Designing fluoroquinolone breakpoints for Streptococcus pneumoniae by using genetics instead of pharmacokinetics-pharmacodynamics.

We determined fluoroquinolone microbiological resistance breakpoints for Streptococcus pneumoniae by using genetic instead of pharmacokinetic-pharmacodynamic parameters. The proposed microbiological breakpoints define resistance as the MIC at which >50% of the isolates carry quinolone resistance-determining region mutations and/or, if data are available, when Monte Carlo simulations demonstrate a <90% chance of bacteriological eradication. The proposed microbiological resistant breakpoints are as follows (in micrograms per milliliter): gatifloxacin, >0.25; gemifloxacin, >0.03; levofloxacin, >1; and moxifloxacin, >0.12. Monte Carlo simulations of the once daily 400-mg doses of gatifloxacin and 750-mg doses levofloxacin demonstrated a high level of target attainment (free-drug area under the concentration-time curve from 0 to 24 h/MIC ratio of 30) by using these new genetically derived breakpoints.

The neuregulin, glial growth factor 2, diminishes autoimmune demyelination and enhances remyelination in a chronic relapsing model for multiple sclerosis.

Glial growth factor 2 (GGF2) is a neuronal signal that promotes the proliferation and survival of the oligodendrocyte, the myelinating cell of the central nervous system (CNS). The present study examined whether recombinant human GGF2 (rhGGF2) could effect clinical recovery and repair to damaged myelin in chronic relapsing experimental autoimmune encephalomyelitis (EAE) in the mouse, a major animal model for the human demyelinating disease, multiple sclerosis. Mice with EAE were treated with rhGGF2 during both the acute and relapsing phases. Clinically, GGF2 treatment delayed signs, decreased severity, and resulted in statistically significant reductions in relapse rate. rhGGF2-treated groups displayed CNS lesions with more remyelination than in controls. This correlated with increased mRNA expression of myelin basic protein exon 2, a marker for remyelination, and with an increase in the CNS of the regulatory cytokine, interleukin 10, at both the RNA and protein levels. Thus, a beneficial effect of a neurotrophic growth factor has been demonstrated on the clinical, pathologic, and molecular manifestations of autoimmune demyelination, an effect that was associated with increased expression of a T helper 2 cytokine. rhGGF2 treatment may represent a novel approach to the treatment of multiple sclerosis.

The SIF binding element confers sis/PDGF inducibility onto the c-fos promoter.

The c-fos proto-oncogene is rapidly and transiently induced by a variety of extracellular stimuli. We have previously shown that conditioned media from v-sis transformed NRK cells rapidly induces a DNA binding protein which binds to a conserved sequence upstream of the human c-fos gene. We now show that purified recombinant c-sis/PDGF can induce this binding activity which we have termed SIF, for sis-inducible factor. Oligonucleotides which bind to the SIF protein will confer sis/PDGF inducibility onto a truncated, unresponsive c-fos promoter. However, sequences lying between -100 and -57 of the c-fos gene are required for this induction. The sis-responsive element functions independently of a region of dyad symmetry previously identified as the serum responsive element (SRE). The time course of c-fos expression driven by the sis-responsive element is similar to that mediated by the SRE. Unlike the SRE, which can respond to signals generated by sis/PDGF, serum or phorbol esters, the SIF binding element mediates c-fos induction only in response to sis/PDGF. The SRE and SIF elements function in an additive manner to stimulate the transcription of the c-fos gene in response to sis/PDGF.

Platelet-derived growth factor stimulates rapid polyphosphoinositide breakdown in fetal human fibroblasts.

The addition of human platelet-derived growth factor (PDGF) to confluent, quiescent cultures of human diploid fibroblasts induced the rapid breakdown of cellular polyphosphoinositides. The levels of 32P-labeled phosphatidylinositol 4,5-bisphosphate (PIP2), phosphatidylinositol 4-phosphate (PIP), and phosphatidylinositol (PI) decreased by 30 to 40% within 1 min after exposure of the cells to PDGF. The levels of PIP and PIP2 returned to their initial values within 3 and 10 min, respectively, after PDGF addition. The level of PI continued to increase after it had returned to control values and was up threefold within 30 min after PDGF addition. In cells prelabeled with myo-[3H]inositol PDGF caused an eightfold increase in the levels of inositol trisphosphate (IP3) within 2 min. Lesser increases, twofold and 1.3-fold, respectively, were seen in levels of inositol bisphosphate (IP2) and inositol monophosphate (IP). Within 10 min after PDGF addition the levels of all three inositol phosphates had decreased to control values. The levels of IP3 measured 2 min after PDGF addition depended on the PDGF concentration and were maximal at 5-10 ng/ml of PDGF. Similar concentrations of PDGF stimulate maximal cell growth and DNA synthesis in these cells.

Immunohistochemical staining of human brain with monoclonal antibodies that identify lymphocytes, monocytes, and the Ia antigen.

Using immunoperoxidase histochemistry, human brain sections obtained at biopsy were labeled with monoclonal antibodies which identify human lymphocyte subsets, monocytes, and the Ia antigen. Staining of a population of cells in white matter was present with the anti-Ia and the anti-M1 (monocyte-associated) antibodies but not with any of the 8 monoclonal antibodies which react with human T-cell subsets (anti-T1, 3, 4, 5, 6, 8, 10 and 12). The Ia antigen was present on 1-2% of cells in white matter, and approximately 5% of cells in white matter were M1-positive. Ia-positive cells demonstrated a pattern of diffuse surface membrane staining, whereas the M1 antigen appeared to cluster at proximal cell processes. Definitive identification of these cells as microglial cells, astrocytes or oligodendrocytes was not possible. These findings demonstrate that: (1) cells which bear the Ia and M1 determinants can be found in histologically normal human white matter, and (2) human oligodendrocytes do not react with monoclonal antibodies (anti-T5 and anti-T8) that identify human suppressor/cytotoxic cells.

CSF cells in multiple sclerosis: monoclonal antibody analysis and relationship to peripheral blood T-cell subsets.

Mononuclear cells were analyzed in CSF and blood of 102 patients with MS. In CSF, the majority (78%) of cells were T lymphocytes (T3+), and the ratio of inducer (T4+) to suppressor/cytotoxic (T8+) cells was 2:1. No characteristic alterations in CSF phenotypes could be related to changes in circulating T8 cells or to disease activity. In a group of 75 patients, CSF cell count was higher in patients with low numbers of circulating T8 cells than in those with normal T8 cells. Thus, decreases in suppressor cells in the blood of MS patients are associated with CSF pleocytosis but not with fluctuations in the ratio of different subsets in CSF. Furthermore, large numbers of T8 cells are not sequestered in CSF when these cells are decreased in peripheral blood.

Immunoregulatory T-cells and lymphocytotoxic antibodies in active multiple sclerosis: weekly analysis over a six-month period.

Immunoregulatory T-cell subsets were measured at weekly intervals over a 4 to 6 month period using monoclonal antibodies (anti-T4 = inducer cell; anti-T8 = suppressor/cytotoxic cell) in a group of 6 patients with multiple sclerosis (MS) and in 4 age- and sex-matched controls. Decreases in the T8 subset and increases in the T4:T8 ratio were present in 4 of the patients with MS but not in controls. Two patients who were neurologically stable during the study period had no changes in the T4:T8 ratio; 2 patients with intermediate disease activity of the relapsing-remitting type had elevated ratios on 3 and 4 occasions respectively; the patient with the most clinically active MS had an abnormal ratio 12 of 27 times. One patient with chronic-progressive MS had an elevated ratio on each occasion tested. No abnormalities in T-cell subsets were present in any of the controls. On three occasions an elevated T4:T8 ratio appeared to precede an acute relapse by 1.5 to 7 days. Lymphocytotoxic antibodies (LCA) against whole lymphocytes or against isolated T-cell subsets were measured in these patients and in a larger group of MS patients, and were not found to correlate with changes in T-cell subsets. This report extends previous findings linking changes in T-cell subsets to disease activity in patients with MS.