Stimulated Lymphocyte Cultures Research Articles

Immunogenicity of a novel anti-allergic vaccine based on house dust mite purified allergens and a combination adjuvant in a murine prophylactic model.

The outer-membrane-derived proteoliposome (PL) of Neisseria meningitidis has been reported as a potent vaccine adjuvant, inducing a Th1-skewed response. This work aimed to assess the immunogenicity of a novel anti-allergic vaccine candidate based on allergens from Dermatophagoides siboney house dust mite and a combination adjuvant containing PL and Alum. In a preventative experimental setting, BALB/c mice were administered with three doses containing 2 µg of Der s1 and 0.4 µg Der s2 allergen, PL and Alum, at 7 days intervals, by subcutaneous route. Furthermore, mice were subjected to an allergen aerosol challenge for 6 consecutive days. Serum IgE, IgG1, and IgG2a allergen-specific antibodies were assessed by ELISA. Cytokine levels in supernatants of D. siboney stimulated lymphocyte cultures and in bronchoalveolar lavage (BAL) were measured by ELISA. Lung tissues were subjected to histological examination. The vaccine prevented the development of both, systemic (IgE) and local allergic responses (featuring lower IL-4, and IL-5 levels in BAL) upon allergen exposure by the inhalant route. Histological examination showed also a diminished allergic inflammatory response in the lungs. After the allergen challenge, cytokine levels in stimulated lymphocyte cultures showed lower values of IL-13 and augmented IFN-γ and IL-10. The vaccine induced a mixed IgG2a/IgG1 antibody response; although only IgG2a was PL-dependent. Both, IgG1/IgE and IgG2a/IgE ratios, showed significantly greater values in vaccinated mice. The findings support a preventative anti-allergic effect associated with the induction of a Th1-like IFN-γ/IL-10 response. IgG1/IgE and IgG2a/IgE ratios could be useful biomarkers for translation into clinical trials.

Jagged-2 enhances immunomodulatory activity in adipose derived mesenchymal stem cells.

Adipose derived Mesenchymal stem cells (AMSCs) are able to expand in vitro and undergo differentiation into multiple cell lineages, yet have low immunogenicity while exhibiting several immunoregulatory characteristics. We sought to investigate the immunomodulatory mechanisms of AMSCs to better understand their immunogenic properties. Following 10 days of chondrogenic differentiation or 48 hours of IFN-γ pretreatment, AMSCs retained low level immunogenicity but prominent immunoregulatory activity and AMSC immunogenicity was enhanced by chondrogenic differentiation or IFN-γ treatment. We found Jagged-2 expression was significantly elevated following chondrogenic differentiation or IFN-γ pretreatment. Jagged-2-RNA interference experiments suggested that Jagged-2-siRNA2 suppresses Jagged-2 expression during chondrogenic differentiation and in IFN-γ pretreated AMSCs. Besides, Jagged-2 interference attenuated immunosuppressive activity by mixed lymphocyte culture and mitogen stimulation experiments. So, the immunoregulatory activity of AMSCs, to some extent dependent upon Jagged-2, might be stronger after multilineage differentiation or influence from inflammatory factors. This may also be why rejection does not occur after allogeneic AMSCs differentiate into committed cells.

Partial deletion of distal long arm encompassing Jacobsen Syndrome

Materials and Methods Segmental aneuploidy or alteration involving #11q arm was detected in 2 cases during conventional cytogenetic analysis carried out in children having multiple congenital anomalies (MCA). Chromosome preparations were obtained from PHA stimulated lymphocyte cultures according to the standard procedure at 500-band level in both patient and their parents. Evaluation of the break point region was performed by 60K oligonucleotide arrayComparative Genomic Hybridization (aCGH) using Agilent platform. Female genomic DNA (Promega Corporation, Madison, WI, USA) was used as a sex-matched reference, which was analyzed with the aCGH analysis software v3.4 (Agilent Technologies Inc., Santa Clara, CA, USA) by applying Z-score segmentation algorithm with a window size of 10 points to identify chromosome aberrations.

The Risk of Polyomavirus-Associated Graft Nephropathy Is Increased by a Combined Suppression of CD8 and CD4 Cell-Dependent Immune Effects

Polyomavirus-associated graft nephropathy (PAN) has emerged as a significant risk factor for kidney graft loss. We analyzed intracellular cytokine responses for possible protective versus permissive immunologic effects on BK-virus replication. One hundred five renal transplant patients included in a prospective single-center study were randomized to receive cyclosporine mycophenolate mofetil (MMF) (CM: n = 31), tacrolimus (Tac)/MMF (TM: n = 32) or Tac/MMF with conversion to everolimus (TErl; n = 32). Ten patients were not randomized (NR) due to contraindications to MMF. The immunosuppressive therapy was monitored pre- and posttransplantation at 4, 12, and 24 months using triple fluorescence flow cytometry for intracellular interleukin (Il)-2 Il-4 and interferon (IFN)-γ production in phorbol myristate acetate- and lipopolysaccharide- stimulated lymphocyte cultures. BK viremia screening was performed by reverse-transcriptase polymerase chain reaction testing on days 0, 14, 30, 60, 90, 120, 180, 270, 360, and 720. Seven of 105 (6.7%) patients developed biopsy-proven PAN (CM: n = 1, TM: n = 3, TErl: n = 2, NR: n = 1), among whom 4 lost their grafts (TM: n = 1, TErl: n = 2, NR: n = 1). Twenty-one of 105 (20.0%) patients had documented BK viremia. BK viremia which preceded PAN in all cases, was significantly associated with TM immunosuppression: 4/31 (12.9%) CM: 11/32 (34.4%) TM; 5/32 (15.6%) TErl, and 1/10 (10.0%) NR patients (P = .034). BK-viremic patients showed significantly diminished CD8+ T-cell Il-2 production at 120 days (P = .011) and 1 year posttransplantation (P = .014) compared with non-BK-viremic patients. Patients with PAN displayed significantly lower CD4+ T-cell Il-4 responses at 1 and 2 years after transplantation (1 year: P = .007; 2 years: P = .001) with diminished IFN-γ responses at 1 year after transplantation (P = .011). Our analysis showed the incidence of BK viremia to be increased among patients with defective cytotoxic CD8+ T-cell -dependent immune reactivity. Recipients who progressed from BK viremia to overt PAN showed an additional immunologic defect in CD4+ T-cell function. Patients on a Tac- plus MMF-based immunosuppression were at higher risk to develop BK viremia.

Combining bacterial-immunotherapy with therapeutic antibodies: A novel therapeutic concept

Immunotherapeutic strategies become more and more important for cancer treatment. Therapeutic monoclonal antibodies (mAbs) like Panitumumab binding and blocking the EGF-receptor are in routine clinical use for the treatment of colorectal carcinoma (CRC). Also, bacterial therapy proved beneficial for experimental treatment of different tumor entities. The latter has been attributed to an activation of the immune system. Here, we describe a combination of both immunotherapeutic approaches in order to develop a novel targeted therapy for CRC. The therapeutic mAbs Trastuzumab and Panitumumab were conjugated to heat-inactivated bacteria expressing protein A or protein G.The potential of the conjugates was tested in comparison to the single components both in vitro and in vivo using a panel of patient-derived CRC cell lines. Antitumoral effects observed in vitro were strictly dependent on the presence of bacteria. Generally, effects could be enhanced by the addition of human lymphocytes. Detailed analysis of effector cells in autologous and allogeneic long-term stimulated lymphocyte cultures revealed the predominance of NK-cell-like cytolytic effectors. Reactivity was observed both against CRC target cells but also against the NK cell target K562. Similarly, in a subsequent in vivo study we observed substantial tumor growth delay accompanied by an increase in circulating NK cells. Contrary to this, the monotherapy with mAb alone caused only marginal effects and the treatment with bacteria was comparable to the mock-treated control.These data demonstrate successful targeting of CRC by bacteria/mAb conjugates. This novel concept may be interesting for future clinical approaches. Additionally, it illustrates the effectiveness of NK cells for cancer immunotherapy.

BRCA1 c.4987-3C>G is a pathogenic mutation

BRCA1 c.4987-3C>G is a pathogenic mutation

Photochemical pathogen inactivation of human serum enables its large-scale application in clinical cell transplantation

Stringent protocols for cGMP, issued by regulatorybodies in Asia, Europe and North America, regulateproduction of cells and tissues for clinical therapy. Dueto the risk of transferring infections, human serum, thepreferred supplement throughout the process of cell cul-ture and transplantation, cannot be used, resulting ininferior quality of released cell products. Photochemicaltreatment (INTERCEPT Cerus Europe BV, Amersfoort,Netherlands), with the psoralen compound, amotosalenHCl and long-wavelength ultraviolet light) has beenshown to inactivate any pathogen of RNA and DNAorigin [1–4]. INTERCEPT treatment of human serumcould allow its use in processing cells and tissues forclinical application.No long-term exposure in vitro of cells to residual amo-tosalen has been performed. However, a carcinogenicitystudy in p53 knockout mice exposed to doses 1000-foldthe clinical dose for 26 weeks, showed no evidence ofcarcogenicity [5].The use of PI-HS compared with C-HS was examined onhuman melanoma cell lines as well as in the production ofclinical grade islets of Langerhans (for the treatment ofType I Diabetes), mesenchymal stem cells (for the MSCs:treatment of GvHD grade III or IV) and T cells (for immunetherapy against malignant melanoma).There was no difference in rate of proliferation during a3-day culture in medium supplemented with either PI-HSor C-HS (10%) for five human melanoma cell-lines(397mel, 526mel, SK23mel, EB81mel, CP64mel).Similarly, there was no difference between islets culturedfor 4 days inCMRL-1066 supplemented with PI-HS orC-HSintermsofinsulinstimulationindexfromadynamicperifu-sion system where the average of the high values dividedwith the average of the low values (14AE7±4AE15, 10AE0±4AE57), the ADP⁄ATP-ratio (0AE010 ± 0AE013, )0AE0004 ±0AE013) and the capacity to cure STZ-diabetic mice (78%,87%). Likewise, no difference was found in intracellularinsulin content (2AE6±1AE14, 2AE3±0AE85 ng⁄ml), expressionof IL-6 (0AE0067 ± 0AE016, 0AE0075 ± 0AE018 pmol⁄lgDNA),IL-8 (0AE264 ± 0AE150, 0AE352 ± 0AE169 pmol⁄lgDNA), MCP-1(0AE164 ± 0AE086, 0AE166 ± 0AE080 pmol⁄lgDNA) or TissueFactor(0AE034 ± 0AE007,0AE087 ± 0AE057 pmol⁄lgDNA).Also, expansion of MSC and the capacity for immuno-suppression in MLC and PHA stimulated lymphocyte cul-tures (71 ± 13, 59 ± 9%) did not differ between MSCgenerated in CMRL-1066 supplemented with PI-HS orC-HS.Finally, no differences in regards of total number of Tcells generated (30AE9±3AE1, 29AE7±1AE5 millions⁄ml), foldexpansion (309AE4±31AE8, 297AE4±15AE2) or expression ofCD25, CD27 or CD26L were observed when T cells were cul-tured in RPMI-1640 supplemented with PI-HS or C-HS.Students t-test was used to evaluate the results between PIand control.The presented technique for PI of human serum providesa solution to an important safety and regulatory problem inmodern cell therapy. PI exerts no negative impact onhuman islets of Langerhans, MSCs, T cells or cell lines andcan even have a positive effect by down-regulation ofinflammatory mediators induced by DNA or RNA strandsreleased from damaged cells. INTERCEPT treatment ofhuman serum allows the routine use of human serum inclinical cell transplantation.In many European countries, the Intercept technology isroutinely used for PI of platelets and plasma for clinicaluse. Applying this already used technique in a new waycould be a new niche for blood banks. They can use this toprovide safer products for clinical cell culture and trans-plantation and by doing this increase the safety for thepatients.

Defining the 10 Year Risk of Disease Progression in Stage A0 CLL.

80% of patients with CLL present with a lymphocytosis detected on a routine blood count. Many studies have identified risk factors for disease progression but there remains uncertainty about the magnitude of risk and which factors are most useful in determining this risk. To address this we have studied a cohort of 122 stage A0 patients who presented with a lymphocytosis and typical CLL immunophenotype before 1996 and whose CLL has either progressed or remained stable over a 10 year period. All patients were reviewed at least annually. Disease progression was defined as the need for anti-leukemic therapy, an increase in stage or a downward trend in haemoglobin or platelet count, progressive lymphadenopathy or splenomegaly or constitutional symptoms. Karyotypic data, from analysis of G banded metaphases derived fromTPA stimulated lymphocyte cultures, were available on all cases at diagnosis. CD38 and ZAP 70 expression were measured on DMSO frozen cells. VH gene usage and mutational status, and telomere length were assessed on frozen DNA samples, while interphase FISH for del 13q14, del 11q and 17p (ATM and p53 loss) and trisomy 12, was undertaken on fixed cell suspensions. All samples had been collected and stored at, or close to the time of diagnosis.50 patients had progressive disease. Of the 72 patients with stable disease, the lymphocyte count remained below 20x109/l in 53 and rose with a lymphocyte doubling time of > 1 year in the remainding 19 (termed slowly progressive). In univariate analysis, a lymphocyte count of > 20x109/l, unmutated VH genes, high expression of CD38 (cut off 30%) and ZAP70, del 11q, abnormal presenting karyotype and short telomere length all predicted for disease progression. There was no difference in VH gene usage between the progressive and stable groups. Among the 107 patients for whom lymphocyte count, VH gene mutational status, CD38 and ZAP 70 expression are all currently available, the number of poor risk factors in the progressive and stable groups is shown in Table 1. There was no difference in the number of risk factors between the stable and slowly progressive groups. Del 11q was found in 9 progressive cases all of whom had 2 or more risk factors. P53 loss was found in a single patient with stable disease and no risk factors.20/122 patients presented with a lymphocyte count < 5.0x109/l, below which patients have been considered to have monoclonal B cell lymphocytosis. Interestingly, 5 of these developed progressive disease. Using only the lymphocyte count, CD38 and ZAP 70 expression, Table 2 shows the risk of disease progression according to the risk factors present.In summary, once ZAP 70 measurement by flow cytometry is standardized, readily available prognostic tests can provide a quantitative estimate of the risk of disease progression. Performing interphase FISH for 11q and p53 loss in poorer risk cases only may provide additional prognostic information.Table 1No. of adverse factors at presentation. (%)01234GroupProgressive11(25)10(23)11(25)9(20)3(7)Stable/Slowly progressive44(70)16(25)3(5)00Table 2Odds of Progression% of Patients ProgressingNo risk factors0.216.9ZAP70 only0.7442.7Lymphs>200.8947.0CD38 only1.5761.1ZAP70+lymphs3.2476.4ZAP70+CD385.7585.2CD38+lymphs6.8587.3All25.196.2

Cytokine profiles in human exposure to metals (IUPAC Technical Report)

Immunosensitization to metal ions through occupational and environmental exposure has been described in earlier papers from this project. Here we discuss the possible role of cytokine profiling in demonstrating and understanding this phenomenon. The cytokines are a large family of polypeptides exerting autocrine, paracrine, and/or endocrine effects. They include interleukins (ILs), interferons (IFNs), and growth factors. They may be grouped as pro-inflammatory (e.g., IL-1, IL-6, IL-12, IL-18, TNF-α), anti-inflammatory (e.g., IL-10), or those regulating T-helper (TH) cell function. The latter are subdivided into those associated with TH1 (e.g., IL-2, IL-12, IFN-γ, TNF-β) or TH2 (e.g., IL-4, IL-5, IL-13) cell function. Because different types of immune reactions (e.g., immediate reaction vs. delayed-type hypersensitivity) differentially involve TH1 and TH2 cells, measurement of cytokine production in response to metal ions can potentially give insight into underlying immune mechanisms and responses. Examples are given for species of Ni, Cr, Co, Hg, Cd, and Be; and in less detail for species of Fe, Pt, Pd, and Rh. Antibodies are available commercially that allow for the determination of many cytokines, and such measurements are most usefully performed with body fluids, supernatants from stimulated lymphocyte cultures, or lysates of lymphocytes or other biopsied cells. The predominant methods include enzyme-linked immunosorbent assay (ELISA) and flow cytometric measurement of the cytokine, bioassay of its activity in cell culture, and polymerase chain reaction (PCR) assessment of its mRNA level. In practice, levels of individual cytokines are highly variable between individuals, and reliable reference values are generally lacking. Ratios of cytokines are more informative than absolute concentrations, and biological variability in cytokine production dictates that repeated testing is necessary to confirm trends. Determining cytokine profiles is presently of questionable diagnostic utility in individual cases of metal sensitization, but is providing mechanistic insights in a research context.

Analysis of IL-2-like factor in lymphocyte culture supernatant of olive flounder,Paralichthys oliveaceus

To study immune mechanism of fish lymphocyte we performed a proliferation assay and ELISA using monoclonal antibody against human IL-2. The result showed that an interleukin-2 (IL-2)-like factor was detected in the supernatant of plant haemoglutinin (PHA)-stimulated lymphocyte culture from peripheral blood, spleen and head kidney of olive flounder,Paralichthys olivaceus. The quantities of IL-2-like factor in the supernatant from different lymphoid tissues were quite different. The IL-2 like factor in the supernatant from cultured head kidney lymphocytes was much higher than those of peripheral blood lymphocytes and spleen lymphocytes (P<0.01). The IL-2 activity was found in either mouse thymocyte proliferation assay or flounder head kidney lymphocyte proliferation assay and shown to have obvious enhancing effect on proliferation of the above two types of cell. The recombinant human IL-2, (rhIL-2) was able to stimulate flounder thymocyte proliferation and used to detect the IL-2 receptor (IL-2R) on the surface of flounder lymphocyte. The cross-reaction between the lymphocytes of flounder peripheral blood and CD25(IL-2R) was detected with flow cytometry and shown that the percentage of CD25-positive cell in peripheral blood was 7.74±0.67%.

Complexde novo cryptic subtelomeric rearrangements in a fetus with multiple ultrasonographic abnormalities and a normal karyotype at amniocentesis

Prenatal diagnosis is usually offered to the majority of pregnancies with fetal structural abnormalities detected by prenatal ultrasound; however, only a small proportion show an abnormal karyotype. We wanted to detect cryptic subtelomeric rearrangements (CSTR) in a fetus with multiple abnormal ultrasonographic findings that revealed a normal karyotype at amniocentesis. Fetal chromosome analysis was performed from amniotic fluid cells. Parental chromosome analysis was done on PHA stimulated lymphocyte cultures. For fluorescence in situ hybridization (FISH) analysis, ToTelVysion multicolor DNA probe mixture was used to hybridize the p and q telomeres of each chromosome. The amniotic fluid chromosome analysis revealed an apparently normal 46,XY karyotype. A follow-up FISH analysis showed three apparently balanced complex translocations involving (1) the chromosome 4p and 22q telomeres (2) 4q and 11q telomeres and (3) 8p, 20p and 20q telomeres. Parental chromosome and subtelomere FISH analysis was found to be normal. To our knowledge, this is the first report of complex de novo cryptic translocations in an abnormal fetus. These CSTR identified by FISH with subtelomere-specific probes are not detected by other cytogenetic and/or molecular cytogenetic approaches. However, to confirm the balanced nature of CSTR, array-CGH can be helpful. Further studies are in progress to determine the frequency of CSTR and its significance in the etiology of fetal abnormalities.

The yield of radiation-induced chromosomal aberrations in first division human lymphocytes depends on the culture time

Purpose: To investigate two long-held beliefs in radiation cytogenetics that were seemingly contradicted by reports that: (a) protracting γ-ray exposures over 0.5 h halves the induced aberration yield compared with acute exposure, and (b) that induced aberration yields in guaranteed first in vitro division metaphases (M1) vary with culture time.Materials and methods: Replicate blood samples were exposed for 3 min to 3.0 Gy γ-rays and standard phytohaemagglutinin stimulated lymphocyte cultures were harvested at 10 times ranging from 45 – 72 h. Forty-eight hour cultures were also made from blood exposed to 3.0 Gy for 30 min. Slides were differentially stained, combining the harlequin method with fluorescent in-situ hybridization (FISH) painting of chromosomes 2, 3 and 5. M1 metaphases were scored for 1- and 2-way translocations involving the painted chromosomes and all unstable aberrations in the full genomes.Results: Dicentric and translocation yields from the 30 min exposure were approximately 10% lower than in 48 h cultures from cells exposed for 3 min, although this reduction is not significant. Dicentric aberration yields from the 3 min exposed cells cultured over the range 45 – 72 h remained constant up to 51 h then rose to a different constant value beyond 60 h. The increase at 60 – 70 h compared with the yield at 48 h was about 50%. A marginal increase at later times was also observed for translocations.Conclusion: The protracted exposure experiment produced results consistent with the G-function hypothesis that models the dose rate effect. Therefore the previous report of a marked departure from this model was not confirmed. The reports of aberration yields increasing with time of arrival at metaphase were confirmed. Possible explanations are discussed; the intercellular distributions of aberrations, or of doses to the cells or heterogeneous radiosensitivity of lymphocyte sub-populations. None alone seems sufficient quantitatively to explain the magnitude of the effect. The implications for biological dosimetry, which employs cultures times of approximately 48 h, are considered to be minor.

Analysis of intracellular regulatory proteins by immunoaffinity capillary electrophoresis coupled with laser-induced fluorescence detection.

Measurement of intracellular regulatory proteins is of great importance in many areas of biomedical research. In this communication we describe an antibody-based capillary electrophoresis system equipped with an on-line laser-induced fluorescence detector capable of measuring intracellular proteins in cultures as low as 100 cells. The system demonstrated a high degree of precision and accuracy, being capable of detecting the fluorochrome-labeled analytes of interest at concentration of approximately 0.5 pg. We have used this instrument to study concentrations of the intracellular regulatory proteins STAT-1 and STAT-3, following stimulation of lymphocyte cultures with the inflammatory cytokine, IL-6. Using a combination of four antibodies specific for either STAT-1 or STAT-3 in both their nonphosphorylated and phosphorylated forms, we were able to demonstrate the differential expression of these proteins over time.

Human hematopoietic progenitors engraft in fetal canine recipients and expand with neonatal injection of fibroblasts expressing human hematopoietic cytokines

The development of large-animal models for human hematopoiesis will facilitate the study of human hematopoietic stem cells and their progenitors in vivo. In previous studies, human hematopoietic progenitors engrafted in fetal dogs and contributed to hematopoiesis for one year. Despite initially high levels of human cells, the proportion declined to less than 0.1% at 6 months, possibly due to inability of the canine hematopoietic microenvironment to support ongoing human hematopoiesis. In the current experiments we examined the potential of co-transplanting fibroblasts expressing human hematopoietic cytokines with the hematopoietic graft to increase the contribution of human progenitors to chimeric hematopoiesis. Mid-gestation canine fetuses were injected with 1-3 x 10(7) human cord blood cells and 1 x 10(7) murine fibroblasts engineered to express human cytokines. Neonatal pups were boosted with additional injections of cytokine-expressing fibroblasts. Human cell engraftment was monitored by PCR amplification of human-specific DNA sequences from recipient hematopoietic tissues. Human hematopoietic cells were detected in 13/15 fetal recipients for at least 7 months. At time points up to 30 weeks of age, human DNA was detected in stimulated lymphocyte cultures, approximately 0.1% of blood leukocytes and 1.5% (85/5757) of myeloid colonies. Eight months postinfusion, 1.7% of colony-forming units (CFUs) were of human origin. By one year 0.5% or less of myeloid colonies and less than 0.01% of blood leukocytes carried human DNA. Following an infusion of cytokine-expressing fibroblasts at one year, the proportion of human myeloid progenitors rose to 11.5% and remained detectable for 8 months. These studies confirm that human hematopoietic progenitors can engraft in fetal pups and contribute to multilineage hematopoiesis. Infusion of cells expressing human cytokines is one approach to stimulate human hematopoietic progenitors in vivo and thus increase their contributions to chimeric hematopoiesis.

Effective depletion of alloreactive lymphocytes from peripheral blood mononuclear cell preparations.

T cells present in an allogeneic bone marrow transplant may produce graft-versus-host disease but also contribute to immune reconstitution and enhance engraftment. Our aim was to separate alloreactive from nonalloreactive T lymphocytes, by performing a mixed lymphocyte culture (MLC) stimulation of donor cells, followed by selective depletion of activated cells expressing the high-affinity interleukin 2 receptor. We then characterized the resulting depleted cell fraction. Donor peripheral blood mononuclear cells were cocultured with irradiated peripheral blood mononuclear cells from HLA-nonidentical recipient stimulators in an MLC. After 3 days, CD25+ lymphocytes (alloreactive cells expressing the alpha chain of the interleukin 2 receptor) were removed by immunomagnetic separation. The depleted donor fraction and untreated cells were then rechallenged in a secondary MLC with the original irradiated stimulator cells or a third party to assess relative alloreactivity. Inhibition of the secondary MLC and of host-specific cytotoxic activities was observed as well as a disappearance of interleukin 2 receptor-positive cells. Alloreactivity against unrelated third-party cells was preserved. Limiting dilution analysis of residual alloantigen-reactive T lymphocytes demonstrated a 1.3 log reduction of antihost reactivity. The depletion largely removed host-specific alloreactive CD4+ cells. This method reduces alloreactivity while retaining reactivity against third-party targets. This approach may allow therapeutic infusion of T cells after HLA-nonidentical allografts with a reduced capacity to produce graft-versus-host disease.

In Vitro Suppression of the Normal Mitogenic T Lymphocyte Response by Steady State Sickle Cell Disease Sera

This study is part of a long term evaluation of sickle cell disease (SCD) as a paradigm for immunosuppression. Serum was obtained from 43 SCD patients during the steady (healthy) state. Peripheral blood mononuclear cells (PBMC), separated by density gradient were obtained from 8 normal healthy donors. PBMC were utilized in assays directly or as a source for obtaining, total T (CD3) and helper T (CD4) cell populations separated by specific T cell columns. Standard in vitro phytohemagglutinin (PHA) stimulation of lymphocyte cultures was done with culture media containing 10% SCD serum, as compared to normal pooled O, Rh+ (O+) serum. Mitogenic responses were expressed as mean counts per minute (cpm) and stimulation index of triplicate cultures. Results revealed PHA responses were positive in all experiments when a standard stimulation index of 10 or greater was used as a test paramater for comparison. Positive results were demonstrated in 43/43 (100%) of triplicate cultures regardless of serum type in all experiments. Conversely, by using mean cpm as the test criterion, suppression of PHA response was shown in SCD serum supplemented cells as follow; 36/43 (84%) of PBMC, 35/43 (81%) of CD3 and 37/43 (86%) of CD4 cultures. The degree of suppression ranged from >10% to 98% in individual experiments, as compared to O+ serum. Inhibitors of normal T lymphocyte in vitro PHA response appear to be present in a significant percentage of SCD sera even during the healthy state of disease. Type 2 cytokines which suppress cell mediated immunity would seem to be the most likely inhibitory agents.

Dendritic-like cells from relB mutant mice.

Mice deficient in the NF-kappa B transcription factor relB appear to have defects in the production of mature dendritic cells, as secondary lymphoid tissues are absent, and spleen cells show a significant loss of antigen presenting function. Moreover, the thymus appears to be impaired in negative selection, and immune responses in vivo are poor. Since dendritic cell precursors such as skin Langerhans cells appear to be normal, we sought information on the nature of the dendritic cell defect in these mice. Cultures of mutant bone marrow in the presence of GM-CSF revealed a delay in the accumulation of cells with dendritic cell features relative to controls; however, these cells were nearly as potent on a per cell basis as wild type cells in the stimulation of allogeneic mixed lymphocyte cultures. Similarly, skin Langerhans cells from mutant mice also showed significant ability to stimulate allogeneic T cells in culture. Since these findings cannot explain the defect in immune responses and the absence of secondary lymphoid tissues, we also looked at the ability of the relB mutant dendritic-like cells to form aggregates in vitro with naive syngeneic T cells. In this case, while wild type dendritic cells generated compact aggregates with T cells, relB mutant cells only formed irregular small aggregates. Thus, while relB mutant dendritic-like cells have some functions of mature dendritic cells, other functions are deficient. Understanding the role of relB in regulation of these functions should lead to a greater understanding of the molecular basis of dendritic cell development and function.

2,3,7,8-TCDD induces cytochrome P450 enzyme activity but not proliferation or phenotypical changes in human peripheral blood lymphocytes

In contrast to the well documented immunosuppressive effects of 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD) in experimental animals, the impact of dioxin on the human immune system remains controversial, although adverse health effects have been reported in humans accidentally or occupationally exposed to dioxin. More recently, a dose-dependent decrease of specific subpopulations of mitogen-activated human peripheral blood lymphocytes (PBL), including helper-inducer/memory cells (CD4 + CD29 +) and B cells (CD20 +), was reported after in vitro treatment with dioxin concentrations as low as 10 −12-10 −14 M TCDD [1]. Therefore, the direct effects of dioxin on human PBL subpopulations have been studied in more detail, in order to assess the availability of a sensitive indicator system for human dioxin exposure. PBL from healthy volunteers were stimulated with pokeweed mitogen (PWM) or anti-CD3 monoclonal antibody (OKT3) and treated with 10 −7–10 −14 M TCDD for 3–4 days. Cytochrome P450 (CYP1A1) enzyme induction was determined by the ethoxy-resorufin- O-deethylase (EROD) assay. Percentages of the different lymphocytes subsets, including CD2 (T cells), CD4, CD45 RA (suppressorinducer/virgin T cells), CD4, CD29, CD8, CD19 (B cells), as well as interleukin 2 (IL-2) receptor (CD25) and class II antigen (human leukocyte antigen HLA-DR) expression, were analyzed by flow cytometry. The proliferative activity was determined by 3H-thymidine uptake after 3–4 days of culture. In the present study, all stimulated lymphocyte cultures showed a significant increase of CYP1A1 activity at dioxin concentrations of 10 −7 and 10 −9 M. In contrast, alterations in surface antigen expression or suppression of proliferative responses did not occur in the mitogen-activated PBL over the whole concentration range of TCDD. These results clearly demonstrate that lymphoproliferation, as well as phenotypes of human PBL, are not affected by dioxin treatment and thus are not useful as sensitive biomarkers in human exposure studies.

Depressed T cell reactivity to recall antigens in Crohn's disease before and after surgical resection.

Earlier studies regarding possible primary immune disturbances participating in the pathogenesis of Crohn's disease yielded conflicting results. Peripheral blood lymphocyte subsets and lymphocyte proliferative responses to five soluble recall antigens and to the polyclonal stimulator phythaemagglutinin were therefore measured in 17 patients with active Crohn's disease, before and six months after surgical resection of the inflamed intestine and in 16 healthy controls. Lymphocyte proliferation in response to all five recall antigens was significantly lower in patients than in controls. No significant differences with controls were detected after surgery. Addition of indomethacin to phythaemagglutinin stimulated lymphocyte cultures had a stronger proliferation enhancing effect in patients than in controls, resulting in comparable proliferative responses in both groups. When both indomethacin and prostaglandin E2 were added, inhibition of reactivity by prostaglandin E2 was stronger in patients' cultures. This suggests a higher sensitivity to inflammatory prostaglandins in Crohn's disease. The degree of lymphocyte stimulation by antigens correlated positively with the percentage of circulating memory T cells (CD 45 RA-). The percentage of activated (HLA-DR+) CD8 cells was higher in patients than in controls. The CD4/CD8 ratio, which was not significantly different between patients and controls, correlated significantly with disease activity and characteristics, even in the postoperative phase. These findings suggest that immune abnormalities in Crohn's disease fluctuate with and are probably secondary to inflammatory activity.

Limited effects of temafloxacin compared with ciprofloxacin on T-lymphocyte function

Temafloxacin increased interleukin-2 production and mRNA levels and enhanced thymidine incorporation in stimulated lymphocyte cultures. Gamma interferon mRNA levels were unaffected. Temafloxacin also stimulated interleukin-2 gene induction, as revealed in a chloramphenicol acetyltransferase reporter gene system. However, temafloxacin exerted significantly weaker effects in these respects than did ciprofloxacin.