Subpopulations Of Peripheral Blood Mononuclear Cells Research Articles

Detection of HBD1 peptide in peripheral blood mononuclear cell subpopulations by intracellular flow cytometry

Production of human β-defensin1 (HBD1) in response to LPS in monocytes, myeloid dendritic cells and plasmacytoid dendritic cells (PDC) was examined. Since PDC make up only 0.1–0.5% of the peripheral blood mononuclear cell population, we developed a method to determine HBD1 peptide levels using four-color flow cytometry, which can examine several cell surface or intracellular markers at once. Coupled with intracellular flow cytometry, we determined that PDC and monocytes only made significant amounts of HBD1 when exposed to >50 ng/ml LPS for 2 h. This response was limited to monocytes when ultrapure LPS was used, and was inhibited in PDC by chloroquine treatment.

In vivo transcription of bovine leukemia virus and bovine immunodeficiency-like virus

Cellular tropism and transcription of bovine leukemia virus (BLV) and bovine immunodeficiency-like virus (BIV) were investigated using peripheral blood mononuclear cells (PBMC) collected from a cow infected with both viruses. Each PBMC subset, purified by magnetic cell sorting, was subjected to PCR and RT-PCR for detection of their integrated proviruses and transcript mRNAs. Both BLV and BIV genomes were detected by nested PCR in CD3 +, CD4 +, CD8 + and γδ T cells, B cells and monocytes. However, BLV tax transcription was only detected in B cells, and only B cells also formed BLV syncytia in CC81 cells. On the other hand, BIV transcript was detected in each subpopulation of PBMC. These results indicated that BLV can infect T cells and monocytes as well as B cells, but can be expressed by transcription only in B cells. In contrast, BIV can express its transcripts in all infected cells.

The varicella-zoster virus induces apoptosis in vitro in subpopulations of primary human peripheral blood mononuclear cells

Varicella-zoster virus (VZV), a member of Herpesviridae, subfamily α-Herpesvirinae, is pathogenic exclusively in the human. Chickenpox is the result of primary infection of VZV. During the viremic stage, VZV infects peripheral blood mononuclear cells (PBMC) and spreads to the periphery. In skin cells it causes typical lesions. Apoptosis has been demonstrated in different cell types by other α-herpesviruses. VZV-infected T lymphocytes, B lymphocytes, and monocytes, respectively, were examined in this in vitro study by flow cytometry, immunofluorescence and electron microscopy. All infected cell types showed signs of apoptosis: a lower DNA content, DNA fragmentation, loss of membrane integrity, and an altered nuclear morphology. The results observed led to the suggestion that VZV can induce apoptosis during infection in vivo in the PBMC subpopulations.

Assessing the feasibility of using neural precursor cells and peripheral blood mononuclear cells for detection of bioactive Sindbis virus

Viruses form a significant class of bio-threat agents. Currently, the only method to determine the bioactivity of viruses in vitro is to measure viral and cellular responses after co-incubation of cells with virus. Our goal is to find biomarkers for classification of agents, establishment of bioactivity, and/or prediction of disease outcomes. To begin development of a cell-based biosensor for detection of bioactive Sindbis virus (SV), our model analyte, we surveyed the outcomes of SV interaction with primary rat neural precursor cells (NPC) and human peripheral blood mononuclear cells (PBMC). Confocal fluorescence analysis of NPC treated with recombinant SV carrying green-fluorescent-protein (SV-GFP) showed that most cells were GFP positive by day 1 post inoculation. 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI) staining of the nucleus showed nuclear condensation and fragmentation, and the percentage of TUNEL positive cells were higher in virus-treated cells than in mock-treated control. Also, there were less BrdU positive cells in virus-treated cells compared to control. Thus, SV infects NPC, decreases cellular proliferation, and induces cell death via apoptosis. PBMC were treated with SV- or UV-inactivated SV. By day 5 post infection, there were fewer adherent cells in SV-treated PBMC compared to UV-inactivated SV treated PBMC. However, the percentage of viable cells remained the same, and virus growth curves showed only clearance of virus. Thus, SV induces detachment of a subpopulation of PBMC while not killing most of the cells. Together, these results indicate that NPC and PBMC respond to bioactive SV inoculation, suggesting potential use as detectors of SV in cell-based biosensor paradigm. These studies also provide the rationale, time-scale, and phenotypic correlates for further studies with gene expression arrays.

Infectibility of separated peripheral blood mononuclear cell subpopulations by varicella-zoster virus (VZV).

Varicella zoster-virus (VZV) is a humanpathogenic alpha-Herpesvirus that causes chickenpox after primary infection. The virus spreads by aerosol or direct contact with infectious vesical fluids, it enters the body via the respiratory tract. In a first viremic stage it replicates in local lymph nodes, followed by a secondary viremic stage. In the course it spreads through the body to endothelial cells in the periphery. During acute viremia of chickenpox viral DNA can be detected in peripheral blood mononuclear cells (PBMC) by PCR and in situ hybridization. Recently published results quantified the viral DNA load in PBMC and subpopulations by real-time PCR. In the animal SCID-hu mouse model system VZV showed a tropism for T-lymphocytes. The aim of this work was the investigation of viral ability to infect and to replicate in purified primary subtypes of PBMC, i.e., T-lymphocytes, B-lymphocytes, and monocytes. These cells were isolated from whole peripheral blood or tonsils and infected with cell-free VZV for different time periods. In all cell types, transcriptional activity was shown by amplification and detection of immediate early (IE) and late (L) viral mRNA by NASBA or RT-PCR. Expression of viral glycoproteins was analyzed and proved in lymphocytes by immunofluorescence microscopy.

Hodgkin and Reed-Sternberg cell–associated autoantigen CLIP-170/restin is a marker for dendritic cells and is involved in the trafficking of macropinosomes to the cytoskeleton, supporting a function-based concept of Hodgkin and Reed-Sternberg cells

Little is known about the distribution in normal cells of CLIP-170, a linkage mediator between endocytic vesicles and microtubules, and restin, a splice variant encoded by the same gene and marker for Hodgkin and Reed-Sternberg (HRS) cells of Hodgkin disease. Although only trace amounts of CLIP-170/restin are present in peripheral blood mononuclear cell subpopulations, monocyte-derived dendritic cells (DCs) and interleukin-4 (IL-4) + CD40L-activated B cells express high levels of CLIP-170/restin. CLIP-170/restin colocalizes preferentially with membranes of intermediate macropinocytic vesicles, suggesting a new function of CLIP-170/restin in the trafficking of macropinosomes to the cytoskeleton, which is a crucial step in antigen presentation. The strong expression of CLIP-170/restin in HRS cells, DCs, and activated B cells underscores their functional similarities supporting a function-based concept of HRS cells as professional antigen-presenting cells.

Human monocytes synthesize hyaluronidase.

The involvement of hyaluronic acid (HA) oligosaccharides and blood-derived mononuclear cells in inflammatory processes prompted us to determine whether peripheral blood mononuclear cells (PBMC) possess hyaluronidase activity. PBMC were incubated with macromolecular-tritiated HA at pH 3.8 and supernatants were analysed by size exclusion chromatography to reveal digestion of HA. This digestion was due to the CD14-positive (CD14+), adherent, non-specific esterase-positive, subpopulation of PBMC. Hyaluronidase activity (72 kDa) was found in aqueous and non-ionic detergent PBMC extracts but not in the medium in which the cells had been cultured. These results indicate that hyaluronidase is, at least in part, linked to the membrane rather than excreted. Hence, monocytes have one or more hyaluronidases that can generate a pool of active HA fragments within tissues. Hyaluronidase activity was also found in 3/3 myelomonocytic lineage leukaemias but not in 3/3 lymphoblastic leukaemias.

Globotriaosylceramide (Gb 3/CD77) is synthesized and surface expressed by bovine lymphocytes upon activation in vitro

Neutral glycosphingolipids (GSLs) are considered activation markers on human lymphocytes, which are fundamental for studying the immune system. For cattle, only a limited number of activation markers has yet been identified. We recently showed that Shiga toxin 1, known to use globotriaosylceramide (Gb 3 syn. CD77) as a cellular receptor, depresses proliferation of activated bovine lymphocytes [Infect. Immunol. 67 (1999b) 2209]. In order to confirm the expression of Gb 3/CD77 on bovine lymphocytes, we flowcytometrically examined a bovine B-lymphoma cell line (BL-3) and bovine peripheral blood mononuclear cells (PBMC) before and after mitogenic stimulation and biochemically characterized neutral GSLs extracted from PBMC. CD77 was detected on the surface of BL-3 cells and cultured PBMC essentially after mitogenic stimulation. Although expressed by all PBMC subpopulations identified, the portion of CD77 + cells was highest for BoCD8 + cells, followed by B-cells and BoCD4 + cells at day 4 of cultivation. Ceramide trihexoside of stimulated PBMC was structurally determined as Gal(α1-4)Gal(1-4)Glc(1-1)ceramide (Gb 3). Biochemically, Gb 3 was also detected within unstimulated PBMC which contained ceramide monohexoside (CMH) and Gb 3 in a ratio of about 4:1. However, stimulation induced an increase of CMH and Gb 3 by a factor of 2.5 and 10, respectively, implicating that bovine lymphocytes regulate surface expression of Gb 3/CD77 predominantly by quantitative changes in the Gb 3 metabolism. This report presents Gb 3/CD77 as the first GSL identified on bovine immune cells and highly recommends this activation dependent antigen as a useful tool to investigate lymphocyte activation within the bovine immune system.

Investigation of varicella-zoster virus DNA in lymphocyte subpopulations by quantitative PCR assay.

To investigate the nature of viremia during the acute phase of varicella, we studied the viral load in nine otherwise healthy children with varicella. Plasma and peripheral blood mononuclear cells (PBMC) were obtained, then PBMC were divided into CD4+T, CD8+T, and B lymphocytes and monocyte/macrophage fractions. The viral DNA in each component was quantified using a real-time quantitative polymerase chain reaction assay. Varicella-zoster virus (VZV) DNA was detected in plasma, PBMC and all subpopulations. The amount of viral DNA was similar in each PBMC subpopulation, suggesting that each lymphocyte fraction and monocytes carry similar amounts of VZV DNA during viremia.

Inflammatory Cytokines and Antigen-Responsive Mononuclear Cells in Peripheral Blood of Cattle Infected with Salmonella Takoradi.

To determine the immunological response in lactating dairy cows infected with Salmonella (S.) Takoradi, the relationships among distributions of peripheral blood mononuclear cell (PBMC) subpopulations, endotoxin concentrations and dynamics of inflammatory cytokines in blood were investigated. The ratio of CD4+ T cells to CD8+ T cells was significantly lower in the affected cattle than in the control cattle (p<0.05) to decrease in the number of CD4+ T cells in the infected cattle. In contrast, the numbers of gammadeltaT cells, MHC class II-positive cells were significantly higher in the affected cattle than in the control cattle (p<0.01 respectively). Endotoxemia was found in all but one of the affected cattle. Serum IL-1 and IL-6 bioactivities were significantly higher in the affected cattle than in the control cattle (IL-1, p<0.05; IL-6. p<0.01). Serum TNF-alpha activities and levels were not detected in the control and affected cattle. The activities of proinflammatory cytokines determined by the bioassay are important to the relationships between concentration of endotoxin, cytokines and clinical signs. such as leukocytosis, leukopenia, fever or bacterial shedding. Serum IL-2 levels were lower in the affected cattle than in the control cattle. Serum IFN-y was not detected in the affected cattle except one. These results by the ELISA seemed to reflect the condition of subpopulation in the PBMCs from the shedding cattle. The present results suggest that cellular immunity is suppressed while the humoral immunity is activated in acute bovine salmonellosis.

HCV in serum, peripheral blood mononuclear cells and lymphocyte subpopulations in C-hepatitis patients

To define the best marker for the follow-up and evaluation of HCV infections we determined anti-HCV antibodies, serum transaminases and HCV RNA in patients diagnosed with chronic hepatitis for C virus and treated with α-interferon. The presence/absence of HCV RNA was determined in serum, peripheral blood mononuclear cells (PBMC) and lymphocyte subpopulations. Samples were submitted to RT-PCR and subsequent nested PCR. Treatment with α-interferon induced a fall in the number of HCV RNA positive patients from an initial 88 to 25% at the end of the treatment. The withdrawal of treatment was associated with a significant increase in the number of HCV RNA positive patients (43% at the 12-month follow-up). In 61% of the patients the PCR analysis of the PBMC population detected the presence of HCV RNA. In 87% of cases the cell fraction identified as CD19 resulted positive in the PCR test and the viral genome was undetectable in PBMC subpopulations in only 13% of cases. In one third of the patients whose serum was negative for PCR the analysis demonstrated the presence of HCV RNA in PBMC. Conclusions: The disappearance of the viral genome in serum, a criterion of treatment response, is not necessarily followed by its disappearance in PBMC. The joint determination of HCV by PCR technique in serum and blood cells should be used as a particular instrument with each patient.

Interleukin-16 stimulates the expression and production of pro-inflammatory cytokines by human monocytes.

Interleukin-16 (IL-16) acts as a chemoattractant for CD4+ cells, as a modulator of T-cell activation, and plays a key role in asthma. This report describes the cytokine-inducing effects of IL-16 on total peripheral blood mononuclear cells (PBMC) and PBMC subpopulations. While CD4+ T lymphocytes did not secrete cytokines in response to rhIL-16, CD14+ CD4+ monocytes and maturing macrophages secrete IL-1beta, IL-6, IL-15 and tumour necrosis factor-alpha (TNF-alpha) upon rhIL-16 stimulation. The mRNA species for these four cytokines were detected as early as 4 hr post-stimulation, with protein being secreted by 24 hr. Secretion of IL-1beta and IL-6 by total PBMC was dose dependent, with maximal secretion being observed using 50 ng/ml rhIL-16. However, for IL-15 or TNF-alpha maximal secretion by total PBMC occurred with all concentrations between 5 ng/ml to 500 ng/ml rhIL-16. Purified monocytes/macrophages secreted maximal concentrations of all four cytokines in the presence of 500 ng/ml rhIL-16, except for monocytes where maximal secretion of IL-15 was, interestingly, observed with only 50 ng/ml rhIL-16. The use of higher concentrations of rhIL-16 (1000 ng/ml) inhibited secretion of all four cytokines. While these IL-16-induced cytokines are likely to be involved in the immune system's response to antigen, the data suggest that IL-16 may play a key role in initiating and/or sustaining an inflammatory response.

Modulation of humoral and cell-mediated immune responses by dietary lutein in cats

The immuno-modulatory role of dietary lutein in domestic cats is unknown. Female Tabby cats (10-month old; n=56) were supplemented daily for 12 weeks with 0, 1, 5 or 10 mg lutein. Blood was collected on Weeks 0, 2, 4, 8 and 12 to assess the following: (1) mitogen-induced peripheral blood mononuclear cells (PBMCs) proliferation, (2) changes in PBMC subpopulations, (3) interleukin-2 (IL-2) production and (4) plasma immunoglobulin (Ig)G production. In addition, delayed-type hypersensitivity (DTH) response to concanavalin A (Con A) or a polyvalent vaccine was performed on Weeks 0, 6 and 12. Dietary lutein increased plasma lutein concentrations in a dose-dependent manner ( p<0.001) and concentrations had not reached steady state after 12 weeks of feeding in cats given 5 or 10 mg lutein. Concentrations of plasma retinol and α-tocopherol were not influenced by diet. The DTH response to vaccine but not to Con A increased ( p<0.05) in a dose-dependent manner on Week 6. Compared to control, cats fed lutein also showed enhanced Con A- and pokeweed mitogen-stimulated PBMCs proliferation. Dietary lutein also increased the percentages of CD4+ and CD21+ lymphocytes on Week 12 but had no significant effect on pan T, CD8 and MHC class II markers. Plasma IgG was higher ( p<0.05) in cats fed 10 mg lutein on Weeks 8 and 12. These results support the immuno-modulatory action of lutein in domestic cats.

Virus antigen expression and alterations in peripheral blood mononuclear cell subpopulations after classical swine fever virus infection

Depletion in the number of lymphocytes and viral persistence are thought to be the most important outcomes of classical swine fever virus (CSFV) infection. To define the change in peripheral blood mononuclear cells (PBMC) and virus replication in leukocytes after CSFV infection, 8-week old pigs were infected with the LPC vaccine strain or virulent CSFV (HCV-YL strain). Changes in the relative number of PBMCs were analyzed by flow cytometry. The results showed a significant increase in the relative percentage of monocytes in PBMCs during acute CSFV infection of naive pigs ( p < 0.05). Monocyte frequencies were not changed in LPC-vaccinated pigs and control pigs. There was also a significant decrease in the number of IgM + cells ( p < 0.05) and a slight decrease in the number of CD4 + lymphocytes after 5 days of infection. There was no change in the frequency of CD8 + lymphocytes in PBMCs after infection. To define which subpopulation of PBMCs was the target for CSFV infection, PBMC populations from CSFV infected pigs were separated and stained for virus antigen expression. Alveolar macrophages (AM) were also studied. The results showed that CSFV replicated in all PBMC subpopulations: CD4 +, CD8 +, and IgM + lymphocytes, and monocytes as well as AMs. However, virus antigen expression was more intense in monocytes and AMs. The infection of lymphocytes may, therefore, contribute to the depletion in their numbers after infection and lead to defective antibody production during virulent CSFV infection.

Shiga toxin 1 from Escherichia coli blocks activation and proliferation of bovine lymphocyte subpopulations in vitro.

Shiga toxin-producing Escherichia coli (STEC) is widespread in the cattle population, but the clinical significance of Shiga toxins (Stx's) for the bovine species remains obscure. Since Stx's exert immunomodulating effects in other species, we examined the effect of purified Stx1 on a bovine B lymphoma cell line (BL-3) and peripheral blood mononuclear cells (PBMC) isolated from adult bovine blood by viability assays and flow cytometry analysis. Stx1 markedly induced apoptosis in stimulated BL-3 cells. The susceptibility of this B-cell-derived cell line was induced only by either lipopolysaccharide (LPS) or pokeweed mitogen, while cultures stimulated with T-cell mitogens were unaffected by the toxin. In contrast, Stx1 did not induce cellular death-neither apoptosis nor necrosis-in primary cultures of PBMC but hindered the mitogen-induced increase in metabolic activity. The influence of Stx1 on single PBMC subpopulations varied with the type of mitogenic stimulus applied. Stimulation with phytohemagglutinin P particularly induced the proliferation of bovine CD8-expressing (BoCD8(+)) cells, and this proliferative response was blocked by Stx1. On the other hand, Stx1 reduced the portion of viable B cells in the presence of LPS. Modulation of activation marker expression (BoCD25 and BoCD71) by Stx1 indicated that the toxin hindered the proliferation of cells by blocking their activation. In conclusion, we assume that Stx1 contributes to the pathogenesis of STEC-associated diarrhea in calves by suppressing the mucosa-associated immune response. The usefulness of cattle as a model in which to study Stx-induced immunomodulation is discussed.

HBV viral load within subpopulations of peripheral blood mononuclear cells in HBV infection using limiting dilution PCR

Extrahepatic viral load in peripheral blood mononuclear cells (PBMCs) of patients with hepatitis B virus (HBV) is still under debate. In this study, HBV infection rates and viral titers were examined within all PBMC subpopulations using limiting dilution-PCR (LD-PCR). PBMCs of patients with acute or chronic hepatitis B were separated by magnetic beads in monocytes, B-cells, CD4+ T-cells, CD8+ T-cells, and NK cells. Using two-round nested PCR, HBV-DNA sequences were detected in all patients examined within each PBMC subpopulation. The frequencies of HBV-positive cells and viral loads were calculated by Poisson analysis of HBV PCR results from serial dilutions of cells and cell lysates. Highest infection rates were found in monocytes and B-cells followed by CD8+ T-cells, NK cells, and CD4+ T-cells. Concerning all subsets, frequencies of HBV-positive cells were 50- to 500-fold higher in chronic than in acute hepatitis B. Viral loads were mostly estimated at about one HBV genome per HBV-positive cell. Moreover, slightly elevated HBV titers were seen in B-cells, CD4+ T-cells, and NK cells in both acute and chronic hepatitis B. It was demonstrated that beside a generally more latent HBV infection in PBMCs, elevated HBV titers point to replication or selective viral uptake within particular PBMC subsets. Therefore, the data suggest that HBV-infected PBMCs may participate in persistence of HBV.

Lipopolysaccharide stimulates butyric acid-induced apoptosis in human peripheral blood mononuclear cells.

We previously reported that butyric acid, an extracellular metabolite from periodontopathic bacteria, induced apoptosis in murine thymocytes, splenic T cells, and human Jurkat T cells. In this study, we examined the ability of butyric acid to induce apoptosis in peripheral blood mononuclear cells (PBMC) and the effect of bacterial lipopolysaccharide (LPS) on this apoptosis. Butyric acid significantly inhibited the anti-CD3 monoclonal antibody- and concanavalin A-induced proliferative responses in a dose-dependent fashion. This inhibition of PBMC growth by butyric acid depended on apoptosis in vitro. It was characterized by internucleosomal DNA digestion and revealed by gel electrophoresis followed by a colorimetric DNA fragmentation assay to occur in a concentration-dependent fashion. Butyric acid-induced PBMC apoptosis was accompanied by caspase-3 protease activity but not by caspase-1 protease activity. LPS potentiated butyric acid-induced PBMC apoptosis in a dose-dependent manner. Flow-cytometric analysis revealed that LPS increased the proportion of sub-G1 cells and the number of late-stage apoptotic cells induced by butyric acid. Annexin V binding experiments with fractionated subpopulations of PBMC in flow cytometory revealed that LPS accelerated the butyric acid-induced CD3(+)-T-cell apoptosis followed by similar levels of both CD4(+)- and CD8(+)-T-cell apoptosis. The addition of LPS to PBMC cultures did not cause DNA fragmentation, suggesting that LPS was unable to induce PBMC apoptosis directly. These data suggest that LPS, in combination with butyric acid, potentiates CD3(+) PBMC T-cell apoptosis and plays a role in the apoptotic depletion of CD4(+) and CD8(+) cells.

A receptor for the lipocalin placental protein 14 on human monocytes

Human placental protein 14 (PP14), a member of the lipocalin structural superfamily, is an abundant amniotic fluid glycoprotein with documented immunoinhibitory activities. While receptors have been characterized for several other lipocalins, none have been reported to date for PP14. In the present study, two-color immunofluorescence and flow cytometry was used to screen peripheral blood mononuclear cell subpopulations for their capacity to engage fluoresceinated recombinant PP14. The tagged PP14 bound strongly in a specific and saturable fashion to CD14 + (monocyte lineage) cells, but not to CD20 + (B cell lineage) or CD3 + (T cell lineage) cells. This binding was both pH- and temperature-sensitive, and was reduced by proteolytic pre-digestion of the cells with trypsin or proteinase K. Scatchard analysis demonstrated a single class of receptors on CD14 + cells, with a K D of ∼1×10 −8 and ∼10–35 000 receptors per cell. These findings constitute the first report of a cell surface-associated binding protein for PP14 and set the stage for exploring the molecular mechanisms of PP14-mediated signaling and immunomodulation.

Anti-(epidermal growth factor) receptor monoclonal antibodies for the induction of antibody-dependent cell-mediated cytotoxicity against squamous cell carcinoma lines of the head and neck.

Squamous cell carcinomas of the head and neck (SCCHN) frequently display high levels of the epidermal growth factor receptor (EGFR). Since EGFR is expressed on the cell surface it may form a suitable target for anticancer therapy with anti-receptor monoclonal antibodies (mAb). Besides the interference with receptor/ligand interactions, binding of mAb to EGFR leads to immunoglobulin-coated tumour cells that may induce or enhance non-specific immune effector mechanisms like antibody-dependent cell-mediated cytotoxicity (ADCC). In established cell lines of SCCHN (UM-SCC 11B, 14C, 22B, and 8029 NA) we investigated the antitumour activity of allogeneic peripheral blood mononuclear cells (PBMC) in combination with rat (ICR 62), mouse (EMD 55900), and humanized (EMD 72000) anti-EGFR mAb. In addition, autologous PBMC were available for tumour line UD-SCC 4. The EGFR protein content of the tumour cell lines ranged between 170 fmol/mg protein and 8100 fmol/mg protein, and MCF-7 cells served as receptor-negative controls. PBMC activity against SCCHN targets was determined in 96-well microtitre-plate monolayer cultures by the colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay after coincubation for 4 h, 24 h and 72 h at effector target ratios of 1:1, 5:1, 10:1 and 20:1. PBMC subpopulations were obtained by macrophage depletion (plastic adherence) or natural killer (NK) cell enrichment (magnetic bead negative selection). Prolonged time of exposure and increased effector:target ratios revealed marked antitumour activity of PBMC alone. This non-specific immune destruction was enhanced considerably by humanized and rat, but not mouse anti-EGFR mAb. Increased EGFR protein in tumour cells partly correlated with an intensification of ADCC but was accompanied by decreased primary PBMC cytotoxicity. The utilization of PBMC subpopulations suggested a mainly NK-cell-mediated ADCC, which appeared to benefit directly or indirectly, e.g. via the secretion of cytokines, from other PBMC components. In conclusion, humanized (EMD 72000) and rat (ICR 62) anti-EGFR mAb were able to generate strong antitumour ADCC in target monolayers of SCCHN.

Bovine herpesvirus 1-induced apoptosis: phenotypic characterization of susceptible peripheral blood mononuclear cells.

Bovine herpesvirus 1 (BHV-1), a member of the Alphaherpesvirinae, induces apoptotic cell death in peripheral blood mononuclear cells (PBMC). To investigate the process by which BHV-1 induces apoptosis, we determined the susceptibility of the three main PBMC subpopulations to BHV-1-induced apoptosis. This study shows that BHV-1 can induce apoptosis individually in T lymphocytes, B lymphocytes and monocytes. This conclusion is based on the following findings: (i) BHV-1 substantially reduces the percentages of viable T and B lymphocytes in PBMCs. (ii) Concomitant detection of cell phenotype and apoptosis indeed showed higher percentages of apoptotic T lymphocytes and B lymphocytes in BHV-1-infected PBMCs than in mock-infected cells. (iii) Each individual PBMC subpopulations (B lymphocytes, T lymphocytes and monocytes) undergo apoptosis when incubated with BHV-1. These data also suggest that BHV-1 does not require the recruitment of one or more individual PBMC subpopulations (e.g. cytotoxic cells) to induce apoptosis. Finally, we observed that BL-3 cells which have been characterized as bovine tumoral B lymphocytes also undergo apoptosis when incubated with BHV-1. Therefore, the use of the BL-3 cell line provides a new experimental model to investigate the apoptotic process induced by BHV-1 in vitro.