Prospective Randomized Study Comparing the Efficacy of Bioequivalent Doses of glycosylated and nonglycosylated rG-CSF for Mobilizing Peripheral Blood Progenitor Cells

Thirty patients diagnosed with breast cancer were included in a prospective randomized study comparing the in vivo priming effect of bioequivalent doses of glycosylated (lenograstim) and nonglycosylated (filgrastim) rG-CSF administration. Analysis of the efficacy of equivalent biological doses of both rG-CSFs showed no significant differences either in the mobilization of the subpopulations of PBPC considered (CD34⁺, CD34⁺/38⁻, CD34⁺/DR⁻), the content of such CD34⁺ cell subsets in the leukapheresis product, or the cost of the mobilization and collection procedures between both recombinant molecules. These results suggest that priming with bioequivalent doses of the two commercially available forms of glycosylated or nonglycosylated rG-CSF has a similar in vivo effect on PBPC mobilization.     

Characterization and phenotypic analysis of differentiating CD34+human bone marrow cells in liquid culture

Abstract: Our current understanding of human haematopoietic stem cell biology is based in part on the characterization of human CD34⁺ bone marrow cell differentiation in vitro. CD34 is highly expressed on early stem cells and haematopoietic progenitor cells with clonogenic potential and is gradually lost during differentiation and commitment. However, CD71 (transferrin receptor) is expressed at low levels on early stem cells and generally increases during haematopoietic progenitor cell proliferation. We reasoned that the combination of these surface markers would provide a useful framework for the simultaneous analysis of multiple lineage differentiation of CD34⁺ haematopoietic progenitor cells in liquid culture. In this report, we identify the phenotype of distinct subpopulations of myeloid, erythroid and lymphoid cells in liquid suspension culture using differential expression of CD34 vs. CD71 in combination with specific lineage markers. Freshly isolated human CD34⁺ bone marrow cells were introduced into suspension culture and monitored over a 6-d period using 3-colour flow cytometry. This is the first demonstration that differential expression of CD34 vs. CD71 can be used to simultaneously monitor differentiation of multiple haematopoietic cell lineages in liquid suspension culture, facilitating the study of cytokine-, drug- or chemical-induced alterations in haematopoietic progenitor cell differentiation in vitro.     

Ontogeny and cellular expression of MHC and leucocyte antigens in human retina

We have investigated the ontogeny of MHC class I, class II, CD45, and macrophage antigens in wholemounts of normal human fetal retina at 10–25 weeks gestation (WG) using monoclonal antibodies and immunogold histochemistry. MHC class I antigens were expressed on retinal vascular endothelial cells and provided a useful marker of vessel organization from 14–25 WG. Microglial cells expressed immunoreactivity to MHC class I, class II, and CD45 antigens from 10 WG (pre-vascularization) and macrophage S22 (Mac S22) antigen from 14 WG (post-vascularization), although none of the antigens tested were detected on neuronal or macroglial elements. Microglia expressing MHC, CD45, and macrophage antigens occurred in both ramified and rounded forms with no close correlation being observed between morphology and antigenicity. The numbers of immunoreactive cells labeled with each of the four markers increased steadily throughout gestation in all specimens studied. Equivalent numbers of microglia expressed MHC class I, class II, and CD45 antigens in retinae at similar gestational ages; however, our data indicate that microglia expressing Mac S22 antigen comprise approximately 40% or less of the population of MHC and CD45-immunoreactive cells during development. Topographical analyses suggest that MHC class I, class II, and CD45-positive microglia enter the retina from both the peripheral retinal margin and the optic disc from at least 10 WG; Mac S22-positive cells appear in association with the development of the retinal vasculature and enter the retina via the optic disc after 14 WG. © 1995 Wiley-Liss, Inc.     

Peripheral blood CD3 and CD4 T-lymphocyte reduction correlates with severity of liver cirrhosis

Summary Immunological disturbances with impairment of immune function and a higher incidence of lymphoproliferative disorders and other malignancies have been described in liver cirrhosis patients. To investigate the pathogenetic mechanism(s) involved in such associated we looked for a possible imbalance in peripheral blood T-lymphocyte subpopulations in patients with liver cirrhosis of differing severity. Immunophenotyping and counts of peripheral blood T-lymphocyte subpopulations were carried out using monoclonal antibodies conjugated with different fluorochromes in 31 consecutive cirrhotic patients and 23 matched healthy volunteers. Univariate and multivariate analyses of lymphocyte phenotype counts were performed and odds ratios were computed. Statistically significant associations, according to both univariate and multivariate analyses, were found between case/control status and mean CD3 and CD4 T-lymphocyte counts (P<0.0001). A strong correlation was found between the Pugh’s index and CD3 and CD4 lymphocyte counts, with a clear reduction of these phenotypes with increasing liver cirrhosis. Median CD3 and CD4 values were 2,283 and 1,329/μl respectively among controls and 896, 801, and 492/μl and 515, 514, and 307/μl, respectively in categories A, B, and C of Pugh’s classification. Very high odds ratios were found using the median values of CD3 and CD4 as a threshold. There was a statistically significant decrease for each of the T-cell phenotypes studied (CD2, CD3, CD4, CD8, CD16, CD19, CD20, CD56, CD57) between patients and controls (P<0.0001). The progressive and severity-related decrease in mean peripheral blood CD3 and CD4 counts in liver cirrhosis suggests a progressive impairment of protective immune function and may be a factor facilitating malignancy in cirrhotic patients.     

CD5 (OKT1) augments CD3-mediated intracellular signaling events in human T lymphocytes

CD5 is expressed on thymocytes, all mature T cells, and a subset of mature B cells, and probably contributes to T-cell–B-cell adhesion. We assessed whether CD5-crosslinking by mAb augments T-cell stimulation. Plate-bound anti-CD5 or anti-CD3 mAb alone had no effect on any of the assessed activation parameters of resting T cells. However, concomitant signaling through both CD5 and CD3 by plate-bound antibodies resulted in marked increases in T-cell surface CD69 expression and T-cell metabolism, as assessed by the T cell's ability to reduce 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxylmethoxyphenyl)-2-(4-sulphophenyl)-2H-tetrazolium (MTS) to formazen. In addition, simultaneous cross-linking of CD5 and CD3 caused a significant (p < 0.001) increase in phosphatidylinositol hydrolysis in resting T cells compared to stimulation with anti-CD3 mAb alone or anti-CD3 mAb plus anti-CD5 isotype control antibody. These results indicate that CD5 augments signaling through CD3 and consequently functions as a costimulatory molecule for resting T cells.     

Inhibition of HIV-1 by modification of a host membrane protease

While it is clear that CD4 Is the receptor for the gp120 envelopeprotein of HIV-1, substantial evidence suggests that other hostcell proteins are required for successful membrane fusion. Studieswere initiated to examine the potential for a protein receptorwhich has an elastase-like character to participate in fusionof HIV-1 with permissive host cells. A synthetic elastase inhibitorwas shown to significantly reduce HIV-1 infectivity when presentduring, but not after, the initial contact between virus andcells. A human T cell elastase-like membrane component was purifiedand shown to be lipid-associated. By competitive Inhibition,the purified protein was shown to bind gp160 within the HIV-1fusion domain. The binding parameters of whole T cell membraneextract, with a hydrophobic pentapeptide representative of thefusion domain, suggested an elastase-like protein is the single,secondary T cell receptor for HIV-1 (K = 1x10³ M^–1). Thepentapeptide interacted with porcine and human (epithelial andpolymorphonuclear leukocyte), but not murine, elastase isoforms,suggesting its participation In the permissiveness of host cellsto infection.     

CD40 ligand-independent B cell activation revealed by CD40 ligand-deficient T cell clones: evidence for distinct activation requirements for antibody formation and B cell proliferation

We report the capacity of CD40 ligand (CD40L)-negative T cell clones to activate human B cells. CD40L-negative T cells induce a level of B cell proliferation 10–20% of that seen with normal T cells. The signal provided by the negative clones is synergistic with that derived from a CD40L transfectant, and restores B cell proliferation to normal levels, showing that CD40L-negative T cell clones are not inherently inhibitory for B cells. Although their capacity to induce proliferation was much reduced, CD40L-negative T cell clones were still strong inducers of B cell differentiation to plasma cells. This differentiation to plasma cells was inhibited by a CD40L transfectant. The data are discussed with regard to the normal in vivo mechanism for maintaining B cell memory and memory antibody responses to T-dependent antigens.     

Enhanced expression of Fas-associated proteins in decidual and trophoblastic tissues in pregnancy-induced hypertension

PROBLEM: To determine if feto-placental tissues from gestations complicated by pregnancy-induced hypertension (PIH) have altered expression of Fas-associated proteins. METHOD OF STUDY: The expression of several Fas-related proteins was determined in fetal membranes, decidua, and placentas obtained from PIH-affected (n = 12, age range 32-36 weeks) and normal (n = 6, age range 37-41 weeks) gestations. Paraffin-embedded tissue sections were stained with specific monoclonal antibodies to Fas, Fas ligand (FasL), caspase-3, and bax. RESULTS: We observed greater expression of Fas and FasL in amnion and decidua from PIH-affected gestations than in normal controls. Intense staining was observed only in the perivascular endothelium (caspase-3) and in decidual cells (bax) from PIH gestations. CONCLUSION: Differential expression of Fas-related proteins in fetal membranes, decidua, and placentas from PIH-affected gestations is consistent with increased apoptosis, and suggests activation of the Fas/FasL pathway in a tissue-specific manner.