Phytohemagglutinin Stimulation Research Articles

EFFECT OF ANTI-IL-6 AND ANTI-10 MONOCLONAL ANTIBODIES ON THE SUPPRESSION OF THE NORMAL T LYMPHOCYTE MITOGENIC RESPONSE BY STEADY STATE SICKLE CELL DISEASE SERA

Previously published work has shown that sera from healthy sickle cell disease (SCD) patients inhibits normal lymphocyte mitogenic response to phytohemagglutinin (PHA) in vitro. The current study is to attempt to ascertain what effect antibody to type 2 cytokines, interleukin (IL)-6 and 10, have on the suppression of lymphocyte PHA response by SCD sera. Peripheral blood mononuclear cells (PBMC), separated by density gradient were obtained from 2 healthy normal donors. Sera from 50 healthy SCD patients, 50 normal healthy controls and pooled normal O, Rh+ (O+) sera were utilized in standard in vitro PHA stimulation of PBMC cultures. Mitogenic responses were expressed as mean counts per minute (cpm) of triplicate cultures. Fifty triplicate cultures of PHA stimulated normal PBMC were done with 10% normal pooled O+, normal control and SCD steady state sera only. In addition 50 cultures were done with SCD sera containing 1 μg/ml of anti-IL-6 monoclonal antibody, as well as 28 SCD serum cultures containing 1 μg/ml of anti-IL-10 monoclonal antibody. The final 11 SCD serum culture experiments contained a combination of both anti-IL-6 and anti-IL-10 antibody, each at the concentration of 1 μg/ml. Results revealed > 15% suppression of mitogenic response in the SCD sera supplemented cultures as compared to control sera in 47/50 (94%) and in 40/50 (80%) of normal pooled O+, as calculated by mean cpm. The degree of suppression ranged from 17% to 98% in individual experiments. The addition of anti-IL-6 antibody alone significantly improved mean cpm (> 20%) in 19/50 (38%) of SCD serum responses compared to O+ sera and 23/50 (46%) of control sera. Complete correction occurred in 9/50 (18%) of all SCD serum suppressions as compared to O+ sera and 6/50 (12%) when compared to control sera. Similarly, anti-IL-10 antibody decreased suppression of the mean cpm of SCD serum cultures in 18/28 (64%) and completely corrected 3/18 (11%). The combined antibody data revealed > 20% increase in mean cpm in 10/11(91%) experiments. Inhibitors of mitogenic response were present in a significant percentage of the SCD sera utilized in the present study. The significant corrective effects of both monoclonal antibodies would seem to support the original hypothesis that high circulating levels of type 2 cytokines may represent the cell-mediated dependent inhibitory factors expressed in the sera of many healthy SCD patients.

Interferon Gamma and Interleukin-2 Secretion in Whole Blood Cell Cultures from Small-Cell Lung Cancer Patients

Secretion of interleukin-2 (IL-2) and interferon-γ (IFNγ) in whole blood cell cultures after phytohemagglutinin (PHA) and pokeweed mitogen (PWM) stimulation was evaluated in 42 small-cell lung cancer patients before treatment. Blood cultures from patients with extensive and limited disease have lower IFNγ secretions after PWM stimulation and lower IL-2 secretions after PWM and PHA stimulation, but this was not statistically significant. IFNγ but not IL-2 secretion was significantly higher after PWM stimulation in cultures from patients with better clinical performance status (0 + 1, WHO scale). Blood cultures from patients with tumor regression after treatment had a higher secretion of IFNγ after PWM stimulation. In contrast, blood cultures from patients with a poor prognosis had significantly lower IL-2 and IFNγ secretions after stimulation with PWM, and a lower IL-2 secretion after stimulation with PHA.

Synthesis of [ARG14 17 19 20jdeacetyl-thymosin a1 and its immunological effect on the impaired T­ lymphocytes of uremic patients

Abstract: A deacetyl-thymosin cx1analogue containing four arginines at positions 14, 17, 19, and 20, instead of four lysines was synthesized by the manual solid-phase method. The synthetic [Arg14 17 19 20 deacetyl-thymosin cx1 and deacetyl-thymosin cx1 were tested for the effect on impaired T-lymphocyte transformation by phytohemagglutinin in uremic patients suffering from recurrent infectious diseases. The restoring activity on the impaired phytohemagglutinin stimulation of T-lymphocytes was obtained after incubation of peripheral lymphocytes isolated from uremic patients with the synthetic [Arg14•17•19•20] deacetyl-thymosin cx1. This analogue exhibited a far stronger restoring effect than that of our synthetic deacetyl-thymosin ex1. Abbreviations used: Obzl, benzyl estrer; Bzl, benzyl; Boe, tert-butoxycarbonyl; Mts, mesitylene-2-sulfonyl; DCC, N, N' -dicyclohexylcarbodiimide; HOBT, 1-hydro-xybenzotriazole; DMF, dimethylformamide; Et3N, triethylamine; AcOH, acetic acid; TFA, trifluoroacetic acid; TLC, thin-layer chromatography; HPLC, high­ performance liquid chromatography; PHA, phytohemagglutinin; SDS, sodium dodecyl sulfate; PBS, phosphate­ buffered saline; RPMI, Rosewell Park Memorial Institute; FCS, fetal calf serum; E-rosette, a rosette with sheep erythrocytes.

Extracellular pH affects the proliferation of cultured human T cells and their expression of the interleukin-2 receptor.

Ex vivo expansion of T cells is an important aspect of many cellular immunotherapy protocols, and the effects of the culture environment on the cells must be understood to produce large numbers of functional cells. Extracellular pH is a fundamental parameter that has many different effects on cultured cells. In this study, peripheral blood mononuclear cells were stimulated with phytohemagglutinin and cultured at pH values of 7.0, 7.2, or 7.4. The effects of pH on the cells were studied during the 2 to 3 weeks of proliferation resulting from phytohemagglutinin stimulation, in order to examine the culture kinetics over realistic time scales for ex vivo expansion. The proliferation capacity of the T cells increased more than three-fold for the pH 7.0 and 7.2 cultures compared with the pH 7.4 cultures. The culture pH also affected the kinetics of the interleukin-2 receptor down-regulation process. The faster receptor down-regulation in both the pH 7.2 and 7.4 cultures resulted in a more than twofold greater fraction of interleukin-2 receptor(+) cells in the pH 7.0 cultures. Although the fraction of apoptotic cells (using the Annexin V flow-cytometric method) remained less than 10%, we observed 27% more apoptosis in the pH 7.4 cultures than in the 7.2 cultures and 49% more apoptosis in the pH 7.4 cultures than in the 7.0 cultures. These effects on interleukin-2 receptor expression and cellular apoptosis may partially explain the observed effects of pH on T-cell proliferation.

Effects of testosterone on cell-mediated and humoral immunity in non-breeding adult European starlings

One of the primary assumptions of the immunocompetence hypothesis is that testosterone is immunosuppressive. Although many studies in birds and mammals have supported this assumption, conflicting results have been reported in a variety of species. We investigated the effects of testosterone manipulation on both cell-mediated and humoral immunity in adult songbirds, European starlings (Sturnus vulgaris). Male and female starlings were wild-caught, housed in the laboratory, and implanted with either empty silastic capsules or capsules containing testosterone. Six weeks after implantation, humoral immunity was assessed by injecting the birds with a novel antigen, keyhole limpet hemocyanin, and measuring specific antibody responses 10 and 15 days later via an enzyme-linked immunosorbant assay. Cell-mediated immunity was assessed 7 weeks after implantation via intradermal injection of the T-cell mitogen phytohemagglutinin into the wing web and measuring the degree of swelling 24 h later. Antibody responses to antigenic challenge were significantly suppressed in testosterone-treated females 10 days post-injection and in both sexes 15 days post-injection. Furthermore, there was a significant inverse relationship between individual variability in antibody responsiveness and plasma testosterone concentrations. Cell-mediated responses to phytohemagglutinin stimulation were also significantly suppressed in testosterone-treated males compared to same-sex controls. Testosterone treatment significantly increased plasma corticosterone concentrations compared to controls, and the possibility of this effect mediating the immunosuppressive effects of testosterone is discussed. The present study is among the first to demonstrate testosterone-induced suppression of both cell-mediated and humoral immunity in a species of songbird.

Effects of High-Dose Inhaled Fluticasone Propionate via Spacer on Cell-Mediated Immunity in Healthy Volunteers

Systemic corticosteroids are known to alter cell-mediated immunity (CMI). However, the effects of inhaled steroids on CMI are unclear. We therefore sought to assess CMI following high-dose inhaled steroids in healthy subjects. Ten healthy nonasthmatic subjects self-administered fluticasone propionate (FP), 440 microg bid, with a spacer device. CMI was assessed by delayed hypersensitivity skin testing to multiple antigens and in vitro by phytohemagglutinin (PHA) stimulation of peripheral blood T lymphocytes. Percentages of CD3(+), CD4(+), and CD3(+)CD8(+) cells expressing CD69(+) were determined by three-color flow cytometry. Studies were conducted before and after 4 weeks of FP treatment. After 4 weeks of FP treatment, two of nine subjects became anergic, whereas six of nine subjects had reduced skin responses (one subject was excluded). Mean total skin test score fell from 18.4+/-10.9 to 9.1 +/-7.2 mm (p = 0.02). There was a decline in tuberculin responses in all four subjects who were positive prior to FP treatment. Following FP treatment, the percentage of unstimulated (from control subjects receiving saline solution) CD3(+)CD4(+)CD69(+) cells declined from 14.8+/-4.2% to 8. 5+/-4.6% (p = 0.02) and the CD3(+)CD8(+)CD69(+) cells decreased from 29.7+/-12.7% to 17.1 +/-5.0% (p = 0.007). PHA stimulation produced significant increases in the percentage of CD3(+)CD4(+)CD69(+) cells before and after FP treatment (67.0+/-9.1%, p<0.02 before FP; 55.4+/-17.0%, p<0.02 after FP), and in the percentage of CD3(+)CD8(+)CD69(+) cells before and after treatment (79.7+/-9.3%, p<0.03 before FP; 71.2+/-11.4%, p = 0. 008 after FP). High doses of FP suppress the proportion of activated circulating T cells but do not affect the ability of T cells to respond to direct stimulation with PHA. However, depression of skin test responses to antigens following treatment with FP suggests an impairment of in vivo clinical manifestations of T-cell activation by a mechanism that requires further investigation.

Cellular immune profile of patients with advanced cancer before and after taxane treatment.

The purpose of this study is to determine immune recovery and function after treatment with docetaxel or paclitaxel. Peripheral blood mononuclear cells were harvested before chemotherapy and at weekly times afterwards for cycle 1. Leukocyte subsets ICD45hiCD14lo polymorphonuclear neutrophils, CD45hiCD14hi monocytes, CD45hiCD14- lymphocytes, CD3+CD4/CD8+ T cells, CD3-CD19+ B cells, CD3-CD16/CD56+ natural killer (NK) cells], and circulating cytokine levels [tumor necrosis factor-alpha, gamma-interferon (gamma-IFN), and interleukins (IL-2, IL-10, IL-12)] were followed. In addition, T-cell mitogenic function, NK function, and lymphokine activated killer (LAK) function was assessed. Ten patients were entered in the trial. T-cell frequency, B-cell frequency, and CD4/CD8 ratio did not change. IL-10 serum levels significantly decreased in paclitaxel-treated patients (4.4+/-1.3 pg/ml at week 4 versus 7.8+/-2.1 pg/ml at baseline; p < 0.05). IL-2, IL-12, and gamma-IFN levels were not detectable. NK cytotoxic activity decreased in docetaxel-treated patients. LAK cell activity was not altered. Four patients achieved a partial or complete response. They demonstrated higher than normal CD4:CD8 T-cell ratios and an improved phytohemagglutinin stimulation index (SI = 2.5). In conclusion, our findings suggest that immune function was affected more significantly after docetaxel treatment. Investigational approaches, which enhance cellular immunity, may be of greater relevance after treatment with docetaxel. Additional studies monitoring NK function after chemotherapy are recommended.

Cytokine gene expression occurs more rapidly in stimulated peripheral blood mononuclear cells from human immunodeficiency virus-infected persons.

Evaluation of cytokine gene expression following in vitro stimulation is one means of examining the dysregulation of the immune system in human immunodeficiency virus (HIV) infection. We have assessed differences in the immune status of non-HIV-infected (HIV-) and HIV-infected (HIV+) individuals by evaluating the kinetics of the expression of cytokine genes. We compared detailed time courses of cytokine mRNA expression in HIV- and HIV+ peripheral blood mononuclear cells (PBMC) and found that there is a significant shift (P<0.01) for all cytokines examined (interleukin 2 [IL-2], IL-6, IL-10, gamma interferon, and tumor necrosis factor alpha [TNF-alpha]) to an earlier time of mean peak mRNA expression by HIV+ PBMC (between 4 and 8 h) compared to HIV- PBMC (8 h) in response to either phytohemagglutinin (PHA) or anti-CD3 stimulation. Additional studies showed that although PHA-stimulated HIV+ PBMC showed decreased median IL-2, IL-4, and TNF-alpha mRNA levels, they typically demonstrated more rapid kinetics (increased mean 4-h/24-h cytokine mRNA ratios), with significant differences for IL-4 (P<0.05) and TNF-alpha (P<0.005), compared to HIV- PBMC. The use of fresh or frozen cells gave comparable cytokine mRNA data; however, the secretion of some cytokine proteins (IL-2 receptor, IL-10, and TNF-alpha) appeared to be reduced in HIV+ PBMC that had been frozen and thawed. Our studies demonstrate that the kinetics of cytokine gene expression can reveal additional dysregulation of the immune system in HIV infection, suggesting that PBMC of HIV-infected persons exist in an activated state in vivo that permits them to express cytokine genes more rapidly than a normal PBMC.

Nonmyeloablative stem cell transplantation for congenital immunodeficiencies

AbstractThe optimal approach for stem cell transplantation in children with immunodeficiency has yet to be determined. Conditioning therapy is necessary for reliable engraftment and full immune reconstitution; however, the beneficial effect of cytoreductive conditioning is counterbalanced by increased short- and long-term treatment-related toxicity. Whether bone marrow transplantation with a nonmyeloablative preparative regimen was sufficient for the establishment of donor immune reconstitution, with the resultant correction of disease phenotype, was investigated. Eight patients with severe immunodeficiency states underwent T-cell replete bone marrow transplantation from a human leukocyte antigen-matched unrelated (n = 6) or sibling (n = 2) donor with nonmyeloablative conditioning using a fludarabine–melphalan–anti-lymphocyte globulin-based regimen. All patients had severe organ dysfunction that precluded transplantation with conventional conditioning. All patients were engrafted with predominantly donor hematopoiesis, and the duration of neutropenia was brief. Significant acute graft-versus-host disease (GVHD) did not develop, but one patient had limited chronic GVHD. One patient died of disease recurrence, and 3 have stable, mixed chimerism. At a median follow-up of 1 year, all patients have had good recovery of CD3+ T-cell numbers, and 6 of 7 evaluable patients have normal phytohemagglutinin stimulation indices. The rate of immune reconstitution is comparable with that of historical controls undergoing standard myeloablative protocols. Two patients with CD40 ligand deficiency now show significant expression, and a patient with adenosine deaminase deficiency has improved deoxy adenosine triphosphate metabolites. In summary, it has been demonstrated that nonmyeloablative stem cell transplantation permits rapid engraftment from both sibling and unrelated donors with minimal toxicity even in the presence of severe organ dysfunction. If long-term immune reconstitution of patients treated with this protocol is demonstrated, it is believed this approach might offer significant advantages compared with standard protocols by combining adequate immune reconstitution with reduced short- and long-term toxicity.

Nonmyeloablative stem cell transplantation for congenital immunodeficiencies

The optimal approach for stem cell transplantation in children with immunodeficiency has yet to be determined. Conditioning therapy is necessary for reliable engraftment and full immune reconstitution; however, the beneficial effect of cytoreductive conditioning is counterbalanced by increased short- and long-term treatment-related toxicity. Whether bone marrow transplantation with a nonmyeloablative preparative regimen was sufficient for the establishment of donor immune reconstitution, with the resultant correction of disease phenotype, was investigated. Eight patients with severe immunodeficiency states underwent T-cell replete bone marrow transplantation from a human leukocyte antigen-matched unrelated (n = 6) or sibling (n = 2) donor with nonmyeloablative conditioning using a fludarabine–melphalan–anti-lymphocyte globulin-based regimen. All patients had severe organ dysfunction that precluded transplantation with conventional conditioning. All patients were engrafted with predominantly donor hematopoiesis, and the duration of neutropenia was brief. Significant acute graft-versus-host disease (GVHD) did not develop, but one patient had limited chronic GVHD. One patient died of disease recurrence, and 3 have stable, mixed chimerism. At a median follow-up of 1 year, all patients have had good recovery of CD3+ T-cell numbers, and 6 of 7 evaluable patients have normal phytohemagglutinin stimulation indices. The rate of immune reconstitution is comparable with that of historical controls undergoing standard myeloablative protocols. Two patients with CD40 ligand deficiency now show significant expression, and a patient with adenosine deaminase deficiency has improved deoxy adenosine triphosphate metabolites. In summary, it has been demonstrated that nonmyeloablative stem cell transplantation permits rapid engraftment from both sibling and unrelated donors with minimal toxicity even in the presence of severe organ dysfunction. If long-term immune reconstitution of patients treated with this protocol is demonstrated, it is believed this approach might offer significant advantages compared with standard protocols by combining adequate immune reconstitution with reduced short- and long-term toxicity.

Granulocyte colony-stimulating factor perturbs lymphocyte mitochondrial function and inhibits cell cycle progression

Objective Sera from healthy subjects receiving recombinant human granulocyte colony-stimulating factor (rHuG-CSF) to mobilize CD34 + peripheral blood progenitors (PBPC) have been recently shown to induce unresponsiveness of allogeneic lymphocytes to mitogenic challenge. In the present investigation, the effects of rHuG-CSF on the early stages of lymphocyte activation–induced apoptosis and on lymphocyte cell cycle entry were evaluated. Materials and Methods Sera were obtained from HLA-identical donors receiving rHuG-CSF to mobilize CD34 + PBPC for allogeneic transplantation. Normal peripheral blood mononuclear cells (PBMC) were challenged with phytohemagglutinin (PHA) in the presence of serum collected before (preG) or after rHuG-CSF administration (postG). Mitochondrial function, that is, incorporation of 3,3′-dihexyloxacarbocyanine iodide [DiOC 6(3)] and generation of reactive oxygen species (ROS) as well as expression of c-Myc and Bcl-2 family members (Bcl-2, Bcl-X L, Bax) were evaluated by multiparameter flow cytometry. The activation-induced fragmentation of genomic DNA was detected by highly sensitive LM-PCR assay. Results CD4 +DiOC 6(3) low and CD8 +DiOC 6(3) low T lymphocytes increased and reached 32% (range 27%–38%) and 20% (range 15%–23%) of circulating T cells, respectively, on day 4 of rHuG-CSF administration. Hypergeneration of ROS could be demonstrated in 65% (range 58%–82%) of CD4 + T lymphocytes and in 0.4% (range 0.2%–0.8%) of circulating CD8 + T cells. rHuG-CSF determined no alteration of mitochondrial function if added to allogeneic PBMC in vitro, thus suggesting indirect effects mediated by soluble factors; on the contrary, when PBMC were challenged with PHA in the presence of postG serum, both perturbation of mitochondrial transmembrane potential (Δψ m) and hypergeneration of ROS were induced, and lymphocytes were predominantly arrested in a G 0 -like phase of the cell cycle and displayed genomic DNA fragmentation. Interestingly, the preincubation of PBMC with a blocking antibody directed against CD95 abrogated the perturbation of lymphocyte Δψ m, suggesting that the CD95 signaling pathway might play a role in the induction of apoptosis after PHA stimulation in the presence of postG serum. Moreover, Bax protein was overexpressed in postG ( median fluorescence intensity = 180, range 168–186 ) compared with preG cultures ( median fluorescence intensity = 75, range 68–80; p < 0.01) , while no differences in Bcl-2, Bcl-X L, and c-Myc staining intensity were observed. Conclusions Our findings demonstrate a humoral-mediated rHuG-CSF–induced dissipation of lymphocyte mitochondrial Δψ m; these effects might be mediated by Bax overexpression, with imbalance between apoptosis-promoting and apoptosis-inhibiting Bcl-2 family members and with subsequent induction of mitochondrial permeability transition. Whether immune dysfunction will favorably impact on incidence and severity of acute graft vs host disease after allogeneic PBPC transplantation remains to be determined.

Tumor necrosis factor-alpha is undetectable in the plasma of SS patients with elevated Hb F.

Steady-state sickle cell disease (SCD) patients may have increased plasma levels of acute phase reactants and pro-inflammatory cytokines because of subclinical inflammation. We have estimated TNF-alpha levels in the plasma and in supernatants following peripheral blood mononuclear cell (PBMC) activation with phytohemagglutinin (PHA) in a group of Kuwaiti SCD patients using ELISA. The group consisted of 28 SS, 8 Sbeta-thal, and 2 SD patients all in steady state; 5 SS patients were studied during 7 episodes of painful crisis. The subjects were aged 2 to 16 years, with a mean of 7.3 +/- 3.5 years. The beta(S)-globin gene cluster haplotype, alpha-tha1 status, and spleen function were determined in the SS group using standard techniques. Most (82%) were homozygous for the Saudi Arabia/India haplotype and had elevated Hb F levels ranging from 15% to 35%. There were 24 controls (Hb AA or AS), of whom 14 were healthy and 10 were acutely ill at the time of the study. None of the children with SCD (either in steady state or crisis) had detectable plasma TNF-alpha, but four controls (3 acutely ill and one healthy) had levels ranging from 61.7 to 249.8 pg/mL. Following PHA stimulation most subjects responded with high levels of TNF-alpha, with the median level among the steady-state SS patients being significantly higher than that in the controls (both the acutely ill or healthy). It therefore appears that because of the mild disease among our Arab SS children, TNF-alpha is not detectable in their plasma in steady state; these children, however, had a significantly higher response than controls following PBMC activation.

Degradation of DNA Topoisomerase I by a Novel Trypsin-like Serine Protease in Proliferating Human T Lymphocytes

DNA topoisomerase I (Topo I) contributes to various important biological functions, and its activity is therefore likely regulated in response to different physiological conditions. Increases in both the synthesis and degradation of Topo I were previously shown to accompany phytohemagglutinin stimulation of proliferation in human peripheral T lymphocytes. The mechanism of this degradation of Topo I has now been investigated with both in vivo and in vitro assays. The activity of a nuclear protease that specifically degrades Topo I was induced in proliferating T lymphocytes. The full-length Topo I protein (100 kDa) was sequentially degraded to 97- and 82-kDa fragments both in vivo and in vitro. The initial site of proteolytic cleavage was mapped to the NH(2)-terminal region of the enzyme. The degradation of Topo I in vitro was inhibited by aprotinin or soybean trypsin inhibitor, suggesting that the enzyme responsible is a trypsin-like serine protease. Furthermore, Topo I degradation by this protease was Mg(2+)-dependent. The Topo I-specific protease activity induced during T lymphocytes proliferation was not detected in Jurkat (human T cell leukemia) cells and various other tested human cancer cell lines, possibly explaining why the abundance of Topo I is increased in tumor cells.

IL-12 synergizes with IL-18 or IL-1beta for IFN-gamma production from human T cells.

IL-18 is a proinflammatory cytokine that plays an important role in NK cell activation and T(h)1 response. IL-18 has a structural homology to IL-1, particularly IL-1beta. IL-18R, composed of IL-1R-related protein (IL-18Ralpha) and IL-1R accessory protein-like (IL-18Rbeta), belongs to the IL-1R family. Furthermore, IL-18R at least partly shares the signal transducing system with IL-1R. Thus, the IL-18-IL-18R system has a striking similarity to the IL-1-IL-1R system. For this reason, we regarded it important to investigate whether, like IL-18, IL-1beta synergizes with IL-12 in inducing IFN-gamma production from human T cells and plays an important role in the T(h)1 response. Here we show that IL-12 and IL-1beta synergistically induce T cells to proliferate and produce IFN-gamma without their TCR engagement. IL-12 stimulation induced an increase in the proportion of T cells positive for IL-18R. Then, IL-12-stimulated T cells responded to IL-18 or IL-1beta by their proliferation and IFN-gamma production, although levels of IL-1beta-induced responses were lower. CD4(+)CD45RA(+) T cells, although they constitutively expressed IL-18Rbeta mRNA, did not express IL-18Ralpha mRNA. Phytohemagglutinin (PHA) stimulation alone induced IL-18Ralpha mRNA without affecting the expression of IL-18Rbeta mRNA. T(h)1-inducing conditions (PHA, IL-12 and anti-IL-4) further increased this expression. We also show that T(h)1 cells but not T(h)2 cells have increased expression of IL-18R and IL-1R, and produce IFN-gamma in response to IL-18 and/or IL-1beta.

Evaluating changes in stable chromosomal translocation frequency in patients receiving radioimmunotherapy

Purpose: The lack of any consistent correlation between radioimmunotherapy (RIT) dose and observed hematologic toxicity has made it difficult to validate RIT radiation dose estimates to marrow. Stable chromosomal translocations (SCT) which result after radiation exposure may be a biologic parameter that more closely correlates with RIT radiation dose. Increases in the frequency of SCT are observed after radiation exposure and are highly correlated with absorbed radiation dose. SCT are cumulative after multiple radiation doses and conserved through an extended number of cell divisions. The purpose of this study was to evaluate whether increases in SCT frequency were detectable in peripheral lymphocytes after RIT and whether the magnitude of these increases correlated with estimated radiation dose to marrow and whole body. Methods and Materials: Patients entered in a Phase I dose escalation therapy trial each received 1–3 intravenous cycles of the radiolabeled anti–carcinoembryonic antigen (CEA) monoclonal antibody, 90Y-chimeric T84.66. Five mCi of 111In-chimeric T84.66 was co-administered for imaging and biodistribution purposes. Blood samples were collected immediately prior to the start of therapy and 5–6 weeks after each therapy cycle. Peripheral lymphocytes were harvested after 72 hours of phytohemagglutinin stimulation and metaphase spreads prepared. Spreads were then stained by fluorescence in situ hybridization (FISH) using commercially available chromosome paint probes to chromosomes 3 and 4. Approximately 1000 spreads were evaluated for each chromosome sample. Red marrow radiation doses were estimated using the AAPM algorithm and blood clearance curves. Results: Eighteen patients were studied, each receiving at least one cycle of therapy ranging from 5–22 mCi/m 2. Three patients received 2 cycles and two patients received 3 cycles of therapy. Cumulative estimated marrow doses ranged from 9.2 to 310 cGy. Increases in SCT frequencies were observed after each cycle for both chromosomes 3 and 4 in 16 of 18 patients and in at least one chromosome for the remaining 2 patients. Cumulative increases in SCT frequencies ranged from 0.001 to 0.046 with no major differences observed between chromosomes 3 and 4. A linear correlation between cumulative marrow dose and increases in SCT frequencies was observed for chromosome 3 ( R 2 = 0.63) and chromosome 4 ( R 2 = 0.80). A linear correlation was also observed between increases in SCT frequency and whole body radiation dose or administered activity ( R 2 = 0.67–0.89). There was less correlation between observed decrease in wbc or platelet counts and marrow dose, whole body dose, or administered activity ( R 2 = 0.28–0.43). Conclusions: Increases in SCT frequency were detectable in peripheral lymphocytes after low dose-rate RIT irradiation. A linear correlation was observed between increases in SCT and marrow dose, whole body dose, and administered activity. This correlation provides one of the strongest radiation dose–response and activity–response relationships observed with RIT. The detection of SCT may therefore have application as an in situ integrating biodosimeter after RIT. This biologic parameter should prove useful in comparing effects on marrow for different therapeutic radionuclides and in comparing effects of RIT and external beam radiation doses on a cGy per cGy basis. As a result, this should allow for a more direct comparison between different methods of irradiation and in further refinement of radioimmunotherapy dose estimates and dosimetry methodology.

Th1-type cytokines production is decreased in kidney transplant recipients with active cytomegalovirus infection

Cytomegalovirus (CMV) infection is a major complication after kidney transplantation. Despite antiviral therapy the infection contributes significantly to high morbidity. The present study was aimed at determining: (a) the stimulation index (S.I.) of phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC) and (b) the levels of Th1- and Th2- related cytokines in kidney transplant recipients with and without active CMV infection. Thirty-five patients with, and 44 without active CMV infections, as diagnosed by a CMV antigenemia assay, were inducted into this study. After PHA stimulation of PBMC from patients, stimulation index (S.I.) was determined by radioactive thymidine uptake while the production of Th1-type cytokines (interleukin-2 [IL-2], interferon-γ [IFN-γ], and tumor necrosis factor-α [TNF-α]) and Th2-type cytokines (IL-4, IL-10) were measured by enzyme-linked immunosorbent assay. PBMC of patients with active CMV infection showed significantly lower S.I. values than patients without an ongoing CMV infection (P < .0001). Levels of Th2-type cytokines in CMV-infected and uninfected kidney recipients were similar; however, the levels of the Th1-type cytokines were significantly lower in CMV-infected patients. Low levels of Th1-type cytokines seem to correlate well with active CMV infection in kidney recipients. J. Med. Virol. 60:223–229, 2000. © 2000 Wiley-Liss, Inc.

Th1-type cytokines production is decreased in kidney transplant recipients with active cytomegalovirus infection

Cytomegalovirus (CMV) infection is a major complication after kidney transplantation. Despite antiviral therapy the infection contributes significantly to high morbidity. The present study was aimed at determining: (a) the stimulation index (S.I.) of phytohemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (PBMC) and (b) the levels of Th1- and Th2- related cytokines in kidney transplant recipients with and without active CMV infection. Thirty-five patients with, and 44 without active CMV infections, as diagnosed by a CMV antigenemia assay, were inducted into this study. After PHA stimulation of PBMC from patients, stimulation index (S.I.) was determined by radioactive thymidine uptake while the production of Th1-type cytokines (interleukin-2 [IL-2], interferon-gamma [IFN-gamma], and tumor necrosis factor-alpha [TNF-alpha]) and Th2-type cytokines (IL-4, IL-10) were measured by enzyme-linked immunosorbent assay. PBMC of patients with active CMV infection showed significantly lower S.I. values than patients without an ongoing CMV infection (P <.0001). Levels of Th2-type cytokines in CMV-infected and uninfected kidney recipients were similar; however, the levels of the Th1-type cytokines were significantly lower in CMV-infected patients. Low levels of Th1-type cytokines seem to correlate well with active CMV infection in kidney recipients.

Incidence of mosaic cell lines in vivo and malsegregation of chromosome 21 in lymphocytes in vitro of trisomy 21 patients: detection by fluorescence in situ hybridization on binucleated lymphocytes.

In order to detect aneuploidy in interphase human lymphocytes, both in vivo and in vitro, fluorescence in situ hybridization (FISH) was carried out on binucleated cells cytokinesis-blocked by cytochalasin B at the first mitosis after phytohemagglutinin stimulation. A pericentric chromosome-21-specific DNA probe prepared from yeast artificial chromosome clone 881D2 by the polymerase chain reaction was employed. One thousand binucleated cells per individual were scored from cultures from twelve trisomy 21 patients aged 0.01-8.9 years (mean 4.3 years) and 20 normal children of similar age. Of trisomy 21 patients, increased frequencies of disomic cells in vivo (1.690+/-1.070%) and cells containing six signals with nondisjunction (0.822+/-0.554%) were found, compared with those of monosomic 21 cells in vivo (0.265+/-0.130%) and cells containing four signals with nondisjunction in normal children (0.369+/-0.250%; P=0.000 and P=0.000, respectively). These results show that malsegregation of chromosome 21 occurs more often in trisomic 21 cells than in disomic cells from normal children. The frequency of nondisjunction was significantly higher than the loss of chromosome 21 in both cultured trisomic (0.822+/-0.554% vs 0.043+/-0.049%, P=0.000) and disomic (0.369+/-0.250% vs 0.010+/-0.30%, P=0.000) cells. Comparisons of in vivo and in vitro data on aneuploidy indicate that a cell selection mechanism may exist in vivo. All these results show that FISH, with a chromosome-specific probe, on binucleated lymphocytes is a powerful tool for simultaneously detecting mosaic cell lines in vivo and malsegregation (loss and nondisjunction) of a corresponding chromosome in vitro in the same cell population.

Soluble receptors and other substances that regulate proinflammatory cytokines in young and aging humans.

Relatively little is known about changes in soluble receptors and other agonists/antagonists that may regulate cytokine actions in aging humans. We have studied age-associated changes in human subjects of (a) the plasma levels of interleukin-1 soluble receptor (IL-1sRII), interleukin-1 receptor antagonist (IL-1ra), tumor necrosis factor soluble receptor-II(75kDa; TNFsRII), and interleukin-6 soluble receptor (IL-6sR) and (b) the ability of their blood mononuclear cells to produce those soluble factors spontaneously and after phytohemagglutinin (PHA) stimulation. Aging subjects (50-67 years) had significantly higher plasma levels of IL-1ra, significantly lower levels of TNFsRII and IL-6sR than young subjects (25-35 years), and no significant change in the level of IL-1sRIL There was less spontaneous output of IL-1ra and TNFsRII by peripheral blood mononuclear cells (PBMC) of aging than of young subjects, but equivalent output of both factors in response to PHA stimulation. Thus, the basal (homeostatic) output of those two factors declined with age, but the potential of the PBMC to produce the factors on stimulation did not. PHA stimulation of PBMC of either age group significantly inhibited the output of IL-6sR. These differences between the young adult and aging subjects, along with reported changes in the corresponding cytokines, presumably foreshadow changes that become more marked with further aging Therefore, immunological processes that depend on, or are modulated by, proinflammatory cytokines may differ between young and aged subjects as a consequence of the availability of regulatory soluble receptors and related agonists or antagonists. The results of this study highlight the need for further studies of the roles played by soluble receptors, and similar agonists/antagonists, in the immune responses of aged adults.

Effect of Spirulina on the Secretion of Cytokines from Peripheral Blood Mononuclear Cells

ABSTRACT The purpose of this study was to evaluate the immunomodulatory activity of Spirulina, a bluegreen alga used as a food supplement. The effects of Spirulina on the secretion of three cytokines from unstimulated and stimulated human peripheral blood mononuclear cells (PBMC) were examined. In resting PBMC, Spirulina stimulated secretion of interleukin (IL)-1beta, IL-4, and interferon (IFN)-gamma to nearly 2.0, 3.3, and 13.6 times basal levels, respectively. Spirulina induced levels of IFN-gamma (229 +/- 104 pg/ml) that were comparable to those seen after phytohemagglutinin (PHA) stimulation (476 +/- 121 pg/ml). However, it was much less mitogenic than PHA (13.1 +/- 6.9 pg/ml) with respect to the induction of IL-4 secretion (0.34 +/- 0.1 pg/ml). In PHA-stimulated cells, Spirulina enhanced secretion of IL-1beta, IL-4, and IFN-beta by 2.9, 4.0., and 1.6 times, respectively. Although Spirulina stimulates several cytokines, it is clearly more effective in the generation of a Thl-type response. This in vitro study offers additional data for consideration of the potential therapeutic benefits of Spirulina.