An immunocytochemical investigation of glial morphology in the Pacific hagfish: radial and astrocyte-like glia have the same phylogenetic age

Summary This study attempts to reconstruct the early phylogenetic history of macroglial cells among craniates. Since glia does not fossilize, such a reconstruction must be based on a cladistic comparison of glial characters in the Recent craniate taxa (hagfishes, lampreys, and gnathostomes); however, there are only few data on glial morphology and none on glial immunocytochemistry in hagfishes. Therefore, we investigated the presence and localization of various macroglia-specific epitopes in the brain and spinal cord of the Pacific hagfish,Eptatretus stouti (Myxinoidea) by means of immunocytochemistry. Antibodies directed against S100-pfotein and vimentin showed no cross reactivity. Antibodies directed against glial fibrillary acidic protein and glutamine synthetase labelled various glial structures. Glial fibrillary acidic protein-like immunoreactivity was observed in ependymal cells with radially oriented processes in some regions. However, throughout the entire CNS, labelling of non-ependymal cells and their processes prevailed. The processes of these cells made occasional vascular contacts and they also made contacts with neuronal perikarya. Glutamine synthetase-like immunoreactivity was also found in some processes with radial orientation and in ependymal cells; but the antibody stained mainly non-ependymal cells which gave rise to a felt-like meshwork of interdigitating fine and very fine processes penetrating the neuropil of the entire brain. Additionally, there was labelling in the walls of blood vessels and in processes enwrapping individual neurons. The occurrence of glial fibrillary acidic protein- and glutamine synthetase-like immunoreactivity in non-ependymal glial elements in the brain of hagfishes and the relative scarcity of labelling in radial glial elements necessitates a re-interpretation of the evolutionary history of glial cells. Non-ependymal macroglia with immunocytochemical and morphological characters resembling typical (mammalian) astrocytes appears to be as primitive as the various forms of radial ependymal glia.     

Immunoglobulin free light chains in saliva: a potential marker for disease activity in multiple sclerosis

A new procedure was developed and applied to study immunoglobulin free light chains (FLC) in saliva of healthy subjects and patients with multiple sclerosis (MS). The procedure was based on a Western blot analysis for detection and semiquantitative evaluation of monomeric and dimeric FLCs. The FLC indices accounting for the total FLC levels and for the monomer/dimer ratios of κ and λ FLC were calculated, and the cut‐off values of the FLC indices were determined to distinguish healthy state from MS disease. The obtained FLC index values were statistically different in the saliva of three groups: active MS patients, MS patients in remission and healthy subjects groups. Our FLC monomer–dimer analysis allowed differentiation between healthy state and active MS with specificity of 100% and a sensitivity of 88·5%. The developed technique may serve as a new non‐invasive complementary tool to evaluate the disease state by differentiating active MS from remission with sensitivity of 89% and specificity of 80%.     

Assessment of clinical significance of anti-Ge in an untransfused man

A 19-year-old, untransfused Melanesian man from Papua New Guinea was admitted to the hospital for repair of an atrial septal defect. His serum contained an alloantibody that reacted strongly on the indirect antiglobulin test and was identified as anti-Ge. Gerbich-negative blood was transfused following urgent surgery. A 51Cr red cell survival study performed 2 weeks after surgery yielded zero survival of Gerbich-positive cells after 24 hours. A monocyte-driven, antibody-dependent, cell-mediated cytotoxicity assay performed on both pretransfusion and posttransfusion serum samples and on concentrated serum showed less than 1 percent specific lysis of Gerbich-positive cells. This did not correlate with the indication of clinical significance predicted by the 51Cr study. Red cell adherence and phagocytosis, not evident in a monocyte monolayer assay using native serum, were demonstrable in 16 percent of monocytes by the use of concentrated serum.     

Family history of myocardial infarction is an independent risk factor for venous thromboembolism: the Tromsø study

Summary. Background: Recent studies indicate that arterial cardiovascular diseases and venous thromboembolism (VTE) share common risk factors. A family history of myocardial infarction (MI) is a strong and independent risk factor for future MI. Objectives: The purpose of the present study was to determine the impact of cardiovascular risk factors, including family history of MI, on the incidence of VTE in a prospective, population‐based study. Patients and methods: Traditional cardiovascular risk factors and family history of MI were registered in 21 330 subjects, aged 25–96 years, enrolled in the Tromsø study in 1994–95. First‐lifetime VTE events during follow‐up were registered up to 1 September 2007. Results: There were 327 VTE events (1.40 per 1000 person‐years), 138 (42%) unprovoked, during a mean of 10.9 years of follow‐up. In age‐ and gender‐adjusted analysis, age [hazard ratio (HR) per decade, 1.97; 95% confidence interval (CI), 1.82–2.12], gender (men vs. women; HR, 1.25; 95% CI, 1.01–1.55), body mass index (BMI; HR per 3 kg m^?2, 1.21; 95% CI, 1.13–1.31), and family history of MI (HR, 1.31; 95% CI, 1.04–1.65) were significantly associated with VTE. Family history of MI remained a significant risk factor for total VTE (HR, 1.27; 95% CI, 1.01–1.60) and unprovoked VTE (HR, 1.46; 95% CI, 1.03–2.07) in multivariable analysis. Blood pressure, total cholesterol, HDL‐cholesterol, triglycerides, and smoking were not independently associated with total VTE. Conclusions: Family history of MI is a risk factor for both MI and VTE, and provides further evidence of a link between venous and arterial thrombosis.     

Human fibroblasts downregulate plasminogen activator inhibitor type-1 in cultured human macrovascular and microvascular endothelial cells

We have previously reported that plasminogen activator inhibitor type-1 (PAI-1) expression in endothelial cells (ECs) can be modulated differently by smooth muscle cells depending on their origin. Human pulmonary artery smooth muscle cells (HPASMCs) strongly downregulated PAI-1 expression in ECs. Fibroblasts (FBs) are another cell type that could come in close contact with ECs. Therefore, it was the aim of this study to investigate whether FBs could also influence the fibrinolytic potential of ECs. As in the case of HPASMCs, PAI-1 antigen produced by human umbilical vein ECs (HUVECs) cocultured with human skin FBs (HSFBs) was significantly lower as compared with the sum of PAI-1 secreted by the respective cell types cultured separately. Not only HUVECs but also human skin microvascular ECs (HSMECs) responded in a dose-dependent way to serum-free conditioned media (CM) from HSFBs from one individual donor. Similar results were obtained when CM from HSFBs from four other individual donors were used. PAI-1 mRNA decreased in HUVECs incubated for 6 hours with HSFB-CM to 24% to 55% of control, depending on the preparation of HSFBs used. A significant PAI-1 downregulatory effect was only observed when CM from low-passage HSFBs (up to passage no. 5) was used, whereas no reduction in EC PAI-1 production was observed with CM obtained from HSFBs in passage no. 8. This PAI-1 downregulatory activity present in HSFB-CM was heat-labile and had a molecular mass of approximately 5 kD. When CM from HPASMCs was analyzed in the same way, an almost identical elution profile was found. In conclusion, our data showed that FBs can decrease the expression of PAI-1 in ECs. Such an effect could be operative during wound-healing and at other capillary sites where FBs could render ECs profibrinolytic, thereby facilitating processes requiring an increase in proteolytic activity such as EC migration and proliferation.     

Anti-graft-versus-host disease effect of DT390-anti-CD3sFv, a single- chain Fv fusion immunotoxin specifically targeting the CD3 epsilon moiety of the T-cell receptor

In a recent study, we showed that an immunotoxin (IT) made with a conventional monoclonal antibody targeting the CD3 epsilon moiety of the T-cell receptor (TCR) had a potent, but partial, graft-versus-host disease (GVHD) effect (Vallera et al, Blood 86:4367, 1995). Therefore, in this current study, we determined whether a fusion immunotoxin made with anti-CD3 single-chain Fv (sFv), the smallest unit of antibody recognizing antigen, would have anti-GVHD activity. A fusion protein was synthesized from a construct made by splicing sFv cDNA from the hybridoma 145-2C11 to a truncated form of the diphtheria toxin (DT390) gene. DT390 encodes a molecule that retains full enzymatic activity, but excludes the native DT binding domain. The DT390-anti-CD3sFv hybrid gene was cloned into a vector under the control of an inducible promoter. The protein was expressed in Escherichia coli and then purified from inclusion bodies. The DT390 moiety of the protein had full enzymatic activity compared with native DT and DT390-anti-CD3sFv, with an IC50 of 1 to 2 nmol/L against phytohemagglutinin-stimulated and alloantigen-stimulated T cells. Specificity was shown (1) by blocking the IT with parental anti-CD3 antibody, but not with a control antibody; (2) by failure of DT390-anti-CD3sFv to inhibit lipopolysaccharide-stimulated murine B cells; (3) by failure of an Ig control fusion protein, DT390-Fc, to inhibit T-cell responses; and (4) with in vivo immunohistochemisty studies. GVHD was studied in a model in which C57BL/6 (H-2b)-purified lymph node T cells were administered to major histocompatibility complex (MHC) antigen disparate unirradiated C.B.-17 scid (H-2d) mice to assess GVHD effects in the absence of irradiation toxicity. Flow cytometry studies showed that donor T cells were expanded 57-fold and histopathologic analysis showed the hallmarks of a lethal model of GVHD. Control mice receiving phosphate-buffered saline showed 17% survival on day 80 after bone marrow transplantation, and mice receiving 2 micrograms DT390-Fc fusion toxin control administered in 2 daily doses for 6 days (days 0 through 5) had a 43% survival rate. In contrast, 86% of mice receiving the same dose of DT390-anti-CD3sFv were survivors on day 80, a significant improvement, although survivors still showed histopathologic signs of GVHD. These findings suggest that new anti-GVHD agents can be genetically engineered and warrant further investigation of fusion proteins for GVHD treatment.     

A monoclonal antibody-defined membrane antigen complex is required for neutrophil-neutrophil aggregation

We examined the aggregation responses of normal neutrophils treated with the murine monoclonal antibody (MoAb) 60.3. Addition of MoAb 60.3 to normal neutrophils produced dose-dependent inhibition of neutrophil aggregation in response to phorbol myristate acetate, zymosan-activated plasma, and N-formyl-methionylleucylphenylalanine. We conclude that the membrane glycoprotein complex recognized by MoAb 60.3--designated CDw18- -is required for neutrophil-neutrophil aggregation in vitro.     

In vitro exposure to human immunodeficiency virus type 1 induces apoptotic cell death of the factor-dependent TF-1 hematopoietic cell line

In this study, we evaluated the effect of a short-term exposure (2 hours) to two different lymphocytotropic strains of human immunodeficiency virus type 1 (HIV-1; HIVIIIB and ICR-3) on the survival of a factor-dependent CD34+ hematopoietic progenitor cell line (TF-1). At flow cytometry analysis, a significant (P < .05) increase in the frequency of apoptotic cell death was observed in HIV-1-treated TF- 1 cells, supplemented with low doses of either interleukin-3 (IL-3; 0.02 to 1 ng/mL) or granulocyte-macrophage colony-stimulating factor (GM-CSF; 0.02 to 0.2 ng/mL) with respect to mock-treated cells. On the other hand, higher doses of both cytokines or combinations of suboptimal concentrations of IL-3 plus GM-CSF (eg, 0.2, plus 0.2 ng/mL) completely reversed the HIV-1-induced increase of apoptosis. Remarkably, no signs of productive or latent virus replication were ever observed in HIV-1-treated TF-1 cells up to 16 days of liquid culture. In parallel experiments, the in vitro exposure to HIVIIIB induced a significant and progressive increase of apoptotic death in purified bone marrow CD34+ cells, seeded in liquid cultures in the presence of 1 ng/mL IL-3. The HIV-1-induced apoptosis of TF-1 cells was likely triggered by the simple interaction of HIV-1 envelope glycoprotein gp120 with CD4 receptor, which was expressed at a low level on the surface of TF-1 cells. In fact, treatment of TF-1 cells with recombinant gp120 plus a polyclonal anti-gp120 antibody or with anti-CD4 monoclonal antibody plus rabbit antimouse IgG significantly increased the percentage of apoptotic death. These data suggest that HIV-1, and perhaps also free gp120 in the presence of anti-gp120 antibody; could play a direct role in the pathogenesis of peripheral blood cytopenias in acquired immunodeficiency syndrome patients by inducing apoptotic death of hematopoietic progenitor cells without the need of a direct infection.