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PubMed1994

Mice lacking granulocyte colony-stimulating factor have chronic neutropenia, granulocyte and macrophage progenitor cell deficiency, and impaired neutrophil mobilization.

Mice lacking granulocyte colony-stimulating factor (G-CSF) were generated by targeted disruption of the G-CSF gene in embryonal stem cells. G-CSF-deficient mice (genotype G-CSF-/-) are viable, fertile, and superficially healthy, but have a chronic neutropenia. Peripheral blood neutrophil levels were 20% to 30% of wild-type mice (genotype G-CSF+/+) and mice heterozygous for the null mutation had intermediate neutrophil levels, suggesting a gene-dosage effect. In the marrow of G-CSF-/- mice, granulopoietic precursor cells were reduced by 50% and there were reduced levels of granulocyte, macrophage, and blast progenitor cells. Despite G-CSF deficiency, mature neutrophils were still present in the blood and marrow, indicating that other factors can support neutrophil production in vivo. G-CSF-/- mice had reduced numbers of neutrophils available for rapid mobilization into the circulation by a single dose of G-CSF. G-CSF administration reversed the granulopoietic defect of G-CSF-/- mice. One day of G-CSF administration to G-CSF-/- mice elevated circulating neutrophil levels to normal, and after 4 days of G-CSF administration, G-CSF+/+ and G-CSF-/- marrows were morphologically indistinguishable. G-CSF-/- mice had a markedly impaired ability to control infection with Listeria monocytogenes, with diminished neutrophil and delayed monocyte increases in the blood and reduced infection-driven granulopoiesis. Collectively, these observations indicate that G-CSF is indispensible for maintaining the normal quantitative balance of neutrophil production during "steady-state" granulopoiesis in vivo and also implicate G-CSF in "emergency" granulopoiesis during infections.

Animals↗
PubMed1995

In multiple myeloma, clonotypic B lymphocytes are detectable among CD19+ peripheral blood cells expressing CD38, CD56, and monotypic Ig light chain.

Multiple myeloma (MM) is characterized by a plasma cell infiltrate of the bone marrow (BM). However, late-stage monotypic B cells have been detected in the blood. This work analyzes the effects of clinical treatment on late stage CD19+ B cells present in 752 blood samples from 152 MM patients. MM patients have 2 to 8 times as many circulating CD19+ cells as do normal donors. Analysis of the Ig heavy chain (IgH) gene rearrangements using polymerase chain reaction indicates that the CD19+ population includes cells sharing the same clonotypic CDR3 region as is detected in the BM plasma cells, for patients analyzed during chemotherapy or in relapse. They are also monotypic as defined by their cytoplasmic or surface expression of Ig kappa or lambda light chain. The light chain restriction is the same as that of the BM plasma cells. Individual patients observed over 1- to 2-year periods exhibit considerable variation in the number of B cells present in blood; this number does not correlate with the concentration of serum monoclonal Ig. The monoclonal blood CD19+ cells are not eliminated by any of the chemotherapy regimens analyzed and remain at high levels during transient remissions. Patients in the progressive phase of disease or in relapse have significantly higher numbers of B cells than do patients in transient remission or untreated patients. During periods when the quantity of blood B cells approaches normal, phenotypically their quality is highly abnormal, with physical and phenotypic heterogeneity. Most B cells express CD45R0, a high density of CD38, and CD56 characteristic of late-stage B or pre-plasma cells. CD38hi blood B cells had a cyclical presence. We conclude that monoclonal B cells in the blood of myeloma patient populations include drug-resistant reservoirs of clonotypic cells that may underlie relapse.

ADP-ribosyl Cyclase↗
PubMed1995

Transient increase in circulating donor leukocytes after allogeneic transfusions in immunocompetent recipients compatible with donor cell proliferation.

Donor leukocytes in therapeutic blood components are implicated in transfusion-related complications ranging from alloimmunization to graft-versus-host disease (GVHD) to viral transmission and reactivation. To further characterize the kinetics of donor leukocyte clearance after allogeneic transfusion, we developed allele-specific polymerase chain reaction (PCR) assays directed at a single-copy Y chromosome gene and HLA class II alleles. These assays enable sensitive detection and quantitation of donor leukocytes at concentrations ranging from one cell to greater than 1,000 cells per 125 microL of recipient blood. When applied to serial samples from five consecutive orthopedic surgery patients who met study criteria, we observed 99.9% clearance of donor leukocytes over the initial 2 days posttransfusion, followed by a transient, 1-log increase in circulating donor leukocytes on days 3 to 5. This phenomenon was reproduced in a canine transfusion model, where the transient donor leukocyte expansion phase was prevented by gamma irradiation of donor blood, and was not observed after transfusions into alloimmunized dogs. We hypothesize that this transient increase in circulating allogeneic donor cells represents one arm of an in vivo mixed lymphocyte reaction, with activated donor T lymphocytes proliferating in an abortive GVHD reaction to HLA-incompatible recipient cells. Further investigation of this phenomenon should provide insight into the mechanisms involved in donor-recipient leukocyte interactions posttransfusion and the relationship of these interactions to leukocyte-induced complications.

Animals↗
PubMed1995

Regulatory effects of intrinsic IL-10 in IgG immune complex-induced lung injury.

IL-10 has regulatory effects in vitro on cytokine production by activated macrophages. In the IgG immune complex model of lung injury, exogenously administered IL-10 has been shown to suppress in vivo formation of TNF-alpha, up-regulation of vascular ICAM-1, neutrophil recruitment, and ensuing lung injury. In the current study, we sought to determine whether endogenous IL-10 is playing a regulatory role in the lung inflammatory response. On the basis of lung mRNA and ELISA measurements, IL-10 induction was found during development of inflammation in the IgG immune complex model of lung injury. Blocking of IL-10 by Ab resulted in a 52% increase in lung vascular permeability, a 56% increase in TNF-alpha activity in bronchoalveolar lavage fluids, and a 47 to 48% increase in bronchoalveolar lavage neutrophils and lung myeloperoxidase content. These findings suggest that IL-10 is an important natural regulator of lung inflammatory injury after deposition of IgG immune complexes.

Animals↗
PubMed1996

In vivo blockade of TNF-alpha by intravenous infusion of a chimeric monoclonal TNF-alpha antibody in patients with rheumatoid arthritis. Short term cellular and molecular effects.

Due to the unknown etiology of RA, specific treatment is not available. Recently, in a double-blinded, placebo-controlled clinical trial, in vivo blockade of TNF-alpha by a single infusion of a chimeric TNF-alpha-blocking mAb, cA2, has proven to be highly effective in the treatment of RA. In parallel to this trial, we tested the consequences of cA2 infusion in ex vivo and in vitro experiments. In this paper, we describe an increase in CD4+ and CD8+ T lymphocyte counts on day 1 and a marked decrease in monocyte counts preferentially on day 7 after cA2 treatment, without major changes in B lymphocyte or NK cell counts. In addition, we found an increased responsiveness of PBMC to CD28 mAb/PMA, but not to CD3 mAb, superantigen staphylococcus enterotoxin B, or PHA on day 1 after infusion. The increase in DNA synthesis of PBMC was paralleled by increased IL-2 mRNA and IL-4 mRNA expression and IL-2 protein secretion in culture supernatants after in vitro stimulation of PBMC with CD28 mAb/PMA. In PBMC, we did not find any significant changes in mRNA or protein expression of CD28 Ag or CD28 ligands, B7-1 and B7-2. Serum concentrations of IL-1 beta, IL-6, and soluble CD14 were significantly diminished after in vivo TNF-alpha blockade. We did not see relevant changes in granulocyte function in vitro after cA2 infusion. Finally, we observed a statistically significant decrease in slCAM-1 molecules in the serum of patients treated with verum compared with that in the serum of subjects given placebo. This change in slCAM-1 concentration was evident on days 1 and 7 after the infusion of 10 mg/kg cA2, whereas it occurred only on day 7 in the serum of patients treated with the low dose (1 mg/kg) of cA2.

Adult↗
PubMed1996

[Association of cytochrome P-450 1A1 (CYP1A1) gene polymorphism to smoking status and hematologic findings].

Measures to control smoking are important in the field of preventive medicine. In order to clarify differences in susceptibility in individuals to lung cancer of genotypes of CYP1A1, which are considered to be related to lung carcinogenesis were evaluated in 391 healthy males to study relationship to smoking status and hematological findings. No correlation was observed between genotypes of CYP1A1 and smoking status. White blood cell counts in smokers with a Val allele were significantly higher than in those without a Val allele. Multiple regression analysis showed that the genotype of CYP1A1 and daily cigarette consumption had significant relationship with white blood cell count in smokers. However, red blood cell count, hemoglobin, hematocrit, MCV, MCH, or MCHC were not significantly associated with genotype of CYP1A1. There have been no previous reports on the relationship between the genotype of CYP1A1 and hematological findings. In consideration of the epidemiologic findings that many individuals with increased white blood cell counts have cancer or cardiac diseases, and reports that the increase in the white blood count was associated with poor respiratory function, white blood cell count may be candidate to for being a risk marker and thus contribute to prevention of these diseases.

Adult↗
PubMed1996

V beta 17 T cell receptor peptide vaccination in rheumatoid arthritis: results of phase I dose escalation study.

OBJECTIVE: To determine whether modulation of activated T cells occurs in patients with rheumatoid arthritis (RA) after immunization with T cell receptor (TCR) V beta 17 peptides, a phase I trial was initiated to investigate the safety and feasibility of TCR peptide immunization as a therapeutic approach in RA. METHODS: 15 patients with moderate to severe RA were given an intramuscular injection of one of 4 doses (10, 30, 100, and 300 micrograms) of the V beta 17 peptide vaccination, followed by a booster injection of the same dose of vaccine 3 weeks later. Patients were followed for 48 weeks. RESULTS: The product was well tolerated and no serious adverse events attributable to the vaccine were observed. This was an uncontrolled phase I trial, however; decreases in patients joint scores were observed at all followup visits starting at 4 weeks after primary immunization. Activated V beta 17 T cells (IL-2R+) in peripheral blood were decreased (> or = 20%) in 3/5 patients in the 100 micrograms group after initial measurement at Week 2 and 3/4 patients in the 300 micrograms group 3 weeks after immunization. Lymphocyte proliferation in response to the V beta 17 peptide was detected at 6 weeks or later after primary inoculation in 6/15 patients (40%) immunized. CONCLUSION: Further controlled studies are required to assess the biologic and clinical efficacy of this treatment approach.

Adult↗
PubMed1997

Detection of low numbers of neuroblastoma cells in vitro.

Neuroblastoma is a tumour derived from the sympathoadrenal progenitors of the neural crest. It is one of the most malignant solid tumours in childhood with an annual incidence of 9.4 per 10(6) children under 15 years of age. Recent studies suggest that immunocytological detection of neuroblastoma cells in bone marrow and circulating neuroblasts during treatment may predict clinical outcome and correlate with tumour relapse. The present methods of diagnosing metastasis in neuroblastoma include histological, biochemical and immunohistological analysis. Morphological distinction between tumour cells and primitive lymphoblasts in bone marrow is often difficult, and these methods may also not always be sensitive enough for early detection of the residual and minimally circulating tumor cells. A sensitive assay for detection of such residual cells using two tissue-specific markers, NFM and SYN, by reverse transcriptase-polymerase chain reaction (RT-PCR) is reported here. Analysis of the specificity of this assay in three neuroblastoma cell lines, namely IMR 32, SK-N-SH and SY5Y showed positive expression while control peripheral blood mononuclear cells (HL 60) were negative. In reconstituted cell spiking tests, this method has the ability to detect 1-10(3) neuroblastoma cell in 10(7) normal peripheral blood mononuclear cells (PBMC), as shown by serial dilution and limiting dilution. The NFM marker was found to be a more sensitive marker. The specificity and sensitivity of this technique makes it suitable for future application in detection of minimally disseminated tumour cells in neuroblastoma patients.

Base Sequence↗
PubMed1998

Molecular and genetic requirements for preferential recruitment of TCRBV8S2+ T cells in Lewis rat experimental autoimmune encephalomyelitis.

The underlying mechanisms behind the preferential expression of select TCRBV products in certain autoimmune illnesses, such as multiple sclerosis and some models of experimental autoimmune encephalomyelitis (EAE), have principally remained enigmatic. In this study, we examined the mutual role of nonself- vs self-origin of antigenic myelin basic protein (MBP) peptides and given MHC haplotypes in relation to the relative frequency of activated TCRBV8S2+ T lymphocytes in the Lewis (LEW) rat EAE model. Inbred MHC (RT1) congenic LEW rats (LEW (RT1l), LEW.1AV1 (RT1av1), and LEW.1W (RT1u)) were immunized with the 63 to 88 peptide of the guinea pig MBP (MBPGP63-88). Additionally, LEW rats were immunized with the corresponding autologous rat sequence (MBPRAT63-88). Although EAE ensued in all MBP peptide/LEW rat strain combinations, only LEW rats immunized with the heterologous MBPGP63-88 peptide elicited T cell responses encompassing a bias toward TCRBV8S2 expression, as determined by flow cytometric analyses. Reduction of TCRBV8S2+ T cells led to mitigation of disease severity in LEW rats immunized with MBPGP63-88, but not with MBPRAT63-88, indicating that critical encephalitogenic characteristics are associated with this T cell subset. We conclude that the preferential recruitment of TCRBV8S2+ T cells in the LEW rat EAE model is due to selective, high-avidity recognition of the nonself-MBPGP63-88 in the context of the RT1.Bl molecule. This inference lends support to the notion that the highly restricted TCR repertoire of the self-MBP-reactive T cells in certain genetically predisposed multiple sclerosis patients may have its source in a multistep molecular mimicry event.

Amino Acid Sequence↗
PubMed2006

COSMIC 2005.

The Catalogue Of Somatic Mutations In Cancer (COSMIC) database and web site was developed to preserve somatic mutation data and share it with the community. Over the past 25 years, approximately 350 cancer genes have been identified, of which 311 are somatically mutated. COSMIC has been expanded and now holds data previously reported in the scientific literature for 28 known cancer genes. In addition, there is data from the systematic sequencing of 518 protein kinase genes. The total gene count in COSMIC stands at 538; 25 have a mutation frequency above 5% in one or more tumour type, no mutations were found in 333 genes and 180 are rarely mutated with frequencies <5% in any tumour set. The COSMIC web site has been expanded to give more views and summaries of the data and provide faster query routes and downloads. In addition, there is a new section describing mutations found through a screen of known cancer genes in 728 cancer cell lines including the NCI-60 set of cancer cell lines.

Databases, Genetic↗
PubMed2002

Clonal expansion of CD8+ BV8 T lymphocytes in bone marrow characterizes thymoma-associated B lymphopenia.

A subgroup of thymoma patients is affected by severe immunodeficiency clinically resembling an HIV infection (Good syndrome). These individuals are characterized by B lymphopenia with B-lymphopoiesis deficiency. To investigate the pathogenesis of this unique condition, we studied the T-cell repertoire in blood and bone marrow samples by heterogeneity length analysis of CDR3 beta variable regions of the T-cell receptor (spectratyping). While no alterations were found in the peripheral blood, we detected an oligoclonal population of beta variable 8 (BV8) CD8(+) T cells in 5 of 5 bone marrow samples. No lymphocyte expansions were found in the bone marrow of 2 thymoma patients with normal B-cell counts, 2 healthy donors, and 3 patients with diseases unrelated to thymoma. These data suggest that an immune response toward an unknown antigen is taking place in the bone marrow of B-lymphopenic thymoma patients. We propose that BV8 CD8(+) T cells may play a role in the pathogenesis of this immunodeficiency syndrome.

Adult↗
PubMed1976

Empirical relationship between the number of nucleotide substitutions and interspecific identity of amino acid sequences in some proteins.

There are three different methods of estimating the number of nucleotide substitutions between a pair of species from amino acid sequence data, i.e. the Poisson correction method, random evolutionary hit method, and counting the actual but minimum number of nucleotide substitutions. In this paper the relationships among the estimates obtained by these methods are studied empirically. The results obtained indicate that there is a high correlation among these estimates and in practice any of the three methods may be used for constructing evolutionary trees or relating nucleotide substitutions to evolutionary time. The effects of varying rates of nucleotide substition among different sites on the Poisson correction and random evolutionary hit methods are also studied mathematically. It is shown that these two methods are quite insensitive to the variation of the rate of nucleotide substitution.

Amino Acid Sequence↗
PubMed1996

Erythropoietin-like activity in vivo of the fusion protein rhIL-6/IL-2 (CH925).

CH925 is a novel cytokine of a fusion protein interleukin-6 (IL-6)/IL-2 exhibiting erythropoietin (Epo)-like effects in vivo and ex vivo, in addition to its enhanced effects compared to IL-2 and IL-6 reported by us previously, which indicates its potential clinical use. Our present study was undertaken to determine the Epo-like activity of CH925 in vivo. The reticulocyte response was observed in transfusion-induced polycythemic mice by using flow cytometry with pyronin Y staining. On day 2 after injection of CH925, the average number of reticulocytes was 2.11% in the group given 250 micrograms/kg/d and 1.01% for 100 micrograms/kg/d. The mean fluorescence intensity (MFI) also significantly increased. Longitudinal studies of CH925 were performed on days 2, 4, and 10, and reticulocyte counts increased up to a peak on day 4. Activity of CH925 (100 micrograms/kg/d) corresponds to 1 U of standard rhEpo in our study.

Animals↗
PubMed1995

P-selectin/ICAM-1 double mutant mice: acute emigration of neutrophils into the peritoneum is completely absent but is normal into pulmonary alveoli.

Neutrophil emigration during an inflammatory response is mediated through interactions between adhesion molecules on endothelial cells and neutrophils. P-Selectin mediates rolling or slowing of neutrophils, while intercellular adhesion molecule-1 (ICAM-1) contributes to the firm adhesion and emigration of neutrophils. Removing the function of either molecule partially prevents neutrophil emigration. To analyze further the role of P-selectin and ICAM-1, we have generated a line of mice with mutations in both of these molecules. While mice with either mutation alone show a 60-70% reduction in acute neutrophil emigration into the peritoneum during Streptococcus pneumoniae-induced peritonitis, double mutant mice show a complete loss of neutrophil emigration. In contrast, neutrophil emigration into the alveolar spaces during acute S. pneumoniae-induced pneumonia is normal in double mutant mice. These data demonstrate organ-specific differences, since emigration into the peritoneum requires both adhesion molecules while emigration into the lung requires neither. In the peritoneum, P-selectin-independent and ICAM-1-independent adhesive mechanisms permit reduced emigration when one of these molecules is deficient, but P-selectin-independent mechanisms cannot lead to ICAM-1-independent firm adhesion and emigration.

Animals↗
PubMed1996

The utility of FK506-binding protein as a fusion partner in scintillation proximity assays: application to SH2 domains.

Methodology has been developed which gives a specific measure of the interaction of an SH2 domain with a phosphopeptide ligand using scintillation proximity assay (SPA) technology. Recombinant SH2 domains were expressed from a T7 RNA polymerase-based vector in Escherichia coli as fusions to the C-terminus of the FK506-binding protein (FKBP) and purified from freeze-thaw lysates in high yield by affinity chromatography using immobilized phosphopeptides. For binding assays the phosphopeptide ligands were synthesized with a biotin tag and the FKBP fusion proteins were noncovalently radiolabeled with commercially available [3H]dihydroFK506. Complexes of tritiated SH2 fusion protein and biotinyl-phosphopeptide were then captured on streptavidin-coated SPA beads and counted. The modular protocol is an equilibrium technique that does not employ washing steps or specialized radiochemical syntheses required in other binding assays. The utility of the assay has been demonstrated in an examination of the ligand specificity of the SH2 domains of the tyrosine kinases ZAP70, Syk, and Lck. The methodology is potentially generalizable to any receptor-ligand interaction in which one component can be expressed as a fusion partner with FKBP and the other component can be captured on a SPA bead.

Amino Acid Sequence↗
PubMedDate not supplied

Cytotoxic activity of saponins from Camassia leichtlinii against human oral tumor cell lines.

Five steroidal saponins from Camassia leichtlinii showed higher cytotoxicity against human oral squamous cell carcinoma cells HSC-2, as compared to normal human gingival fibroblasts HGF. The tumor specificity of saponins varied considerably from sample to sample, but was generally higher than that of tannins, flavonoids and prenylated compounds such as geranylgeraniol and vitamin K2 (MK-2). Agarose gel electrophoresis showed that the saponins failed to induce internucleosomal DNA fragmentation, but produced large DNA fragments in HSC-2 cells, whereas two saponin samples (compounds 1 and 5) induced internucleosomal DNA fragmentation in human promyelocytic leukemic HL-60 cells. In contrast to epigallocatechin gallate or gallic acid, the cytotoxic activity of saponins was not significantly affected by metals (Co2+, Cu2+, Fe3+) or by antioxidants (sodium ascorbate, N-acetyl-L-cysteine, catalase). Furthermore, the saponins did not produce radicals (detected by ESR spectroscopy) nor oxidation potential (measured by NO monitor). These data suggest that an oxidation-mediated mechanism is not involved in the cytotoxicity induced by the steroidal saponins.

Antineoplastic Agents, Phytogenic↗
PubMed2002

Marginalized kernels for RNA sequence data analysis.

We present novel kernels that measure similarity of two RNA sequences, taking account of their secondary structures. Two types of kernels are presented. One is for RNA sequences with known secondary structures, the other for those without known secondary structures. The latter employs stochastic context-free grammar (SCFG) for estimating the secondary structure. We call the latter the marginalized count kernel (MCK). We show computational experiments for MCK using 74 sets of human tRNA sequence data: (i) kernel principal component analysis (PCA) for visualizing tRNA similarities, (ii) supervised classification with support vector machines (SVMs). Both types of experiment show promising results for MCKs.

Computational Biology↗