In-vivo evaluation of random donor platelet concentrates from pooled buffy coats

 Random-donor platelet concentrates (PC) prepared from pooled buffy coats have recently been described as an alternative method for platelet preparation. We evaluated such PCs in the clinical setting compared with a standard PC from platelet apheresis. PCs were prepared either from pools of buffy coats (BC-PC) or from single donors (SD-PC) with the cell separator CS-3000 plus. PCs were stored for up to 5 days before transfusion. We compared fresh PC (day 1) with stored (day 2–3) and long-stored PC (day 4–5). For analysis, platelet increment in the recipient was determined immediately and 16–22 h (mean 20 h) after transfusion, corrected for total body area and transfused platelets (CCI). A total of 316 PCs were administered to 36 thrombocytopenic patients suffering from various hematological disorders. Patients with detectable HLA or platelet-specific antibodies or splenomegaly were excluded from the study. Mean platelet content of the PC was 262×10⁹ for BC-PC and 251×10⁹ for SD-PC. The 20-h CCI after transfusion of fresh PC was slightly higher with BC-PC than with SD-PC (14.5 versus 11.9;p=0.19), but values did not differ significantly between the two types of PC on any day of storage. For BC-PC, 20-h CCI decreased with further storage by 30% (10.2;p=0.02). For SD-PC a decrease by 9% was not significant. In conclusion, platelet concentrates prepared from pools of buffy coats showed excellent transfusion results when administered fresh, but storage decreased the CCI by 30%. No significant difference from PCs from plateletpheresis was observed on any day of storage. Both types of platelet concentrates were capable of sufficient platelet increment even when stored for up to 5 days. Received: 28 December 1995 / Accepted: 14 May 1996     

Expression of selectin ligands on murine effector and IL-10-producing CD4+ T cells from non-infected and infected tissues

Endothelial selectins are crucial for the recruitment of leukocytes into sites of inflammation. On T cells, ligands for selectins become induced upon differentiation into the effector/memory stage. Initial in vitro studies suggested a correlation between the Th1 phenotype and ligand expression, but whether this also holds true in vivo remained uncertain. We here analyzed selectin ligands on CD4+ T cells producing IFN-gamma, IL-4 or IL-10, prototypic cytokines of the Th1, Th2 and Tr1 subset, respectively. We analyzed mice infected with influenza virus, the bacterium Listeria, and the parasites Toxoplasma (all Th1 models) or Nippostrongylus (Th2 model). A link between the Th1 phenotype and ligand expression was not found in vivo. Surprisingly, the potentially regulatory IL-10-producing T cells displayed the highest frequency of ligand-positive cells in general. Within the inflamed tissues, the frequencies of P-selectin-binding cells increased in the dominant subset, either Th1 or Th2. Up-regulation was also found for E-selectin ligands during influenza, but not Nippostrongylus infection. In conclusion, conditions driving T cell polarization into either Th1 or Th2 in vivo do not affect the expression of selectin ligands, but acquisition of P-selectin binding and hence migration into inflamed tissues is boosted by an inflammatory milieu.     

Genetic and phylogeographic structure of populations of<Emphasis Type="Italic"> Pulex simulans</Emphasis> (Siphonaptera) in Peru inferred from two genes (<Emphasis Type="Italic">CytB</Emphasis> and<Emphasis Type="Italic"> CoII</Emphasis>)

In this paper we discuss the potential usefulness of determining the phylogeographic and phylogenetic patterns of a vector for understanding the spread of pathogens or insecticide resistance. We do so using the example of Pulex simulans in Peru. Six populations from six different localities were investigated. Mitochondrial DNA sequences were obtained and branching patterns were inferred using phylogenetic reconstruction methods and nested clade analyses. Ten different haplotypes were discovered. Phylogenetic analysis revealed P. simulans in Peru as a monophyletic group, containing clades that were generally not geographically correlated. The data suggest that P. simulans is not a single genetic entity but rather that this species shows a high degree of intraspecific variation. Restricted gene flow with long distance dispersal coupled with range expansion and long distance colonization are likely to have contributed to the observed patterns of variation.     

Is auditory evoked potential augmenting/reducing affected by acute tryptophan depletion?

Several lines of evidence suggest that the auditory evoked potential (AEP) augmenting/reducing slope may serve as a biological marker of central serotonergic activity. According to Hegerl and Juckel (Biol. Psychiatry, 33, 1993, 173), reduced serotonergic activity is hypothesized to increase the slope of the AEP amplitude stimulus intensity function (ASF-slope). Hints for this hypothesis were investigated by employing the acute tryptophan depletion paradigm in 18 healthy females. A within-subject, placebo controlled double-blind cross over design was used for that purpose. Subjects ingested both a 50 g amino-acid drink with (placebo condition) and without tryptophan (depletion condition). With respect to the N1/P2-slope, test-retest reliability of a 1 week interval ranged between r=0.56 and 0.58 for the pre-ingestion baseline recording sessions. Affect was not altered by tryptophan depletion and not related to the ASF-slope. The comparison between placebo and depletion conditions did not reveal significant alterations of the ASF-slope, neither after 5 nor 6 h post-ingestion. Thus, the results do not support the assumption of the ASF-slope reflecting central serotonergic function.     

The effect of ventilation on spectral analysis of heart rate and blood pressure variability during exercise

Heart rate variability (HRV) and systolic blood pressure variability (BPV) during incremental exercise at 50, 75, and 100% of previously determined ventilatory threshold (VT) were compared to that of resting controlled breathing (CB) in 12 healthy subjects. CB was matched with exercise-associated respiratory rate, tidal volume, and end-tidal CO(2) for all stages of exercise. Power in the low frequency (LF, 0.04-0.15 Hz) and high frequency (HF, >0.15-0.4 Hz) for HRV and BPV were calculated, using time-frequency domain analysis, from beat-to-beat ECG and non-invasive radial artery blood pressure, respectively. During CB absolute and normalized power in the LF and HF of HRV and BPV were not significantly changed from baseline to maximal breathing. Conversely, during exercise HRV, LF and HF power significantly decreased from baseline to 100% VT while BPV, LF and HF power significantly increased for the same period. These findings suggest that the increases in ventilation associated with incremental exercise do not significantly affect spectral analysis of cardiovascular autonomic modulation in healthy subjects.     

Non-heparan sulfate-binding interactions of endostatin/collagen XVIII in murine development.

Knobloch syndrome is characterized by a congenital generalized eye disease and cranial defect. Pathogenic mutations preferentially lead to a deletion or functional alteration of collagen XVIII's most C-terminal endostatin domain. Endostatin can be released from collagen XVIII and is a potent inhibitor of angiogenesis and tumor growth. We show differential expression of binding partners for endostatin, vascular endothelial growth factor (VEGF), and the collagen XV endostatin homologue in murine embryonal development using a set of alkaline phosphatase fusion proteins. Consistent with the human phenotype, vascular mesenchyme in the developing eye was identified as endostatin's primary target. While endostatin predominantly bound to blood vessels, the VEGF164 affinity probe labeled nonvascular tissues such as forebrain, hindbrain, the optic nerve, and the surface ectoderm of the future cornea. Strikingly increased staining specificity was observed with a non-heparin/heparan sulfate-binding endostatin probe. In contrast, elimination of the heparan sulfate binding site from VEGF led to complete loss of binding. The collagen XV endostatin homologue showed a highly restricted binding pattern. Oligomerization with endogenous endostatin was ruled out by use of collagen XVIII knockout mice. Our data provide strong evidence that collagen XVIII's C-terminal endostatin domain harbors a prominent tissue-binding site and that binding can occur in the absence of heparan sulfates in situ.     

Clinical utility of an enhanced human immunodeficiency virus type 1 p24 antigen capture assay

The presence of p24 core antigen in the serum of individuals with human acquired immunodeficiency syndrome has been used as one of the important prognostic markers of HIV-1 infection and also as an end point in evaluating antiviral drugs and vaccines. Unfortunately the majority of p24 antigen present in serum exists as an antigenantibody complex and is not detected with the commercial kits currently available to measure p24 antigen. In this study, we report a simple procedure utilizing treatment of serum samples with glycine buffer (pH 1.85) to dissociate antigen-antibody complexes prior to assaying for p24 antigen. A 300% increase in the number of p24-reactive samples and a 3- to 12-fold increase in the quantity of antigen detected were observed when samples were pretreated with 1.5M glycine buffer (pH 1.85) for 1 hr. Glycine treatment of samples did not result in nonspecific positive tests and samples previously shown to be reactive remained positive. In reconstruction experiments the release of antigen was found to be inversely proportional to the amount of p24 antibody present in the serum. The percentage of HIV-1-infected patients positive for p24 antigen was clearly a function of CD4 count. Forty-nine percent of patients with more than 500 CD4 cells and 100% of patients with less than 200 CD4 were p24 positive. The improved sensitivity for detection of p24 provided by this procedure enhances our understanding of the pathogenesis of AIDS by showing that the majority of patients with HIV-1 infection is p24 positive and facilitates the analysis of data obtained in clinical trials involving anti-HIV compounds.     

Glial cell expression of hepatocyte growth factor in vitreoretinal proliferative disease

The hepatocyte growth factor (HGF) has been crucially implicated in the development of proliferative retinal diseases; however, it is unclear whether retinal glial cells express or respond to HGF. Therefore, we examined the expression of HGF and of the receptor for HGF, c-Met, by immunohistochemical costaining with glial fibrillary acidic protein (GFAP) in epiretinal membranes of patients with proliferative vitreoretinopathy (PVR) and proliferative diabetic retinopathy (PDR), respectively. Furthermore, it was determined whether cells of the human retinal glial cell line, MIO-M1, secrete HGF protein, and whether HGF stimulates proliferation and chemotaxis, and secretion of the vascular endothelial growth factor (VEGF). Neuroretinas of patients with PVR express elevated mRNA level for HGF in comparison to control retinas. In epiretinal membranes of patients with PVR or PDR, immunoreactivity for HGF and for c-Met, respectively, partially colocalized with immunoreactivity for GFAP. Fetal bovine serum and basic fibroblast growth factor, but not heparin-binding epidermal or platelet-derived growth factors, evoked HGF secretion by cultured retinal glial cells. HGF displayed only a marginal effect on cell proliferation while it stimulated chemotaxis. HGF promoted the secretion of VEGF, via activation of the phosphatidylinositol-3 kinase. It is concluded that glial cells in epiretinal membranes express both HGF protein and c-Met receptors. The results suggest an autocrine/paracrine role of HGF in glial cell responses during proliferative vitreoretinal disorders as well as in retinal neovascularization, by stimulating of VEGF release.