Eva Niederer

Identification and Functional Characterization of N-Terminally Acetylated Proteins in Drosophila melanogaster

A new study reveals a functional rule for N-terminal acetylation in higher eukaryotes called the (X)PX rule and describes a generic method that prevents this modification to allow the study of N-terminal acetylation in any given protein.

Genotypic, phenotypic and functional analysis of CD4+CD7+ and CD4+CD7- T lymphocyte subsets in Sézary syndrome

Abstract  The expansion of CD4 + CD7 – T cells in the peripheral blood of Sézary syndrome (SS) is well known. It remains unclear whether this population contains the dominant T cell clone. Peripheral blood mononuclear cells (PBMC) of five SS patients were sorted by fluorescence-activated cell sorting into CD4 + CD7 – and CD4 + CD7 + populations. These populations were analysed separately for clonality of the T cell receptor γ chain (TCR-γ) by PCR-DGGE. The cytokine profile of both populations was investigated by RT-PCR ELISA for IFN-γ, IL-2, IL-4, IL-5, IL-10, IL-13 and IL-15. In three other patients with known Vβ-usage, the dominant T cell clones were phenotypically characterized by double staining. PCR-DGGE of TCR-γ demonstrated that all patients had a clonal population in their blood and that this population was present in CD4 + CD7 – and CD4 + CD7 + populations. Concerning mRNA cytokine transcription, the two populations did not show any consistent differences. In three patients with identified clones (Vβ 3.1, 5.3 and 6.7), double staining revealed positivity for CD2, CD3, CD4, CD5, CD45RO and CD7 in a significant proportion (at least 35%). We conclude that the CD4 + CD7 – population does not represent the dominant T cell clone in patients with SS. An increase in this population of PBMC in SS might account for deviations in the T cell functions of the patients.

Immunization-induced decrease of the CD4+:CD8+ ratio in cats experimentally infected with feline immunodeficiency virus

In a previous experiment a group of 15 specified pathogen free (SPF) cats were experimentally infected with a Swiss isolate of feline immunodeficiency virus (FIV). A group of 15 SPF cats served as FIV negative controls. Nine cats of each group were vaccinated with a recombinant feline leukemia virus (FeLV) vaccine, six cats in each group with a placebo vaccine. All vaccinated cats developed high antibody titers to FeLV and were protected against subsequent FeLV challenge infection. In both control groups five of six cats became persistently infected with FeLV. Unexpectedly, the primary immune response to the vaccine antigen was significantly higher in the FIV positive group than in the FIV negative. The secondary response was stronger in the FIV negative cats. The goal of the present investigation was to further study the immune response in these 30 cats. They were immunized twice with the synthetic peptide L-tyrosine-L-glutamic acid-poly(DL-alanine)-poly(L-lysine) (TGAL) 21 days apart. Blood samples were collected on four occasions during the immunization process. They were tested for antibodies to TGAL, complete blood cell counts and CD4+, CD8+ and pan-T-lymphocyte counts. The following observations were made: (1) in contrast to the FeLV vaccine experiment, the primary immune response to TGAL was not significantly stronger in the FIV positive cats when tested by enzyme-linked immunosorbent assay (2). The absolute size of the CD4+ lymphocyte population was distinctly smaller in the FIV positive than in the FIV negative cats. The lowest CD4+ values were found in the dually FIV/FeLV infected cats. (3) A population of CD8+ lymphocytes was identified that was characterized by a distinctly weaker fluorescence. The size of this population increased in FIV positive and decreased in FIV negative cats during the TGAL immunization experiment. (4) The CD4+:CD8+ ratio increased in FIV negative cats during TGAL immunization from 1.9 to 2.3. In contrast, in FIV positive animals the CD4+:CD8+ ratio decreased significantly from 1.9 to 1.3 during the same period. From these and earlier data it was concluded that in short-term FIV infection the immune response to T-cell dependent antigens may be increased over that of the controls. Immune suppression develops gradually with duration of the infection. The significant drop of the CD4+:CD8+ ratio over a 5 week immunization period suggests that antigenic stimulation may accelerate the development of immune suppression in FIV positive cats. If this is a general feature, FIV infection may provide a particularly interesting model for studying the pathogenesis of AIDS.

The use of anti-T-cell receptor-Vβ antibodies for the estimation of treatment success and phenotypic characterization of clonal T-cell populations in cutaneous T-cell lymphomas

Summary. Sézary syndrome and Mycosis fungoides are the most common forms of cutaneous T‐cell lymphomas. To assess the response to different therapies especially in Sézary syndrome, it is helpful to monitor the percentage of circulating tumour cells in the blood. The use of T‐cell receptor (TCR)‐Vβ specific monoclonal antibodies provides a suitable tool for detecting Sézary cells. In this study, we analysed the levels of clonal CD4+Vβ+ cells of seven patients with various treatment modalities using flow cytometry and investigated the immunophenotype of the clonal cells by double staining with a panel of antibodies recognizing lymphatic surface markers. Additionally, a polymerase chain reaction‐denaturing gradient gel electrophoresis assay was performed on clonal CD4+Vβ2+ cells, showing that these cells carry a Vγ10/11, JγP1/2 TCR rearrangement. Follow‐up studies revealed close association of the Vβ+ clone developmentwith the clinical response to different therapiesinsixpatients. Intwo cases, the CD4+Vβ+ cells decreased accompanied by partial regression or even complete remission. In four cases, a stable or increasing clonal CD4+Vβ+ population reflected well a stable or progressing course of the disease. Double staining of Vβ+ cells revealed the following pattern, CD3+, CD5+, CD7+, CD28+, CD80–, CD86+ and human leucocyte antigen (HLA) class I+. In contrast, HLA‐DR was heterogeneously expressed. We conclude that identification and monitoring of CD4+Vβ+ clonal T cells by fluorescence‐activated cell sorting with double staining is a suitable method to assess clinical responses to different therapies.

Human CD8 T cells of the peripheral blood contain a low CD8 expressing cytotoxic/effector subpopulation

Heterogeneity of lymphocyte populations demonstrates the diversity of cellular immune responses and provide a better understanding of the immune system. CD3+ CD8+ T cells exhibit a low CD8 expressing (CD8low) population in flow cytometric analysis of peripheral blood T cells. In healthy donors, this population consists of 0·2–7·0% of all CD8 T cells. The majority of the CD8low T cell population showed an elevated expression of CD25, CD45RA, and CD95L, and low levels of CD28, CD62L and CD45RO. Circulating CD8low T cells resemble cytotoxic effector cells because they express cytolytic mediators and are able to execute cytotoxicity. A restricted T cell receptor profile with increased Vβ9, Vβ14 and Vβ23 expression was observed and the CD8low T cell population contain Epstein–Barr virus‐specific T cells. Therefore, the CD8low population represent a subset of activated CD8 effector T cells, resulting most probably from a continous and/or balanced immune response to intracellular pathogens.

Analysis of the phenotype and phagocytic activity of monocytes/macrophages from cattle infected with the bovine leukaemia virus.

The bovine leukaemia virus (BLV) is a retrovirus that infects mainly B lymphocytes of cattle, but proviral DNA can also be isolated from monocytes/macrophages. This study investigated the effect of BLV infection on surface antigens on freshly isolated peripheral blood monocytes and cultured monocyte-derived macrophages, with and without lipopolysaccharide (LPS) stimulation. The effect of BLV infection on phagocytic activity of CD14+ monocytes was also assessed. The percentage of monocytes expressing the surface antigens CD11b, CD32 (FcgammaRII), MHC class II and the surface antigen recognised by mAb DH59B were increased in BLV-positive cattle. In contrast, expression intensity of all markers was low in samples from BLV-positive cattle. CD14+ monocytes from BLV-positive cattle showed less Fcgamma-receptor-mediated phagocytosis compared to monocytes from BLV-negative cattle. After 7 days in culture, there was evidence for shedding/downregulation of surface antigens on monocyte-derived macrophages, in particular on cells from BLV-positive cattle. LPS stimulation decreased the percentage of cells expressing the measured markers in monocyte-derived macrophages taken from BLV-negative cattle, but not in cultures derived from BLV-positive cattle. The results provide further evidence for an altered function of monocytes and macrophages in BLV-infected cattle.

SEZARY SYNDROME, T-HELPER 2 CYTOKINES AND ACCESSORY FACTOR-1 (AF-1)

Mycosis fungoides and the Sézary Syndrome are characterized by clonal accumulation of well differentiated T-helper memory cells in the skin and, in the case of the Sézary Syndrome, also in the blood and lymph nodes. Well known immunological abnormalities in patients with MF and SS include reduced delayed type hypersensitivity, decreased proliferation upon stimulation with mitogens of the peripheral blood mononuclear cells, elevated IgE or IgA serum levels and eosinophilia. These abnormalities can be explained by the predominance of T-helper 2 cytokines. Clonal T-lymphocytes purified from the peripheral blood of SS patients transcribe mainly IL-10 and IL-5. They can be CD7 positive or negative. These clonal T cells express the accessory factor-1 (AF-1) or Interferon gamma receptor beta-chain that is essential for Interferon gamma signalling. These results imply perspectives for new therapeutical approaches, such as IL-12 or chimeric fusion proteins.

Dominance of highly divergent feline leukemia virus A progeny variants in a cat with recurrent viremia and fatal lymphoma.

BackgroundIn a cat that had ostensibly recovered from feline leukemia virus (FeLV) infection, we observed the reappearance of the virus and the development of fatal lymphoma 8.5 years after the initial experimental exposure to FeLV-A/Glasgow-1. The goals of the present study were to investigate this FeLV reoccurrence and molecularly characterize the progeny viruses.ResultsThe FeLV reoccurrence was detected by the presence of FeLV antigen and RNA in the blood and saliva. The cat was feline immunodeficiency virus positive and showed CD4+ T-cell depletion, severe leukopenia, anemia and a multicentric monoclonal B-cell lymphoma. FeLV-A, but not -B or -C, was detectable. Sequencing of the envelope gene revealed three FeLV variants that were highly divergent from the virus that was originally inoculated (89-91% identity to FeLV-A/Glasgow-1). In the long terminal repeat 31 point mutations, some previously described in cats with lymphomas, were detected. The FeLV variant tissue provirus and viral RNA loads were significantly higher than the FeLV-A/Glasgow-1 loads. Moreover, the variant loads were significantly higher in lymphoma positive compared to lymphoma negative tissues. An increase in the variant provirus blood load was observed at the time of FeLV reoccurrence.ConclusionsOur results demonstrate that ostensibly recovered FeLV provirus-positive cats may act as a source of infection following FeLV reactivation. The virus variants that had largely replaced the inoculation strain had unusually heavily mutated envelopes. The mutations may have led to increased viral fitness and/or changed the mutagenic characteristics of the virus.

Separation of a cysteine-rich surface antigen-expressing variant from a cloned Giardia isolate by fluorescence-activated cell sorting

Clonally derived trophozoites (clone O2-4A1, from a sheep) of the morphologically defined group ofGiardia duodenalis change their cysteine-rich surface proteins (CRISPs) spontaneously during in vitro culture. This phenomenon constitutes a serious obstacle for studies that rely on a pure population of cells bearing a particular variant CRISP. We describe herein the successful separation and quantitation of a trophozoite subpopulation expressing a 90-kDa major surface antigen (CRISP-90) from a heterologous population using fluorescence-activated cell sorting (FACS) on the basis of fluorescein-tagged antibodies directed specifically against O2-4A1 CRISP-90. During subsequent in vitro culture, the purified cell population exhibited a progressive decline in the proportion of cells labeled by CRISP-90 specific antibodies. After 46 generations, only one-third of the total trophozoite population reacted with the anti-CRISP-90 antibodies.

Single pericytes and pericytes in suspension are stimulated in a similar way by low-density lipoprotein

Background Pericytes are regarded as the microvascular counterpart of smooth muscle cells and implicated in the regulation of blood pressure at the microvascular level. Ca2+ plays an important role in biochemical processes involved in blood pressure regulation and can be activated by low-density lipoprotein (LDL) cholesterol. Objective To determine whether stimulation either of single cells or of cells in suspension by LDL would produce any difference in the increase in cytosolic free calcium levels ([Ca2+]i). Design and methods Single pericytes were loaded with 2 μmol/l of the Ca2+-sensitive dye Indo-1/AM. The Indo-1 fluorescence was recorded at 405 nm (Ca2+-bound) and 485 nm (Ca2+-free) after stimulation with LDL. Pericytes in suspension were loaded with 2 μmol/l of the Ca2+-sensitive dye FURA-2/AM. The FURA-2 fluorescence kinetics were recorded at 340–380 nm. Ratios of fluorescence at the two wavelengths were transformed to [Ca2+]i. Results Basal [Ca2+]i levels appeared to be higher in single cells (148 ± 13 nmol/l, n = 20) than they were in cells in suspension (128 ± 8 nmol/l, n = 25; P = 0.0078). After stimulation with LDL the increase in [Ca2+]i in both systems was about 220% above baseline. A clear dose dependency was seen for both systems. Conclusions Single pericytes and pericytes in suspension increase their [Ca2+]i after stimulation with LDL dose-dependently. Even though single-cell measurements revealed some technical limitations, their responses were comparable to those obtained in a cell suspension. In analogy to aortic smooth muscle cells, our results indicate that LDL might also play a blood-pressure-regulatory role in the microvasculature.