Oligodeoxynucleotides containing unmethylated cytosine-guanine motifs are effective immunostimulants against pneumococcal meningitis in the immunocompetent and neutropenic host.

BACKGROUND: Bacterial meningitis is a fatal disease with a mortality up to 30% and neurological sequelae in one fourth of survivors. Available vaccines do not fully protect against this lethal disease. Here, we report the protective effect of synthetic oligodeoxynucleotides containing unmethylated cytosine-guanine motifs (CpG ODN) against the most frequent form of bacterial meningitis caused by Streptococcus pneumoniae. METHODS: Three days prior to the induction of meningitis by intracerebral injection of S. pneumoniae D39, wild-type and Toll-like receptor (TLR9)-/- mice received an intraperitoneal injection of 100 µg CpG ODN or vehicle. To render mice neutropenic, anti-Ly-6G monoclonal antibody was daily administrated starting 4 days before infection with a total of 7 injections. Kaplan-Meier survival analyses and bacteriological studies, in which mice were sacrificed 24 h and 36 h after infection, were performed. RESULTS: Pre-treatment with 100 µg CpG ODN prolonged survival of immunocompetent and neutropenic wild-type mice but not of TLR9-/- mice. There was a trend towards lower mortality in CpG ODN-treated immunocompetent and neutropenic wild-type mice. CpG ODN caused an increase of IL-12/IL-23p40 levels in the spleen and serum in uninfected animals. The effects of CpG ODN on bacterial concentrations and development of clinical symptoms were associated with an increased number of microglia in the CNS during the early phase of infection. Elevated concentrations of IL-12/IL-23p40 and MIP-1α correlated with lower bacterial concentrations in the blood and spleen during infection. CONCLUSIONS: Pre-conditioning with CpG ODN strengthened the resistance of neutropenic and immunocompetent mice against S. pneumoniae meningitis in the presence of TLR9. Administration of CpG ODN decreased bacterial burden in the cerebellum and reduced the degree of bacteremia. Systemic administration of CpG ODN may help to prevent or slow the progression to sepsis of bacterial CNS infections in healthy and immunocompromised individuals even after direct inoculation of bacteria into the intracranial compartments, which can occur after sinusitis, mastoiditis, open head trauma, and surgery, including placement of an external ventricular drain.

[Acute disseminated encephalomyelitis. Pathogenesis, diagnosis, treatment, and prognosis]. / Akute disseminierte Enzephalomyelitis. Pathogenese, Diagnose, Behandlung und Prognose.

Acute disseminated encephalomyelitis (ADEM) is typically a monophasic, demyelinating disease of the CNS that predominantly affects children. Typically, its clinical symptoms follow an infection or vaccination. In this regard, numerous viral and bacterial pathogens as well as several vaccinations have been associated with ADEM. Studies from animal models suggest that primary and secondary autoimmune responses may contribute to CNS inflammation and demyelination in ADEM. The diagnosis of ADEM is strongly suggested by a close temporal relationship between a viral infection or immunization and the onset of neurologic symptoms, and it is supported by extensive, multifocal, subcortical white-matter disease on brain magnetic resonance imaging. While mild lymphocytic pleocytosis and elevated proteins are detectable in the CSF in ADEM, oligoclonal bands are not always present. Treatment of this disorder consists of anti-inflammatory and immunosuppressive therapies, and the prognosis is generally considered favorable.

CD45 isoform expression in autoimmune myasthenia gravis.

In myasthenia gravis (MG), humoral and cellular immune mechanisms are involved in the autoimmune pathogenesis. In this study, we investigated the role of the CD45 molecule in MG, having recently reported an association in multiple sclerosis. CD45, a protein-tyrosine phophatase receptor type C (PTPRC), is essential for both thymic selection and peripheral activation of T and B cells. Our aims were to determine (a) the prevalence of a functional mutation in the CD45 gene (exon 4 77C --> G; prevalence analysis), and (b) the distribution of memory (CD45RO+) and naive (CD45RA+) T cells in the peripheral blood (subset analysis). T cells from 78 patients with generalised MG were stained with monoclonal antibodies against CD45RO, CD45RA, CD4 and CD8 and quantified by four-colour flow cytometry. The control panel for the prevalence analysis (a) consisted of 303 healthy individuals. (b) From those, 67 age- and sex-matched probands were randomly selected as controls for the subset analysis. Patients were stratified according to their MG onset age, thymic pathology and immunosuppressive treatment. Statistical analysis was performed by Fisher's exact test, asymptotic chi2 test, the two-sided Mann-Whitney test and Spearman's correlation coefficient. As a result, the 77C --> G mutation in exon 4 of the CD45 gene was found in 1 of 78 patients versus none of the 303 controls. Thus, no association was detected with this single nucleotide polymorphism in MG patients overall. Surprisingly, however, ratios of CD45RO+ to CD45RA+ T cells were lower among CD8+ T cells from patients with late-onset MG (P = 0.023). Thymoma patients also showed a similar trend among CD4+ and CD8+ T-cells, as expected. These differences were not related to immunosuppressive drug treatment or thymectomy (in the 67 informative patients). Since there is no other evidence for increased thymopoiesis in late-onset MG, we propose an altered subset balance in the circulation.

[New approaches in research of therapy of multiple sclerosis]. / Neue Forschungsansätze zur Therapie der multiplen Sklerose.

BACKGROUND: Multiple sclerosis is a chronic inflammatory demyelinating disease of the central nervous system. With a prevalence of 0.1-0.15% in Germany multiple sclerosis is the most common cause of severe disability in young adults. PATHOGENESIS: Epidemiological and family studies demonstrate the role of environmental and genetic factors in the pathogenesis of multiple sclerosis. Based on those observations and findings in experimental animal models, it is believed that multiple sclerosis is caused by an autoimmune process. However, target antigens and mechanisms leading to tissue destruction are largely unknown. THERAPY: Since the efficacy of current immunomodulatory and immunosuppressive therapies (beta-interferons, glatiramer acetate, mitoxantrone) is limited, it is necessary to develop new strategies for the treatment of multiple sclerosis. To reach this goal, a much better understanding of disease pathogenesis is necessary which takes into account the clinical, paraclincial and histopathological heterogeneity of the disease. CONCLUSION: Only further intensive research activity on basic mechanisms of disease pathogenesis and a consequent development of resulting therapeutic strategies--from animal models to phase III studies--will result in significant improvement of the long-term course of multiple sclerosis.

X-ray structure of HPr kinase: a bacterial protein kinase with a P-loop nucleotide-binding domain.

HPr kinase/phosphatase (HprK/P) is a key regulatory enzyme controlling carbon metabolism in Gram- positive bacteria. It catalyses the ATP-dependent phosphorylation of Ser46 in HPr, a protein of the phosphotransferase system, and also its dephosphorylation. HprK/P is unrelated to eukaryotic protein kinases, but contains the Walker motif A characteristic of nucleotide-binding proteins. We report here the X-ray structure of an active fragment of Lactobacillus casei HprK/P at 2.8 A resolution, solved by the multiwavelength anomalous dispersion method on a seleniated protein (PDB code 1jb1). The protein is a hexamer, with each subunit containing an ATP-binding domain similar to nucleoside/nucleotide kinases, and a putative HPr-binding domain unrelated to the substrate-binding domains of other kinases. The Walker motif A forms a typical P-loop which binds inorganic phosphate in the crystal. We modelled ATP binding by comparison with adenylate kinase, and designed a tentative model of the complex with HPr based on a docking simulation. The results confirm that HprK/P represents a new family of protein kinases, first identified in bacteria, but which may also have members in eukaryotes.

Mutations lowering the phosphatase activity of HPr kinase/phosphatase switch off carbon metabolism.

The oligomeric bifunctional HPr kinase/P-Ser-HPr phosphatase (HprK/P) regulates many metabolic functions in Gram-positive bacteria by phosphorylating the phosphocarrier protein HPr at Ser46. We isolated Lactobacillus casei hprK alleles encoding mutant HprK/Ps exhibiting strongly reduced phosphatase, but almost normal kinase activity. Two mutations affected the Walker motif A of HprK/P and four a conserved C-terminal region in contact with the ATP-binding site of an adjacent subunit in the hexamer. Kinase and phosphatase activity appeared to be closely associated and linked to the Walker motif A, but dephosphorylation of seryl-phosphorylated HPr (P-Ser-HPr) is not simply a reversal of the kinase reaction. When the hprKV267F allele was expressed in Bacillus subtilis, the strongly reduced phosphatase activity of the mutant enzyme led to increased amounts of P-Ser-HPr. The hprKV267F mutant was unable to grow on carbohydrates transported by the phosphoenolpyruvate:glycose phosphotransferase system (PTS) and on most non-PTS carbohydrates. Disrupting ccpA relieved the growth defect only on non-PTS sugars, whereas replacing Ser46 in HPr with alanine also restored growth on PTS substrates.

The HPr kinase from Bacillus subtilis is a homo-oligomeric enzyme which exhibits strong positive cooperativity for nucleotide and fructose 1,6-bisphosphate binding.

Carbon catabolite repression allows bacteria to rapidly alter the expression of catabolic genes in response to the availability of metabolizable carbon sources. In Bacillus subtilis, this phenomenon is controlled by the HPr kinase (HprK) that catalyzes ATP-dependent phosphorylation of either HPr (histidine containing protein) or Crh (catabolite repression HPr) on residue Ser-46. We report here that B. subtilis HprK forms homo-oligomers constituted most likely of eight subunits. Related to this complex structure, the enzyme displays strong positive cooperativity for the binding of its allosteric activator, fructose 1,6-bisphosphate, as evidenced by either kinetics of its phosphorylation activity or the intrinsic fluorescence properties of its unique tryptophan residue, Trp-235. It is further shown that activation of HPr phosphorylation by fructose 1,6-bisphosphate essentially occurs at low ATP and enzyme concentrations. A positive cooperativity was also detected for the binding of natural nucleotides or their 2'(3')-N-methylanthraniloyl derivatives, in either phosphorylation or fluorescence experiments. Most interestingly, quenching of the HprK tryptophan fluorescence by using either iodide or acrylamide revealed a heterogeneity of tryptophan residues within the population of oligomers, suggesting that the enzyme exists in two different conformations. This result suggests a concerted-symmetry model for the catalytic mechanism of positive cooperativity displayed by HprK.

p55-hGRF, a short natural form of the Ras-GDP exchange factor high yield production and characterization.

p55-hGRF, a natural short form of the guanine-nucleotide-releasing factor for p21-Ras from human brain, was expressed at high level in Escherichia coli as well as an engineered truncated form, p39-hGRF. A T7 polymerase expression system was used, resulting in the formation of insoluble cytoplasmic protein aggregates. The recombinant products were resolubilized, renatured and purified to homogeneity. The exchange activity of the refolded hGRF samples on H-Ras was comparable with that published for the soluble catalytic domain of the mouse counterpart, CDC25 Mm. Both p55-hGRF and p39-hGRF form dimers. We established a procedure to prepare and purify the complex with Ras. The results of the characterization study are consistent with a stoichiometry of 1:1 and an equilibrium between dimeric and monomeric forms of the complex.

Dimerization of Cdc25p, the guanine-nucleotide exchange factor for Ras from Saccharomyces cerevisiae, and its interaction with Sdc25p.

The oligomerization state of Cdc25p, the guanine nucleotide exchange factor for ras from yeast, was analyzed using different complementary approaches. The two-hybrid system showed that the C-terminal part of Cdc25p (Cdc25-Ct) can interact with itself but also with Sdc25p-Ct, the corresponding part of Sdc25p, the other guanine exchange factor from yeast. The homotropic interaction of Cdc25p-Ct has been confirmed in yeast using immunoprecipitation experiments with epitope-tagged and beta-galactosidase-fused polypeptides. No other component was required for this interaction, since dimerization was shown to occur with material synthesized in vitro. The size of Cdc25-Ct produced in Escherichia coli has been directly measured on gel filtration columns and corresponds to a dimer. The dimerization domain is localized in the same part of the molecule as the catalytic domain and the portion responsible for membrane localization. The biological relevance of dimerization is still an open question, however by allowing heterodimerization with Sdc25p it could permit a more complex combinatorial regulation of ras in yeast.

Evidence for a dimeric intermediate on the crystallization pathway of ribonuclease A.

Early steps in the crystallization process of pancreatic ribonuclease have been investigated by time-dependent fluorescence anisotropy, using a labeled protein as a fluorescent probe. Previous experiments have shown that steady-state fluorescence anisotropy is sensitive to protein-protein interactions and can be used to find new crystallization conditions. The present work describes an attempt, by means of time-resolved experiments, to detect and characterize species appearing in the early stages of the crystallization pathway. Fluorescence anisotropy decay was measured with synchrotron radiation as a light source under a variety of conditions where it is known that the solutions tend towards crystallization; the decay was analyzed by a maximum-entropy method that calculates a rotational correlation-time distribution. Fluorescence anisotropy originates in the Brownian rotatory motion of macromolecules and the values of the correlation times are related to the size and shape of different species present in the solution. In the presence of high salt concentrations, a bimodal distribution is always observed. Whereas a peak of protein monomer is still present, a second peak appears as a stable intermediate in the crystallization pathway. The correlation time of this new species varies between two and three times the correlation time of the monomer. The second peak is possibly the symmetrical dimer of the ribonuclease molecules commonly observed in all the high-salt crystal forms.

Crystallization of D-lactate dehydrogenase from Lactobacillus bulgaricus.

The D-lactate dehydrogenase (D-LDH) from Lactobacillus bulgaricus has been purified and co-crystallized with its cofactor NAD+. Crystals suitable for X-ray diffraction experiments have been obtained from an ammonium sulfate solution by the hanging-drop method. The crystals belong to the orthorhombic space group C222 (or C222(1)) with cell dimensions a = 76.5 A, b = 93.3 A, c = 118.4 A and one monomer of 37,000 daltons per asymmetric unit. They diffract beyond 3.0 A resolution. Sequence comparison suggests that D-LDHs have no evolutionary relationship to L-LDHs and belong instead to the family of the D-2-hydroxyacid dehydrogenases. The X-ray crystallographic structure of the D-LDH from Lactobacillus bulgaricus will be a decisive test of this hypothesis.

Crystal structure of a fluorescent derivative of RNase A.

The crystal structure of RNase A chemically modified with the fluorescent probe, N-[[(iodoacetyl)-amino]ethyl]-5-naphthylamine-1-sulfonic acid (1,5-IAENS), has been solved and refined to high resolution. It yields information on the mode of binding, the mobility of a probe commonly used in spectroscopic studies, and anion binding sites in RNase A. Trigonal crystals of the fluorescent derivative grown in sodium or cesium chloride and ammonium sulfate, pH 5.1, were nearly isomorphous with those of a semisynthetic RNase [DeMel, et al. (1992) J. Biol. Chem. 267, 247-256]. Refinement starting from semisynthetic RNase led to a model with R = 20% against 1.7-A diffraction data from crystals in ammonium sulfate and another model with R = 17% against 1.9-A data taken in the presence of 3 M NaCl. The second model contains three chloride ions: one is at the active site, and the other two are at molecular interfaces. Otherwise, the two models are very similar. The fluorophore has very little effect on the protein conformation. It is found to be covalently attached to the active site His-12 with the naphthyl group stacked on the imidazole ring of His-119. It remains largely accessible to solvent and in a polar environment on the protein surface, even though the fluorescence emission spectrum is blue shifted as it is in nonpolar solvents.