Unstimulated Peripheral Blood Lymphocytes Research Articles

IL-23 Gene Expression in PBMCs of Patients with Coronary Artery Disease

Objective: Both adaptive and innate immune systems are involved in coronary artery disease (CAD). The aim of this study was to evaluate TH17 cytokines expression profiles in un-stimulated peripheral blood lymphocytes (PBMCs) of patients with coronary artery disease. Methods: Expression profiles of IL-17, IL-23, and TGF-β1 were determined in individuals with and without CAD using Real-time PCR. Results: A significant decrease in IL-23 gene expression in un-stimulated PBMCs of patients with CAD compared to those without CAD was found (p=0.003, OR=0.045, 95% CI: 0.006–0.355). Conclusion: Our data reinforce the potential role of the IL-23 as a critical regulatory molecule that bridges the innate and adaptive arms of the immune system in the complex mechanisms associated with the development of atherosclerosis.

IL-23 gene expression in PBMCs of patients with coronary artery disease.

Objective: Both adaptive and innate immune systems are involved in coronary artery disease (CAD). The aim of this study was to evaluate TH17 cytokines expression profiles in un-stimulated peripheral blood lymphocytes (PBMCs) of patients with coronary artery disease. Methods: Expression profiles of IL-17, IL-23, and TGF-β1 were determined in individuals with and without CAD using Real-time PCR. Results: A significant decrease in IL-23 gene expression in un-stimulated PBMCs of patients with CAD compared to those without CAD was found (p=0.003, OR=0.045, 95% CI: 0.006–0.355). Conclusion: Our data reinforce the potential role of the IL-23 as a critical regulatory molecule that bridges the innate and adaptive arms of the immune system in the complex mechanisms associated with the development of atherosclerosis.

Phosphorylation Levels of Extracellular-Signal Regulated Kinase (ERK) and AKT in Circulating Lymphocytes Predict Response to Targeted Therapy with Kinase Inhibitors in Refractory/Relapsed Hodgkin Lymphoma Patients,

Abstract 3705 INTRODUCTION:Identification of predictive biomarkers of response to molecularly targeted therapy represents a challenging issue. Therefore, we conducted a pharmacodynamic study evaluating phosphorylation levels of ERK (pERK) and AKT (pAKT) in circulating lymphocytes from patients with refractory/relapsed Hodgkin lymphoma (HL) enrolled in two consecutive phase II trials evaluating activity and safety of the multikinase inhibitor Sorafenib (Nexavar®, Bayer) used as single agent, or in combination with the AKT inhibitor Perifosine (Æterna Zentaris GmBH, Germany). METHODS:Between July 2008 and May 2011, 25 patients (15 males and 10 females; median age 36 years, range, 18–73 years) with relapsed (n = 3) or refractory (n = 22) HL were analyzed. In one trial, patients received Sorafenib alone (400 mg BID, per os), and in the second trial Perifosine (50 mg BID, per os) for 4 weeks followed by combination therapy of Perifosine plus Sorafenib (400 mg BID, per os) until disease progression or clinically significant toxicity. Tumor responses were assessed according to the revised response criteria for malignant lymphoma of the International Working Group. Peripheral blood lymphocytes (PBL) collected prior to therapy initiation and monthly thereafter were analyzed for pERK and pAKT expression by flow cytometry. Unstimulated or phorbol myristate acetate (PMA)-stimulated PBL were fixed with paraformaldehyde, permeabilized with methanol and stained with Alexa Fluor-conjugated Anti-ERK1/2 (pT202/pY204) and anti-Akt (pS473) monoclonal antibodies (MoAbs) or the appropriate isotype controls (all from Becton-Dickinson). PBL from normal donors were also included as internal control. Results were expressed as percentage of cells stained with the relevant MoAb in PMA-stimulated cultures after subtraction of staining detected in unstimulated PBL. The study was approved by the Institutional Ethical Committee. RESULTS:Eight out of 25 patients (32%) achieved objective responses, including complete response (CR, n = 1) and partial response (PR, n = 7), whereas 9/25 patients achieved stable disease (SD), and 8/25 patients experienced PD. Median overall survival (OS), progression-free survival (PFS) and duration of response (DOR) were 16, 5 and 4 months, respectively. Data of pERK and pAKT were available for 23 and 20 patients, respectively. At baseline, the median pERK level was 43% (range, 6 – 82). Baseline pERK levels were significantly higher in responsive patients, i.e., those achieving CR, PR and SD (n = 14) as compared to unresponsive patients (n = 9), i.e., those experiencing PD, (55 ± 18% vs 22 ± 9%, P =.0001). The median baseline value of pERK efficiently discriminated responsive and unresponsive patients. In fact, 12/12 patients with baseline pERK levels ≥43% achieved CR, PR or SD, whereas 9/11 patients (82%) with baseline pERK <43% experienced PD (P <.0001). Moreover, median PFS was 9 months for patients with baseline pERK ≥43%, and 3 months for patients with baseline pERK <43% (P <.0007). After 2 months of therapy, pERK levels were significantly reduced in responsive patients (P =.0001), whereas they were unchanged in unresponsive patients. Similarly, significantly higher baseline levels of pAKT were observed in responsive (n = 12) as compared to unresponsive (n = 8) patients (42 ± 14% vs 15 ± 6%, P ≤.0001), with a significant reduction of pAKT levels being observed after 2 months of therapy in responsive patients only. The median baseline value of pAKT discriminated responsive and unresponsive patients (P =.02). In fact, patients with pAKT levels ≥23% (n = 10) had significantly longer PFS than patients with baseline pAKT levels <23% (7 vs 3 months, P ≤.008). Assuming the median baseline levels of pERK (43%) and pAKT (23%) as cut-off, the positive predictive values were 100% for pERK and 90% for pAKT, and the negative predictive values were 82% for pERK and 70% for pAKT. Sensitivity of pERK ≥43% and pAKT ≥23% were 86% and 75%, respectively, with a specificity of 100% and 87%, respectively. CONCLUSIONS:Clinical data from phase II studies using Perifosine and/or Sorafenib in patients with relapsed/refractory HL patients suggest a correlation of baseline pERK and pAKT levels with objective responses and time to tumor progression. Whether these parameters will prove to be useful predictive biomarkers of response in HL patients receiving kinase inhibitors will need to be validated in prospective studies. Disclosures:Off Label Use: Sorafenib in Hodgkin Lymphoma Patients.

Expression of IL-10, IL-4 and interferon-gamma in unstimulated and mitogen-stimulated peripheral blood lymphocytes from HIV-seropositive patients.

Infection of immune cells with HIV induces dysregulation of cytokines which may play a vital role in HIV pathogenesis. We analysed the expression of T helper type 1 (Th1) (interferon-gamma (IFN-gamma)) and Th2 (IL-4, IL-10) type cytokines in peripheral blood lymphocytes (PBL) from HIV+ patients. The semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) analysis revealed that IFN-gamma mRNA in unstimulated PBL was significantly decreased and IL-10 mRNA was significantly upregulated in patients with < 400 CD4+ T cells/mm3 (n = 30) as compared to patients with > 400 CD4+ T cells/mm3 (n = 6) and normal controls (n = 16). In addition, IL-10 mRNA levels were inversely associated with IFN-gamma expression. Similar results were obtained by measuring IL-10 production in the supernatants of PBL cultured in vitro without stimulation by employing an enzyme immunosorbent assay (ELISA). However, the levels of IL-4 and IFN-gamma produced by unstimulated PBL were undetectable by ELISA. Mitogen stimulation of PBL revealed two groups of HIV+ individuals based on IL-10 production. PBL from one set of individuals produced low levels of IL-10 (low IL-10 producers) whereas the other group produced IL-10 comparable to that of normal controls (IL-10 producers). Production of IL-4 was significantly reduced in HIV+ individuals with < 400 CD4+ T cells/mm3 as compared to the normal controls. However, ability to produce IFN-gamma by mitogen-stimulated total PBL and CD4+ purified cells was not impaired in HIV+ individuals. These results suggest that unstimulated and mitogen-stimulated PBL of HIV+ individuals exhibit dysregulation of Th2 type cytokines which may play a role in HIV immunopathogenesis.

Timing of apoptosis onset depends on cell cycle progression in peripheral blood lymphocytes and lymphocytic leukemia cells

Apoptosis results in cell death within 10 min after initiation by Bcl-2 family proteins and mitochondria; however, cells enter the apoptotic pathway at different elapsed times after being triggered. Intrinsic factors related to chemical or physical cell damage can initiate apoptosis at a specific cell cycle phase; it is not clear whether cells insulted via an extrinsic pathway also die at a specific cell cycle phase, or how apoptosis is related to cell cycle progression in cells. To illustrate the kinetic changes of apoptosis during cell cycle progression, we examined both intrinsically and extrinsically induced apoptosis in MOLT-4 and Jurkat lymphocytic leukemia cells and in cultured peripheral blood lymphocytes (PBLs) using a recently modified annexin V and propidium iodide method, which detects cell cycle-specific apoptosis. Apoptosis predominantly occurred at a specific cell cycle phase. Leukemia cells were sensitive to induction by both intrinsic (X-rays, UV light, camptothecin, arsenic trioxide, and the traditional Chinese medicine Jinke, which is an extract of Auricularia auricula) and extrinsic factors (via Fas and TNF receptor pathways). The phase at which leukemia cells entered apoptosis depended on the nature of the insult (X-ray or UV, G1-phase; camptothecin, S-phase; arsenic, G1/S phases; Jinke, G1/S phases; and TNF or Fas ligand, G1/S phases), whereas PBLs did not exhibit such insult-dependent differences. PHA-stimulated PBLs entered apoptosis, and additional cells were recruited following additional insults. Unstimulated PBLs remained unresponsive to apoptosis, and proliferating cells became insensitive to the insults after the cell cycle checkpoint was abolished by caffeine. Confluent or starving PBLs were also unresponsive to apoptotic triggers. Thus, apoptotic cell death is a cell cycle event with most, if not all, apoptosis being initiated during a particular cell cycle phase, and changes in the cell cycle result in changes in the apoptotic pattern and schedule. The coordination of apoptosis and proliferation in cells offers a mechanism for the integration of both cell cycle and apoptotic signals.

Lentivirus-mediated gene transfer and expression in established human tumor antigen-specific cytotoxic T cells and primary unstimulated T cells.

In this report, we evaluated the efficiency of stable gene transfer into established CD8(+) human tumor antigen-specific cytotoxic T cell (CTL) lines and peripheral blood lymphocytes (PBL) by oncoretroviral and lentiviral vectors. In the oncoretroviral vector, the green fluorescent protein (GFP) reporter gene was regulated by the murine stem cell virus (MSCV) promoter. In three human immunodeficiency virus type 1 (HIV-1)-based lentiviral vectors, the GFP transgene was regulated by either a chimeric MSCV/HIV-1 promoter, or cellular promoters from human housekeeping genes PGK and EF1 alpha. We found that several lines of proliferating tumor-specific CTL were poorly (=2%) transduced by the oncoretroviral vector that transduced Jurkat T cell line efficiently (=80%). In contrast, three lentiviral vectors transduced 38-63% of these proliferating CTL. More interestingly, all lentiviral vectors packaged without the HIV-1 accessory proteins transduced human bulk PBL and purified CD4(+) and CD8(+) lymphocyte subsets without prior stimulation. Detailed analysis indicated that the lentiviral vectors containing the EF1 alpha or PGK ubiquitous promoter can transduce unstimulated PBL and achieve low-level transgene expression in the absence of any T-cell activation. However, T-cell activation subsequent to the transduction of unstimulated PBL is required for high-level transgene expression. Transduced PBL expressing transgene delivered by the lentiviral vectors still preserved resting and naïve cell phenotypes. Taken together, prior T cell stimulation and HIV-1 accessory proteins are dispensable for lentivirus-mediated gene transfer into resting naïve and memory T lymphocytes. These results will have significant implications for the study of T-cell biology and for the improvement of clinical gene therapies of acquired immune deficiency syndrome (AIDS) and cancer.

Human choriocarcinoma cell resistance to natural killer lysis due to defective triggering of natural killer cells.

The trophoblast, the outermost layer of the human placenta, lacks expression of the classical human leukocyte antigen (HLA) class I molecules. This prevents allorecognition by T cells but raises the question of what protects the trophoblast from natural killer (NK) cells. In a previous study, we have shown that choriocarcinoma cell (CC) resistance to NK lysis was mainly independent of HLA class I molecules. In the present study, we postulated that CC may prevent activation of NK cells by failing to stimulate their triggering receptors (TR). To test this hypothesis, we evaluated the lysis of JAR and JEG-3 CC after effective cross-linking and activation of NK cells by means of lectins or antibodies. Our results show that NK-resistant CC were sensitive to lysis by unstimulated peripheral blood lymphocytes in the presence of phytohemagglutin (PHA), to antibody-dependent cell cytotoxicity in presence of anti-Tja antibodies, and to monoclonal antibody redirected killing using anti-TR antibodies anti-CD16 and anti-CD244/2B4. Finally, CC fail to express CD48, the ligand for CD244/2B4. These results indicate that the resistance of CC to lysis results primarily from defective NK cell activation, at least partially due to the lack of expression of ligands, such as CD48, involved in the triggering of NK cells.

Aberrant Fas Ligand Expression in Lymphocytes in Patients with Behçet’s Disease

Background: Defects in immune responses have been reported in patients with Behçet’s disease (BD). To further characterize the immune dysfunction and its contribution to the pathogenesis, we have studied Fas ligand (FasL) expression in peripheral blood lymphocytes (PBL) and mononuclear cells in the skin lesions in patients with BD. Methods: FasL expression in PBL was studied with RT-PCR and immunoblotting with rabbit anti-human FasL antibody. We studied the expression of FasL in cryostat sections of biopsy specimens of erythema nodosum lesions from 4 patients with BD and of a genital ulcer lesion in another patient using immunohistochemical staining. Apoptotic cell death was detected with the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method. Results: We found that FasL mRNA and FasL protein expression was detected marginally in the unstimulated PBL, and was induced upon activation in normal individuals. PBL from patients with BD exhibited an enhanced expression of FasL mRNA and FasL protein without in vitro stimulation. Moreover, mitogen stimulation failed to augment FasL expression of their lymphocytes, suggesting a dysregulation of FasL expression of PBL in patients with BD. The skin biopsy specimens revealed that cells infiltrating into skin lesions expressed FasL and there were several TUNEL staining-positive cells in the lesions, suggesting that Fas/FasL-mediated apoptosis is involved in the development of the skin lesion and thus may be associated with the pathogenesis. Conclusions: We found an excessive expression of FasL in circulating as well as skin-infiltrating lymphocytes and the presence of apoptotic cells in the skin lesions, suggesting that lymphocytes expressing FasL aberrantly may play a role in the development and pathogenesis of BD.

DNA repair capacity after γ-irradiation and expression profiles of DNA repair genes in resting and proliferating human peripheral blood lymphocytes

DNA repair plays an important role in maintaining genomic integrity, and deficiencies in repair function are known to promote cancer development. Several studies have used the individual capacity to repair DNA damage in peripheral blood lymphocytes (PBLs) as a cancer risk marker. As the cell’s ability to remove DNA damage may be correlated with proliferative activity, it is an important question whether quiescent or dividing cells should be used in such studies. The aim of our study was to compare DNA repair capacity and expression profiles of 70 known DNA repair genes, both in resting and phytohemagglutinin (PHA) stimulated human PBLs. Using the comet assay, γ-radiation-induced DNA damage and repair in lymphocytes was analyzed. No difference, neither in the rate of radiation-induced DNA damage nor in DNA repair capacity between PHA-stimulated and non-stimulated PBLs was observed. Stimulated cells, however, showed significantly elevated values for background damage. Transcriptional profiles of repair genes were analyzed using cDNA arrays. Hybridization experiments were performed with mRNA isolated from both unstimulated and PHA-stimulated PBLs. More than 70% of all evaluated genes had constant expression levels. Twelve genes responded with a more than two-fold increase of transcripts to the mitogenic stimulus. Most of the up-regulated repair enzymes are also known to play a role in DNA replication. In conclusion, the data presented here suggest that all repair proteins needed for the repair of γ-irradiation induced DNA-damage, that can be detected by the alkaline comet assay, are already present in G 0 cells at sufficient amounts and do not need to be induced once lymphocytes are stimulated to start cycling. Our results thus do not support a general increase in DNA repair activity of PBLs by PHA stimulation, and the use of stimulated PBLs in molecular epidemiological studies on DNA repair of γ-irradiation induced DNA damage seems not to be mandatory.

Histidine Decarboxylase Expression in Human Melanoma

Histamine has been implicated as one of the mediators involved in regulation of proliferation in both normal and neoplastic tissues. Histidine decarboxylase, the only enzyme that catalyzes the formation of histamine from L-histidine, is an essential regulator of histamine levels. In this study, we investigated the gene and protein expression of histidine decarboxylase in melanoma. Reverse transcriptase polymerase chain reaction and in situ hybridization studies of WM-35, WM-983/B, HT-168, and M1 human melanoma cell lines both resulted in positive signals for histidine decarboxylase messenger RNA. A polyclonal chicken antibody was developed against human histidine decarboxylase and protein expression was confirmed by western blot analysis of the cell lysates, revealing a predominant immunoreactive band at approximately 54 kDa corresponding to monomeric histidine decarboxylase. Protein expression of histidine decarboxylase was also shown by flow cytometric analysis and strong punctate cytoplasmic staining of melanoma cell lines. Moreover, both primary and metastatic human melanoma tissues were brightly stained for histidine decarboxylase. When compared with the very weak or no reactions on cultivated human melanocytes both western blot and immunohistochemical studies showed much stronger histidine decarboxylase expression in melanoma cells. These findings suggest that expression of histidine decarboxylase is elevated in human melanoma.

Induction of AIDS virus-specific CTL activity in fresh, unstimulated peripheral blood lymphocytes from rhesus macaques vaccinated with a DNA prime/modified vaccinia virus Ankara boost regimen.

The observed role of CTL in the containment of AIDS virus replication suggests that an effective HIV vaccine will be required to generate strong CTL responses. Because epitope-based vaccines offer several potential advantages for inducing strong, multispecific CTL responses, we tested the ability of an epitope-based DNA prime/modified vaccinia virus Ankara (MVA) boost vaccine to induce CTL responses against a single SIVgag CTL epitope. As assessed using both 51Cr release assays and tetramer staining of in vitro stimulated PBMC, DNA vaccinations administered to the skin with the gene gun induced and progressively increased p11C, C-->M (CTPYDINQM)-specific CD8+ T lymphocyte responses in six of six Mamu-A*01+ rhesus macaques. Tetramer staining of fresh, unstimulated PBMC from two of the DNA-vaccinated animals indicated that as much as 0.4% of all CD3+/CD8alpha+ T lymphocytes were specific for the SIVgag CTL epitope. Administration of MVA expressing the SIVgag CTL epitope further boosted these responses, such that 0.8-20.0% of CD3+/CD8alpha+ T lymphocytes in fresh, unstimulated PBMC were now Ag specific. Enzyme-linked immunospot assays confirmed this high frequency of Ag-specific cells, and intracellular IFN-gamma staining demonstrated that the majority of these cells produced IFN-gamma after peptide stimulation. Moreover, direct ex vivo SIV-specific cytotoxic activity could be detected in PBMC from five of the six DNA/MVA-vaccinated animals, indicating that this epitope-based DNA prime/MVA boost regimen represents a potent method for inducing high levels of functionally active, Ag-specific CD8+ T lymphocytes in non-human primates.

Effects of deoxyribonucleosides and cell-stimulation on frequencies of ultraviolet-B-induced micronuclei in human peripheral blood lymphocytes

In the present study the involvement of deoxyribonucleotides (dNTPs) in the clastogenicity of ultraviolet-B (UVB) in unstimulated peripheral blood lymphocytes (G0-PBLs) was investigated. This was studied by analyzing the frequency of UVB-induced micronuclei (MN), either after adding a cocktail of the four deoxyribonucleosides to the PBLs immediately after exposure to UVB, or by stimulating the cells before exposure. In total, PBLs obtained from two different donors were investigated. For both donors, it could be demonstrated that addition of deoxyribonucleosides to UVB-irradiated G0-PBLs resulted in a significant reduction of the clastogenic effect of UVB. A gradual reduction of the clastogenic effect of UVB could also be realized by irradiating PBLs that were progressively more stimulated with the lectin PHA before exposure. The latter finding is explained by upregulation of intracellular pool sizes of dNTPs in stimulated PBLs.

Rapid and Efficient Cell-to-Cell Transmission of Human Immunodeficiency Virus Infection from Monocyte-Derived Macrophages to Peripheral Blood Lymphocytes

Macrophages are considered of central importance in cell-to-cell transmission of human immunodeficiency virus (HIV) infection in vivo. In this report, we describe a novel cell-to-cell transmission model using HIV-infected monocyte-derived macrophages (MDMs) as donor cells and peripheral blood lymphocytes (PBLs) as recipients. Virus was transmitted during a 2-h coincubation period from intracellular or tightly cell-associated viral stores in adherent infected MDMs to nonadherent CD3+ PBLs. Transmission required cell contact, but syncytia formation was not observed. HIV cell-to-cell transmission occurred in both allogeneic and autologous systems, and replication was higher in phytohemagglutinin (PHA)-stimulated than unstimulated recipient PBLs. In contrast, transmission of infection by cell-free virus was barely detectable without PHA stimulation of recipients, suggesting the cell–cell interaction may have provided stimuli to recipient cells in the cell-to-cell system. Viral DNA levels increased 5–24 h postmixing, and this increase was inhibited by pretreatment of cells with the reverse transcription inhibitor azidothymidine, indicating de novo reverse transcription was involved. Cell-to-cell transmission was more efficient than infection with cell-free virus released from donor MDMs, or 0.1 TCID50/cell cell-free viral challenge. This model provides a system to further investigate the mechanisms and characteristics of HIV cell-to-cell transmission between relevant primary cells that may be analogous to this important mode of virus spread in vivo.

Up-regulation of interleukin-4 and CD23/FcepsilonRII in minimal change nephrotic syndrome.

Although the pathogenesis of childhood minimal change nephrotic syndrome (MCNS) has not been clearly defined, the current hypothesis favors an involvement of T cell dysfunction. The symptom onset and the relapse of MCNS are frequently associated with allergy and increased IgE levels in sera. Since a T cell-derived cytokine interleukin-4 (IL-4) plays a key role in the regulation of IgE production and allergic response, we investigated the role of IL-4 in the pathophysiology of MCNS. Using fluorescence-activated cell scanning we observed a significantly higher expression of CD23, the type II IgE receptor (FcepsilonRII), on fresh B cells from active MCNS patients (n=22) compared with age-matched healthy normal controls (n=12). The upregulation of CD23 correlates with greater IL-4 activity in the culture supernatant of MCNS peripheral blood lymphocytes (PBLs) than normal PBLs stimulated by mitogens, as assessed by the CD23-inducing effect of the PBL supernatant on tonsillar B cells. Furthermore, Northern blot and reverse transcription-based polymerase chain reaction analysis have revealed significantly elevated levels of IL-4 mRNAs both in mitogen-stimulated and unstimulated MCNS PBLs, compared with healthy normals or disease controls with other renal disorders. Together these results strongly suggest that the upregulation of IL-4 in T cells may be part of the T cell dysfunction involved in MCNS.

Expression and function of Fas and Fas ligand on peripheral blood lymphocytes in normal subjects

We investigated the expression and function of Fas and Fas ligand (FasL) on peripheral blood lymphocytes (PBLs). The cells were stimulated with various cytokines or 12-0-tetradecanoyl phorbol 13-acetate (PMA) plus ionomycin. About 30% of unstimulated PBLs expressed Fas, and the expression was augmented by interleukin-1β (IL-1β), IL-2, tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), or PMA plus ionomycin. Although only minimal FasL expression was detected on unstimulated PBLs, FasL expression was markedly induced by IL-2 or PMA plus ionomycin, suggesting that Fas and FasL were both expressed on IL-2-stimulated or PMA-plusionomycin-stimulated PBLs. Although IL-2-stimulated or PMA-plus-ionomycin-stimulated PBLs were positive for both Fas and FasL, no significant increase in apoptosis was demonstrated in these activated PBLs. In addition, treatment of PBLs with IL-2 or PMA plus ionomycin did not change anti-Fas-induced apoptosis, although these activated PBLs expressed Fas strongly when compared with unstimulated PBLs. Only IL-2-stimulated or PMA-plus-ionomycin-stimulated PBLs killed Fas + target cells efficiently via the interaction of Fas on target cells with FasL of PBLs. Bcl-2 was constitutively expressed on unstimulated PBLs, but its expression was significantly augmented by IL-2 or PMA plus ionomycin. The expression of Box was clearly induced only on IL-2-stimulated or PMA-plus-ionomycin-stimulated PBLs and that of other Bcl-2 family proteins such as Bcl-x and Bad could not be detected on human PBLs, including IL-2-stimulated or PMA-plus-ionomycin-stimulated PBLs. Our results suggest that PBLs activated by IL-2 or PMA plus ionomycin express both Fas and FasL and that they kill Fas + target cells by using FasL on the surface. The resistance of these activated PBLs to Fas-mediated apoptosis may be due to the augmented Bcl-2 expression or the presence of Bcl-2:Bax heterodimers on these cells.

In vitro effect of swainsonine on bovine and ovine lymphoblastogenesis.

Peripheral blood lymphocytes (PBL) were obtained from heifers and wethers to assess the in vitro effects of swainsonine. Stimulated and unstimulated PBL (2x10(5)/well; 96 well plates) were incubated in the presence of swainsonine (2, .2, .02, .002, and 0 microg/mL; n = 32) for 72 h. Mitogens used to stimulate PBL were concanavalin A (ConA; 5 microg/mL), phytohemagglutinin-P (PHA-P; 5 microg/mL), phytohemagglutinin-M (PHA-M; 5 microg/mL), pokeweed mitogen (PWM; 5 microg/mL), and lipopolysaccharide (LPS; 10 microg/mL). Controls received no swainsonine or mitogen. Effects of swainsonine are expressed as a percentage of stimulation relative to control (stimulation index; SI). Unstimulated bovine PBL treated with 2 microg/mL of swainsonine exhibited a depressed (P<.001; SI of 87 and 100 for 2 microg/mL and control, respectively; SE = 2.7) SI. At concentrations of .2 and 2 microg/mL, swainsonine inhibited (P<.001; SI of 351, 310, and 464 for .2, 2, and 0 microg/mL, respectively; SE = 27.1) bovine PBL proliferative response to PHA-P. Swainsonine had a mitogenic effect on unstimulated ovine PBL at .2 and 2 microg/mL (P<.05; SI of 118, 113, and 100 for .2 and 2 microg/mL and control, respectively; SE = 4.4). Swainsonine inhibited ovine proliferative responses to PHA-P at .2 and 2 microg/mL (P<.005; SI of 190, 178, and 228 for .2, 2, and 0 microg/mL, respectively; SE = 9.4) and to PHA-M at .002 and 2 microg/mL (P<.03; SI of 165, 167, and 192 for .002, 2, and 0 microg/mL, respectively; SE = 7.9). The opposing responses of unstimulated ovine (mitogenic) and bovine (antiproliferative) PBL to swainsonine indicates a differential species response. Swainsonine suppression of PHA-P-induced proliferation would indicate a negative effect on ovine and bovine T-cell function.

Protein kinase C isotypes theta, delta and eta in human lymphocytes: differential responses to signalling through the T-cell receptor and phorbol esters.

The repertoire of novel and atypical protein kinase C (PKC) isotypes present in human T cells and their subcellular localization have not been fully characterized. We detected calcium-independent PKC activity in whole cell fractions from unstimulated peripheral blood lymphocytes (PBL). Towards an understanding of the role of PKC isoforms in lymphocyte activation we have studied the expression of calcium-independent PKC isoforms theta, delta and eta in PBL. With isoformspecific antibodies we detected the presence of PKC theta and delta in whole cell fractions from unstimulated human PBL by Western blot analysis. In addition, immunocytochemical analysis confirmed the presence of the novel PKC isoform PKC eta in PBL. Using immunocytochemistry, PKC theta, delta and eta had distinct patterns of redistribution following activation by phorbol myristate acetate (PMA). However, signalling through the T-cell receptor (TCR) did not appear to induce such changes in isoenzyme redistribution. These findings indicate that activation of lymphocytes either through the TCR-CD3 complex or with PMA induces different signalling pathways with respect to calcium-independent isoenzymes. Signalling through receptors other than the CD3 complex may be involved in activation of these isotypes.

THE ROLE OF TRANSFERRIN RECEPTOR (CD71) IN PHOTODYNAMIC THERAPY OF ACTIVATED AND MALIGNANT LYMPHOCYTES USING THE HEME PRECURSOR δ‐AMINOLEVULINIC ACID (ALA)

Endogenously generated protoporphyrin IX (PpIX) from exogenous ALA can be an effective photosensitizer. PpIX accumulation is inversely dependent on available intracellular iron, which is required for the conversion of PpIX to heme. Iron also is necessary for cell replication. Since iron can be toxic, intracellular iron levels are tightly controlled. Activated and proliferating cells respond to the demand for intracellular iron by upregulating membrane expression of the transferrin receptor (CD71) which is needed for iron uptake. We predicted that activated lymphocytes (CD71+) would preferentially accumulate PpIX because of their lower intracellular iron levels and because of competition for iron between ALA-induced heme production and cellular growth processes. Thus, the CD71+ cells could serve as PDT targets. Stimulation of human peripheral blood lymphocytes (PBL) with the mitogens, phytohemagglutinin A, concanavalin A and pokeweed prior to incubation with ALA results in PpIX accumulation correlating with level of activation. Activated lymphocytes expressing high levels of surface CD71 transferrin receptors generated more PpIX than those with low CD71 expression. Incubating activated cells in transferrin depleted medium (thereby decreasing the iron availability) further increased PpIX levels. Malignant, CD71+ T lymphocytes from a patient with cutaneous T-cell lymphoma (CTCL)/Sezary syndrome also accumulated increased PpIX levels in comparison to normal lymphocytes. PDT of activated lymphocytes and Sezary cells after ALA incubation demonstrated preferential killing compared to normal, unstimulated PBL. These findings suggest a possible mechanism for the selectivity of ALA PDT for activated CD71+ cells. They also indicate a clinical use for ALA-PDT in therapy directed towards the malignant lymphocytes in leukemias and lymphomas, and as animmunomodulatory agent.

Horse Trophoblasts Produce Tumor Necrosis Factor α but not Interleukin 2, Interleukin 4, or Interferon γ1

The distribution of four cytokines was analyzed in the endometrium and trophoblast of the horse between Days 30 and 55 of gestation. Endometrial tissues, invasive trophoblast (chorionic girdle), and noninvasive trophoblast (chorion and allantochorion) were examined separately. Cytokine expression was determined by amplification of specific mRNA via the reverse transcriptase polymerase chain reaction (RT-PCR). Messenger RNA for interleukin 2 (IL-2), interleukin 4 (IL-4), and interferon gamma (IFN gamma) was detected in endometrial tissues, unstimulated peripheral blood lymphocytes, and control kidney tissue, but not in trophoblasts. leukocytes resident in the endometrium or traversing the uterus via blood vessels might be the source of these cytokines. Endometrial tissues and invasive and noninvasive trophoblasts expressed mRNA for tumor necrosis factor alpha TNF alpha). Immuonoreactive TNF alpha protein was detected in different cell types of the endometrium and in the invasive and noninvasive trophoblast. The ubiquitous expression of TNF alpha by the endometrium and trophoblasts suggests that this cytokine might have an important role in regulating placental growth and differentiation or maternal leukocyte responses to trophoblasts. IL-2, IL-4, and IFN gamma might have important immunoregulatory roles within the endometrium.

Deficient nucleotide excision repair activity in protein extracts from normal human lymphocytes.

DNA repair activity in human peripheral blood lymphocytes (PBL) has been investigated by various techniques. Here, we report the use of an in vitro assay in order to assess nucleotide excision repair activity (NER). The mechanism of this major repair process relies on two broad steps: first, recognition, incision and excision of the damaged DNA; second, repair synthesis on the gapped DNA. Briefly, damaged plasmids were incubated with whole cell extracts which allows one to quantify DNA repair synthesis. When NER was determined on plasmid DNA damaged with UV-light or cisplatin, PBL extracts showed no repair synthesis for unstimulated lymphocytes. Using a new in vitro assay measuring only the damage-specific DNA incision activity in cell extracts, we found that the incision step in the repair reaction was blocked in unstimulated PBL. By mixing PBL with XP (group A, B, C, D) extracts, no restoration of NER activity was observed. In addition, these lymphocytes also lacked DNA replication activity as determined with pre-incised plasmid substrate. However, a phytohemagglutinin treatment of PBL led to an extent of repair synthesis similar to that observed with extracts from lymphoblastoid cells. When lymphocytes were incubated in 20% serum medium with and without phytohemagglutinin, the repair activity increased dramatically after 24 h. During the activation of lymphocytes, the extent of repair synthesis was proportional to the percentage of cells in S phase of the cell cycle. Our results suggest that the blockage of the cell cycle in G0/G1 in PBL may be responsible for their lack of NER activity.