatory and adhesion molecules, CD45RA+ subset-derived clones recognize epitopes with higher functional Ag avidity. These findings point to a central role of the naive CD45RA+ T cell subset as the source for immunodominant, potentially pathogenic effector CD4+ T cell responses in humans.

Differential effects of phosphodiesterase type 4-specific inhibition on human autoreactive myelin-specific T cell clones.

Proinflammatory cytokines, secreted by autoreactive CD4+ T lymphocytes may contribute to the pathogenesis of several human autoimmune diseases, including multiple sclerosis (MS). Since the antigen specificities of these T cells are not known at present, therapeutic strategies aiming at common effector pathways, in particular cytokine secretion, may be more feasible in the near future. We have studied the influence of the isoenzyme-specific phosphodiesterase inhibitor rolipram on the proliferation and cytokine secretion of human myelin basic protein-specific T cell clones. The inhibition of proliferation correlated with interference with the IL-2/IL-2 receptor system, while the effects of rolipram on several T helper 1-(TNF-alpha, TNF-beta, IFN-gamma) and T helper 2-like cytokines (IL-4, IL-13) as well as IL-10 revealed an interesting drug profile, with preferential inhibition of TNF-beta, TNF-alpha and IL-10. This profile suggest that rolipram differs from other currently used immunomodulatory drugs.

Interferon-beta interferes with the proliferation but not with the cytokine secretion of myelin basic protein-specific, T-helper type 1 lymphocytes.

Interferon-beta (IFN-beta) has beneficial effects on the frequency and severity of relapses, as well as on disease progression in patients suffering from relapsing-remitting MS. Its mode of action, however, is not completely understood. Previous studies on T-lymphocyte bulk cultures and T-lymphocyte lines with specificity for different antigens suggested that the drug might partially act via suppression of T-cell proliferation and secretion of proinflammatory cytokines like interferon-gamma (IFN-gamma) and/or tumor necrosis factor-alpha (TNF-alpha). In this study we investigated the effects of human recombinant IFN-beta 1b on proliferation, interleukin 2 (IL-2) receptor (IL-2R) alpha-chain upregulation, and cytokine and chemokine secretion of myelin basic protein-reactive, MS patient-derived T-cell clones secreting T-helper type 1 (Th1) cytokines. IFN-beta partially suppressed both antigen- and IL-2-driven proliferation of these cells without affecting the expression of either IL-2 or IL-2R alpha-chain. There was no inhibitory effect on the secretion of IFN-gamma, TNF-alpha, and macrophage inflammatory protein (MIP)-1 alpha, but release was rather slightly enhanced. In conclusion, while IFN-beta does reduce proliferation of Th1-like, MBP-specific T cells in vitro, the drug does not result in overall dysfunction of these cells. Therefore, the effect of IFN-beta on MS may not depend on a primary inhibition of potentially encephalitogenic T lymphocytes.

In vitro modulation of human, autoreactive MBP-specific CD4 + T-cell clones by cyclosporin A.

Cyclosporin A (CsA) is a potent immunosuppressant affecting many components of cellular and humoral immunity. Its main action probably results from inhibition of T-lymphocyte activation and interference with secretion of cytokines like IL-2, IL-4, IFN-gamma and TNF-alpha. Correspondingly, CsA has beneficial effects on the course of several autoimmune diseases thought to be mediated by T-lymphocytes, including a mild effect on multiple sclerosis. We exposed CD4 + cytotoxic T-lymphocytes specific for myelin basic protein, a putative target autoantigen in MS, to CsA in vitro, and determined the drug's effects on proliferation, expression of high affinity IL-2R, secretion of the proinflammatory cytokines IFN-gamma and TNF-alpha as well as on the secretion of the chemokines MIP-1 alpha and MIP-1 beta. In all instances, we observed a partial to complete inhibition. In contrast, the response of activated cells to IL-2 was resistant to CsA. Our observations are in line with results obtained in different experimental systems. The discrepancy between the profound inhibition of T-cells and the modest therapeutic effects on MS is discussed.

Quantification of cytokine mRNA expression by RT PCR in samples of previously frozen blood.

In order to facilitate cytokine mRNA detection in blood cells, we have developed a highly reproducible and easily performed RNA isolation method for use with whole blood. Previously frozen human whole blood samples were lysed in guanidine thiocyanate solution to isolate total RNA. After reverse transcription a PCR method was applied to detect beta-actin and cytokine mRNA expression (interleukin-(IL)2, IL4, IL10, tumor necrosis factor alpha (TNF alpha) and interferon gamma (IFN gamma)). The presence of cDNA was confirmed by agarose gel electrophoresis and quantitated on-line using sequence-specific fluorochrome labeled internal oligonucleotide probes. This quantitative method is based on the cleavage of fluorescent dye labeled probes by the 5' --> 3' endonuclease activity of the Taq DNA polymerase during PCR and measurement of fluorescence intensity by a Sequence Detector System. The signal generated was directly proportional to the starting copy number of target molecules in the sample over 6 log concentrations and quantitative analysis of cDNA concentrations was performed in comparison to beta-actin or cytokine cDNA standards. mRNAs coding for beta-actin and TNF alpha were readily detectable in cDNAs prepared from the whole blood of eight healthy donors, while the other cytokines were expressed in lower amounts (IFN gamma, IL10) or were undetectable (IL2, IL4). The assay described is highly reproducible, requires no post PCR manipulation of the amplicons and permits the analysis of several hundred PCR reactions per day. Using this method it is possible to detect and quantify cytokine mRNA expression reliably in small amounts of previously frozen blood even after storage of samples for at least several months.

T cell antigenic and neuritogenic activity of recombinant human peripheral myelin P2 protein.

The major neuritogenic protein of peripheral nerve myelin is the P2 protein. Human P2 is a candidate autoantigen in inflammatory demyelinating diseases of the peripheral nervous system. Since human P2 is not readily available, we produced full-length recombinant human P2 protein (rhP2) in Escherichia coli. RhP2 was recognized by neuritogenic rat T cell lines and induced experimental autoimmune neuritis in Lewis rats. Production of rhP2 allowed the generation of human T cell lines reactive to the autologous protein. Studies of human T cell autoreactivity as well as efforts to use hP2 as a tolerogen will be facilitated by the large-scale expression of rhP2.

Mafosfamide induces DNA fragmentation and apoptosis in human T-lymphocytes. A possible mechanism of its immunosuppressive action.

Cyclophosphamide, an alkylating agent belonging to the family of nitrogen mustards, is commonly used to treat progressive autoimmune diseases in humans. At the molecular level, its cytotoxicity results from DNA double strand crosslinks and, at higher concentrations, from DNA strand breaks. At the cellular level, cyclophosphamide may selectively affect mature lymphocytes with relative sparing of the respective precursor cells. In this study, we show that 4-hydroxycyclophosphamide (4-OH-CP), the active metabolite of cyclophosphamide, induces apoptosis in mature human lymphocytes at concentrations that are achieved in vivo. Since cyclophosphamide requires enzymatic conversion in the liver to yield its active metabolite, 4-OH-CP was generated in vitro by non-enzymatic hydrolysis of mafosfamide. Apoptotic cell death of lymphocytes was characterized by typical morphological changes, nucleosomal DNA fragmentation, and quantified by 3'-OH end labeling of fragmented DNA. The percentage of apoptotic cells both depended on drug concentration and time of exposure. Cycloheximide or ZnSO4 did not suppress 4-OH-CP induced apoptosis. Etoposide, a topoisomerase II inhibitor known to induce apoptosis in human tumor cell lines like 4-OH-CP, did induce detectable DNA fragmentation in only a minor proportion of T-lymphocytes but suppressed T-cell proliferation.

T lymphocyte recognition sites on peripheral nerve myelin P0 protein.

Synthetic peptides corresponding to the extracellular and cytoplasmic domain of bovine (b) or rat (r) peripheral myelin P0 protein were used to establish a total of 50 short-term T cell lines (TCL) from blood of eight healthy subjects. Despite expressing different HLA-DR and HLA-DQ specificities, one or more TCL (range 1-16) specific for peptide bovine P0 19-38 could be isolated from the blood of each donor. Therefore, this peptide covers an immunodominant T cell recognition site in humans. However, when testing seven bP0-19-38-specific TCL derived from blood of two healthy subjects for recognition of the corresponding human P0 sequence, no TCL showed any proliferative response. Bovine P0-19-38 differs in only two amino acid residues from the human peptide. This observation stresses the necessity for using homologous antigens when screening for T cell-mediated autoreactivity to myelin antigens in humans. Unexpectedly, we failed to establish a single P0 peptide-specific TCL from blood of four patients with acute Guillain-Barré syndrome (GBS), in which P0 is considered a putative target autoantigen. As already suggested by others, this could indicate that T cell responses to P0 do not play a pathogenic role in all GBS cases. Alternatively, in these four patients neuritogenic P0-specific T lymphocytes may have been sequestrated to peripheral nerves.

Human T lymphocytes distinguish bovine from human P2 peripheral myelin protein: implications for immunological studies on inflammatory demyelinating neuropathies.

In patients with inflammatory demyelinating neuropathy, which is possibly mediated by autoreactive, myelin-specific T lymphocytes, most studies focusing on immune responses to the major neuritogenic myelin protein P2 have been performed with bovine P2. However, the primary structure of bovine P2 differs from the human protein by nine amino acid residues that may profoundly influence the antigen recognition by T lymphocytes. We purified bovine and human P2 from peripheral nervous tissue and established a total of 19 T cell lines (TCL) reactive with bovine P2 from blood of two patients with acute Guillain-Barré syndrome (n = 5 TCL) and from six healthy individuals. Only four of these TCL, all raised from the blood of the GBS patients, transiently cross-recognized human P2 protein. Our results suggest that the use of human autoantigen may be crucial for the characterization of T cellular immune responses against P2 protein both in patients with inflammatory demyelinating neuropathy and in healthy controls.

Measles virus-directed responses of CD4+ T lymphocytes in MS patients and healthy individuals.

To analyze the antigen specificities of measles virus (MV)-reactive CD4+ T cells in multiple sclerosis (MS) patients as compared with those of healthy donors, we established 492 MV-reactive short-term T-cell lines (TCL) from blood of 12 MS patients (n = 243 TCL) and 12 healthy subjects (n = 249 TCL). We determined antigen specificities of these TCL by proliferative responses to optimal concentrations of recombinant MV structural proteins (MV-SP) and human myelin basic protein (MBP). In both donor groups, there was a dominant reactivity against the MV fusion protein and the MV nucleocapsid protein. However, there was a substantial heterogeneity of T-cellular polypeptide specificities among MS patients as well as among healthy individuals, which was true even in subjects sharing identical HLA-DR and HLA-DQ haplotypes. By comparing the T-cell antigen specificity patterns obtained in both donor populations, we found decreased percentages of TCL reactive with the MV fusion protein, the hemagglutinin, and the phosphoprotein in MS patients, but these differences failed to reach statistical significance. None of the 492 MV-specific TCL, nor an additional 276 TCL, showed any reactivity to MBP. Therefore, we did not detect any MS-specific pattern of T-cell responses to MV-SP. Furthermore, our study suggests that mechanisms other than molecular mimicry between MV-SP and MBP may cause myelin-directed autoimmunity.

The spectrum of immune responses to Campylobacter jejuni and glycoconjugates in Guillain-Barré syndrome and in other neuroimmunological disorders.

An acute infectious illness frequently precedes the Guillain-Barré syndrome. Recently, Campylobacter jejuni was claimed to be a predominant precipitating agent that may also trigger a humoral immune response to glycoconjugates of peripheral myelin in Guillain-Barré syndrome. Because of conflicting reports, we determined the frequency of a recent infection with C. jejuni in 38 patients with Guillain-Barré syndrome using a highly sensitive and specific immunoblot technique, and of the presence of circulating antibodies to gangliosides. We detected IgM and/or IgG C. jejuni directed antibodies in 15 of 38 patients with Guillain-Barré syndrome. In contrast, only 7 of 39 healthy control subjects, 3 of 20 patients with multiple sclerosis, and 2 of 72 patients with neuroborreliosis showed IgA or IgM antibody responses to C. jejuni. In Guillain-Barré syndrome, C. jejuni-specific antibodies were predominantly directed to outer membrane proteins of one specific serotype, Lior 11, whereas the most common serotype associated with enteritis in Germany is Lior 4. Two of 27 patients with Guillain-Barré syndrome had ganglioside-specific IgA antibodies; 1 of 32 patients, antibodies of IgM; and 4 of 31 patients, antibodies of IgG class. There was no correlation between severity, type (axonal versus demyelinating), and outcome of the disease and the presence or absence of a humoral immune response to C. jejuni or to glycoconjugates. Our findings do not support previous suggestions that a preceding C. jejuni infection heralds a poorer outcome or that antibodies to gangliosides carry prognostic significance.(ABSTRACT TRUNCATED AT 250 WORDS)