Peter Schlenke

Expression and kinetics of cytokines determined by intracellular staining using flow cytometry

Cytokine production by human lymphocytes was assessed by a flow cytometric procedure involving staining of intracellular cytokines by the paraformaldehyde-saponin procedure. The production of interleukin 2 (IL2), interferon gamma (IFNgamma) and tumor necrosis factor alpha (TNFalpha) was determined in T-helper (Th) and cytotoxic T-cells (CTL) as well as in naive and memory cells after stimulation with phorbol-12-myristate-13-acetate (PMA) and ionomycin under the influence of monensin. Kinetic studies on IL2, IFNgamma and TNFalpha in lymphocyte subpopulations showed that TNFalpha was the first cytokine produced by T-cells. Production of this cytokine peaked at 2 h and declined rapidly thereafter. The peak of IL2 and IFNgamma production was at 8 h, and production of IL2 exceeded that of IFNgamma in T-cells at all times. IL2 production declined markedly after 8 h, while IFNgamma remained relatively stable for 24 h. IL2 and TNFalpha were mainly produced by Th cells while CTL primarily expressed IFNgamma. At all times a higher percentage of memory cells stained cytokine positive compared to naive cells and production of cytokines increased more rapidly in the memory cells. Naive cells produced primarily IL2, while memory cells expressed all the studied cytokines in substantial amounts. Kinetic studies between 1 and 24 h showed that 5 h was the optimal time point for evaluating the cytokines studied; hence normal values obtained from 50 healthy blood donors were evaluated after 5 h continuous PMA and ionomycin stimulation.

Anti‐myeloma activity of natural killer lymphocytes

Summary. Natural killer (NK) cells are assumed to contribute to a graft‐versus‐leukaemia effect. In vitro experiments have shown that many leukaemic cells are NK‐cell sensitive. Nevertheless, no data concerning the influence of purified NK cells on malignant myeloma (MM) cells exist. We co‐incubated NK cells with three different MM cell lines and fresh bone marrow samples of nine MM patients. The proportion of vital MM cells was determined before and after co‐cultivation by a flow‐cytometry‐based assay. All MM cells tested, with the exception of one cell line (NCI H929), were susceptible to a NK‐cell attack even without exogenous interleukin 2 (IL‐2). The mean killing of the native MM samples was 23·1 ± 5·4% and 34·5 ± 6·5% at 10:1 and 20:1 effector:target ratio respectively, This corresponded to about 2/3 of those values obtained with the highly sensitive line K562. In contrast, CD34‐positive haematopoietic stem cells as well as peripheral mononuclear cells were completely resistant under similar experimental conditions (1·3% killing). To elucidate the underlying triggering mechanisms, we measured human leucocyte antigen (HLA)‐class I expression of the MM cells. No evidence for HLA loss, which could have explained the NK‐cell recognition if it occurred, was demonstrated. These findings may contribute to the understanding of in vivo NK‐cell activation and encourage clinical applications of NK cells for MM patients.

Increased numbers of CCR5+ interferon‐γ– and tumor necrosis factor‐α–secreting T lymphocytes in multiple sclerosis patients

To determine the frequency of in vivo activated TH1 lymphocytes, T‐cell subsets of 9 multiple sclerosis patients with active disease and 17 healthy controls were analyzed by immunostaining for CCR5, CD26, and their expression of interleukin‐2, interferon‐γ, and tumor necrosis factor‐α. The numbers of CCR5+ interferon‐γ– and tumor necrosis factor‐α–producing T cells were significantly increased in the peripheral blood of multiple sclerosis patients. CCR5 expression may be a useful marker to identify effector cells in multiple sclerosis and could be used as a tool for monitoring disease activity. Ann Neurol 2000;47:269–273

In vivo matrix-guided human mesenchymal stem cells

Abstract.Microfracture of subchondral bone results in intrinsic repair of cartilage defects. Stem or progenitor cells from bone marrow have been proposed to be involved in this regenerative process. Here, we demonstrate for the first time that mesenchymal stem (MS) cells can in fact be recovered from matrix material saturated with cells from bone marrow after microfracture. This also introduces a new technique for MS cell isolation during arthroscopic treatment. MS cells were phenotyped using specific cell surface antibodies. Differentiation of the MS cells into the adipogenic, chondrogenic and osteogenic lineage could be demonstrated by cultivation of MS cells as a monolayer, as micromass bodies or mesenchymal microspheres. This study demonstrates that MS cells can be attracted to a cartilage defect by guidance of a collagenous matrix after perforating subchondral bone. Protocols for application of MS cells in restoration of cartilage tissue include an initial invasive biopsy to obtain the MS cells and time-wasting in vitro proliferation and possibly differentiation of the cells before implantation. The new technique already includes attraction of MS cells to sites of cartilage defects and therefore may overcome the necessity of in vitro proliferation and differentiation of MS cells prior to transplantation.

Isolation and Characterization of Adult Stem Cells from Human Salivary Glands

Currently, adult stem cells are attracting significant interest in regenerative medicine and tissue engineering. These cells have been isolated from various tissue sources; however, in most cases, adult stem cells useful for tissue engineering and regeneration are present at a low frequency. High numbers of stem cells with an effective and reliable potential for differentiation are needed for clinical applications. Thus, the identification of new stem cell sources and the establishment of optimized cell culture conditions that allow for the amplification of stem cells are of utmost relevance. In addition, the isolation procedure should ideally be minimally invasive and possibly be performed under local anesthesia. We report here for the first time on the identification of adult stem cells with mesenchymal characteristics in human parotid gland tissue. Cells were isolated from freshly resected specimens of parotid glands using enzymatic digestion and plastic adhesion protocols. Following an initial proliferation period and short-term culture for four passages, immunophenotyping revealed the presence of mesenchymal stem cell markers. In the presence of tissue-specificinduction medium, stem cells could be differentiated into adipogenic, osteogenic, and chondrogenic cell types. Tissue-specific differentiation was confirmed by histochemical and immunocytochemical staining as well as by RT-PCR for defined marker genes. This study is, to the best of our knowledge, the first report on the isolation and differentiation of stem cells from adult human parotid glands. Although isolated from an endodermal tissue source, these stem cells share many characteristics with MSCs. Easy accessibility and a high differentiation potential make salivary gland-derived stem cells a promising source for future applications in regenerative medicine.

Molecular Parameters for Precise Diagnosis of Asymptomatic Epstein-Barr Virus Reactivation in Healthy Carriers

ABSTRACT Asymptomatic Epstein-Barr virus (EBV) reactivations periodically occur in oral mucosa-associated lymphoid tissues. Until now, EBV reactivation has been diagnosed by serologic profiles that suggest virus replication. Serologic responses, however, are delayed and do not necessarily indicate ongoing replicative activity. The aim of the present study was to establish in healthy carriers parameters for a molecular diagnosis of reactivated EBV infection. Recent studies emphasized the association of an increase in peripheral-B-cell viral load with replicative activity at remote sites. Therefore, real-time PCR was used to quantitate EBV genomes in the peripheral blood mononuclear cells (PBMC) (viral load) and plasma samples (viremia) of 22 healthy EBV-seropositive blood donors over a period of 15 months. Furthermore, transcription of the immediate-early gene encoding BZLF1 was investigated in the PBMC of all volunteers. Serology suggested reactivation in nine donors, of whom all but one showed at least once a significant increase in viral load. Another five individuals also exhibited significant changes in viral load but no serologic response. Of the 13 volunteers with significant increases in viral load, 6 had a period of viremia accompanying the rise in viral load. A stable viral load without viremia and negative serology was seen in eight adults. BZLF1 mRNA was undetectable throughout. We conclude that for healthy subjects serology underestimates the frequency of asymptomatic EBV reactivations. Prospective examination of peripheral viral load and viremia is suitable for the exact diagnosis of EBV reactivation, which might be of advantage for immunocompromised patients in whom EBV reactivations are considerably harmful.

Mesenchymal Stem or Stromal Cells: Toward a Better Understanding of Their Biology?

The adult bone marrow has been generally considered to be composed of hematopoietic tissue and the associated supporting stroma. Within the latter compartment, a subset of cells with multipotent differentiation capacity exists, usually referred to as mesenchymal stem cells. Mesenchymal stem cells can easily be expanded ex vivo and induced to differentiate into several cell types, including osteoblasts, adipocytes and chondrocytes. Up to now, mesenchymal stem cells have gained wide popularity. Despite the rapid growth in this field, irritations remain with respect to the defining characteristics of these cells, including their differentiation potency, self-renewal and in vivo properties. As a consequence, there is a growing tendency to challenge the term mesenchymal stem cell, especially with respect to the stem cell characteristics. Here, we revisit the experimental origins of mesenchymal stem cells, their classical differentiation capacity into mesodermal lineages and their immunophenotype in order to assess their stemness and function. Based on these essentials, it has to be revisited if the designation as a stem cell remains an appropriate term.

Pathogen Inactivation Technologies for Cellular Blood Components: an Update

Nowadays patients receiving blood components are exposed to much less transfusion-transmitted infectious diseases than three decades before when among others HIV was identified as causative agent for the acquired immunodeficiency syndrome and the transmission by blood or coagulation factors became evident. Since that time the implementation of measures for risk prevention and safety precaution was socially and politically accepted. Currently emerging pathogens like arboviruses and the well-known bacterial contamination of platelet concentrates still remain major concerns of blood safety with important clinical consequences, but very rarely with fatal outcome for the blood recipient. In contrast to the well-established pathogen inactivation strategies for fresh frozen plasma using the solvent-detergent procedure or methylene blue and visible light, the bench-to-bedside translation of novel pathogen inactivation technologies for cell-containing blood components such as platelets and red blood cells are still underway. This review summarizes the pharmacological/toxicological assessment and the inactivation efficacy against viruses, bacteria, and protozoa of each of the currently available pathogen inactivation technologies and highlights the impact of the results obtained from several randomized clinical trials and hemovigilance data. Until now in some European countries pathogen inactivation technologies are in in routine use for single-donor plasma and platelets. The invention and adaption of pathogen inactivation technologies for red blood cell units and whole blood donations suggest the universal applicability of these technologies and foster a paradigm shift in the manufacturing of safe blood.

The repertoire of HLA-Cw-specific NK cell receptors CD158 a/b (EB6 and GL183) in individuals with different HLA phenotypes.

Over the last few years, natural killer (NK) cells have been shown to express major histocompatibility complex (MHC) molecules recognizing receptors that are thought to function primarily as negative signalling receptors. Much attention has been focused on the NK cell receptors CD158a (EB6) and CD158b (Gl 183), which recognize two alternative epitopes on the HLA‐Cw locus. In order to investigate whether HLA type affects the CD158a/b repertoire, expressed as percentage positive cells for a particular receptor and mean expression on this population of NK cells, peripheral blood lymphocytes of 47 HLA‐typed donors were examined. Peripheral blood samples were examined by flow cytometric analysis to investigate the expression of CD158a and CD158b receptors on the surface of NK cells. In parallel, we determined each individual’s HLA phenotype. There was a great heterogeneity in CD158 expression; nevertheless all individuals had NK cells belonging either to the CD158 a+b−, a−b+ or a−b− populations. No positive or inverse correlations could be shown between either receptor expression intensity or proportion of positive cells, and presence of the appropriate ligand. Thus no association between an individual’s NK receptor repertoire and HLA serotype could be demonstrated. It is concluded that CD158 is expressed on NK cells in a highly redundant fashion. Our data do not support either a positive selection mechanism or the receptor calibration model.

A flow-cytometry based cytotoxicity assay using stained effector cells in combination with native target cells

Flow-cytometry based assays for cellular cytotoxicity have established themselves widely over the last years. Discrimination of target and effector cells is critical for such assays. If scatter properties are not informative, the standard approach until now has been to label the target cells with a suitable fluorescent dye. However, this cannot be applied to a number of experimental settings, e.g. if one effector cell type is tested against several target cells, or if target cells do not incorporate the dye properly. Therefore, our goal was to develop a protocol based on the labelling of effector cells. For this purpose, we came around to using a membrane dye, DIOC18, which is not commonly used for flow-cytometric applications. This dye showed very stable membrane integration properties that allowed long-term coincubation periods (24 h) without leakage to neighbouring cells. The vitality and cytotoxic activity of the effector cells were not altered by staining. For the detection of dead cells, the intercalating DNA-dye 7-AAD was used. The spectral emission wavelengths of this combination also enable the additional use of PE-conjugated antibodies to surface antigens in three-color cytometry devices. Cytotoxicity values obtained by our protocol were highly correlated with values obtained by the chromium release assay at different E/T ratios and using several target cell lines. All in all, we present here an easy to handle protocol, which enables the precise determination of cellular cytotoxicity in various experimental settings.