Lymphocyte Culture Supernatants Research Articles

Evaluation of a Typhoid/Paratyphoid Diagnostic Assay (TPTest) Detecting Anti-Salmonella IgA in Secretions of Peripheral Blood Lymphocytes in Patients in Dhaka, Bangladesh

BackgroundRapid and reliable diagnostic assays for enteric (typhoid and paratyphoid) fever are urgently needed. We report the characterization of novel approach utilizing lymphocyte secretions, for diagnosing patients with enteric fever by the TPTest procedure.MethodologyTPTest detects Salmonella-specific IgA responses in lymphocyte culture supernatant. We utilized TPTest in patients with suspected enteric fever, patients with other illnesses, and healthy controls. We also evaluated simplified modifications of TPTest for adaptation in laboratories with limited facilities and equipment.Principal FindingsTPTest was positive in 39 (27 typhoid and 12 paratyphoid A) patients confirmed by blood culture and was negative in 74 healthy individuals. Among 32 individuals with other illnesses, 29 were negative by TPTest. Of 204 individuals with suspected enteric fever who were negative by blood culture, 44 were positive by TPTest and the patients were clinically indistinguishable from patients with confirmed bacteremia, except they were more likely to be under 5 years of age. We evaluated simplifications in TPTest, including showing that lymphocytes could be recovered using lysis buffer or buffy coat method as opposed to centrifugation, that incubation of cells at 37°C did not require supplemental CO2, and that results were available for majority of samples within 24 hours. Positive results by TPTest are transient and revert to negative during convalescence, supporting use of the test in endemic areas. The results can also be read using immunodot blot approach as opposed to ELISA. Since no true gold standard currently exists, we used a number of definitions of true positives and negatives. TPTest had sensitivity of 100% compared to blood culture, and specificity that ranged from 78–97% (73–100, 95% CI), depending on definition of true negative.ConclusionThe TPTest is useful for identification of patients with enteric fever in an endemic area, and additional development of simplified TPTest is warranted.

Immunotherapy – 2068. Immunomodulatory and safety profile of a novel anti-allergic vaccine based on allergens from dermatophagoides siboney and a combination adjuvant, in sensitized mice

This article was originally published online on 23 April 2013 Th1/Tr1 promoting adjuvants, in allergen-specific vaccines, could be a valuable alternative to the current allergen immunotherapy. It is now the pro-Th1 adjuvant effect of Neisseria meningitidis proteoliposome (PL). Previous studies in a prophylactic murine model of respiratory allergy the vaccine showed its immunogenicity and safety. To assess the immunogenicity and non-clinical safety profile of a novel anti-allergic vaccine candidate based on purified allergens from Dermatophagoides siboney and a combination adjuvant containing PL and Alum, in sensitized mice. In therapeutic experimental setting C57/Bl6 mice were first sensitized administering D. siboney allergen by IP route and exposing mice to allergen aerosols. Later, allergic mice were treated with 3 doses of the adjuvanted vaccine (2µg of Der s1) by subcutaneous route. Further, mice were subjected to inhalation allergen challenge. IgE, IgG1, and IgG2a allergen-specific antibody response was measured by ELISA. The specific cellular response (IL-13, IL-10 and INF-g), were evaluated in supernatants of Lymphocyte cultures stimulated with the allergen, by eBioscience kit for FACS. Systemic toxicity was assessed measuring body weight and macroscopic and histological examination of several organs and injection site. The experimental model reproduces the allergic condition with allergen-specific IgE production; increased blood eosinophils and Histology of lung tissues show all the characteristic of allergy inflammation. As in the previously tested preventive model, the vaccine induces a strong IgG and IgG1 antibody responses significantly higher than control groups, and moderate levels of IFN-g, although together with Tr1 (IL-10) and Th2 (Il-13) cytokines. After challenge, it was noted a significant increase (p<0.05) of the IgG/IgE ratio, with a decrease in bloods eosinophils and allergic inflammation in lung tissues as compared to placebo. No significant differences between body weights and differential leukocyte count in treated mice and placebo group was observed. Normal histology was observed. Local tolerance at the injection site suggests that granulomes in vaccinated subjects are caused by alum. The adjuvanted vaccine does not exacerbate the allergic response nor promote Th1 inflammation, supporting a satisfactory safety profile for further clinical trials in humans. This immunomodulatory effect suggests clinical benefits both in cellular and blocking antibody responses.

INTERLEUKIN -2 ( IL-2 ) AND GAMMA INTERFERON ( IFN γ ) OF LYMPHOCYTE CULTURE SUPERNATANT IN IRON DEFICIENCY ANEMIA PATIENTS WITH INFECTION

Iron is an essential nutrient for every living cells because of it role as molecule fortransport of oxygen, as well as DNA synthesis through synthesis of ribonucleotidereductase. Iron deficiency anemia patients, especially pregnant women and children aremore susceptible to infection because of deterioration of their immune response. This wassupported by findings of decreased in phagocytic activities of white blood cells and Tcelllymphocyte proliferation impairment. Iron deficiency anemia (IDA) patients alsoaffect working capacities hence diminishing working outcomes. Although the underlyingmechanism of immune defect in iron deficiency anemia is not clearly understood,multifactor events considered play their contributing roles such as abnormality ofribonucleotide reductase enzym, impairment of T-cell proliferation and activities, alteredcytokine production of IL-2 and IFNγ .The study was done to asses the relationship of IL-2 and gamma IFN withinfection in IDA patients on lymphocyte culture supernatant of IDA patients. Study wasconducted on cross-sectional analytic design. Sixty-four iron deficiency anemia patientstreated in Sanglah General Teaching Hospital were recruited, and 31 (48.4%) out of 64IDA patients were man and 33 (51.6%) women, have been selected for the study. Thisstudy found 17 (26.7%) IDA patients with infection, aged 38 ± 14.48 years and 47(73.3%) IDA patients without infection, with age average of 40.5 ± 14.4 years. Allvariables of data characteristics examined did not indicate any statistical significantdifference between group of IDA patients with infection and those without infection. Theaverage level of hemoglobin between the two groups did not differ statistically. Similarresult was obtained if samples were differentiated into severe (Hb&lt; 7g/dl) and mildanemia. The study also revealed that there were no differences of cytokine level observedbetween older and younger age (upper and below 44.5 years) in IDA patients withinfection and without infection. Furthermore, no differences of cytokine level were foundbased on gender between IDA male 10.9 (8.60 – 12,65) (pg/l) patients and IDA femalepatients 10.6 (7.50 – 13.43) (pg/l) with Z -0.490, p =0.624. Nevertheless, significantdifferences were noted between supernatant of IL-2 and IFNγ in IDA patients withinfection when compared to IDA patients without infection (Z= - 2.509, p= 0.012 forsupernatant IL-2; and Z= -2.569, p= 0.010 for supernatant IFNγ ).The study conclusion is that level of IL-2 and IFNγ from lymphocyte culturesupernatant of patient suffered from IDA with infection is significantly lower whencompared to IDA patient without infection. It therefore summarized that lower level ofIL-2 and gamma IFN in patients suffered from iron deficiency impaired their immune response to certain infections therefore this findings support the theory that IDA patientsmore susceptible to get infected.

T-regulatory cells in severe atopic dermatitis: alterations related to cytokines and other lymphocyte subpopulations

The changes in lymphocyte subpopulations in atopic dermatitis (AD) concern also T-regulatory cells. We investigated the expression of various surface receptors on CD3+CD4+CD25highFoxP3+ T-regulatory cells and the activation CD28+ receptor and the inhibitory CD152+ receptor on helper/inducer as well as cytotoxic/suppressor T cells. Peripheral blood lymphocytes of 15 AD patients and 20 healthy subjects were analyzed by flow cytometry using monoclonal antibodies. The concentrations of IL-6, IL-10 and TGF-β were determined in the serum and the supernatant of ConA-stimulated CD4+ lymphocytes. In AD patients the percentage of CD4+CD25highFoxP3+ as well as CD3+CD8+ cells increased, which positively correlated with SCORAD index (r = 0.55, p = 0.03). The concentrations of IL-10 in the CD4+ lymphocyte culture supernatants and the concentrations of TGF-β in the sera and the supernatant negatively correlated with the severity of AD (p < 0.01, r = −0.63; p < 0.02, r = −0.64 and p < 0.03, r = −0.58, respectively), whereas the serum concentration of IL-6 correlated positively (p < 0.003, r = 0.71). The regulatory cells expressed more CD62L and CD134 surface markers but less CD95. Reduced expression of the apoptotic CD95 receptor suggests that survival time of these cells is prolonged. Since CD62L and CD134 were upregulated, the enhanced modulatory effect of CD4+CD25highFoxP3+ cells seemed to be suggested, which may result in increased co-expression of CD28/CD152 on both CD4+ and CD8+ subpopulations.

Resveratrol modulates murine collagen-induced arthritis by inhibiting Th17 and B-cell function

BackgroundAlcohol intake is inversely related to rheumatoid arthritis (RA) disease incidence and severity. Resveratrol, a safe, well-described plant-derived compound, possesses anti-inflammation and immune-regulatory properties and is present in red wine....

薬剤アレルギー性肝内胆汁うっ滞症の胆汁うっ滞因子 (lymphokines) について

薬剤アレルギー性肝内胆汁うっ滞症の胆汁うっ滞因子 (lymphokines) について

Effects of Bifidobacterium bifidum on adaptive immune senescence in aging mice

Bifidobacteria are a natural component of the bacterial flora of the human body and have a symbiotic bacteria-host relationship with human beings. Aging is associated with reduced numbers of beneficial colonic Bifidobacteria and impaired immunity. The possible anti-senescence effects of Bifidobacteria are presently unknown. The aims of the present study were to investigate possible anti-senescence effects of B. bifidum on naturally senescent mice and to explore their mechanisms. After treatment with B. bifidum, mice were killed and samples collected. Cytokine production in serum and lymphocyte culture supernatant, anti-oxidation activity and gene expression were measured. B. bifidum significantly increased cytokine IL-2 and IFN-γ levels but decreased proinflammatory cytokines IL-6 and TNF-α concentrations. Moreover, B. bifidum improved anti-oxidation activity and reduced lipid peroxidation in thymus and spleen. In addition, B. bifidum down-regulated p16 expression in thymus and spleen. Taken together, the results indicate, for the first time, that B. bifidum delays senescence by several mechanisms, including enhancement of anti-oxidation activity in thymus and spleen, alteration of gene expression and improvement in immune function.

Abnormal regulation of soluble and anchored IL-6 receptor in monocytes from patients with essential thrombocythemia

In a previous study, we found increased plasma soluble receptor for interleukin-6 (sIL-6R) levels in patients with essential thrombocythemia (ET) that could promote megakaryopoiesis through IL-6 binding and further interaction with the signal transducer gp130. Here we have searched for the cell source of sIL-6R within mononuclear cells in these patients and the underlying abnormalities involved in its overproduction. Thirty patients with the diagnosis of ET were studied. sIL-6R levels were measured by enzyme-linked immunosorbent assay technique in the supernatants of peripheral monocyte and lymphocyte cultures. Expression of membrane-anchored IL-6R was determined by flow cytometry. In order to study the mechanism of sIL-6R production, tumor necrosis factor-α protease inhibitor was added to specifically block IL-6R shedding. Gene expression of sIL-6R levels were evaluated by reverse transcription polymerase chain reaction. Monocytes were the main source of sIL-6R. Besides, in ET patients, monocyte sIL-6R release was higher than that of controls (p = 0.0014). Lymphocytes enhanced monocyte sIL-6R production by cell-mediated contact in normal controls, but this cooperation could not be seen in patients. Membrane expression of IL-6R was increased after monocyte adhesion in ET. sIL-6R synthesis was upregulated in most patients, while messenger RNA was normal. Our results indicate that ET monocytes are responsible for sIL-6R overproduction within mononuclear cells through synthesis upregulation. In addition, the lack of cooperation of lymphocytes in monocyte sIL-6R production in ET could be due to a monocyte abnormality. The agonistic effect of sIL-6R on IL-6 action could contribute to the exacerbated megakaryocytic growth in ET.

Pathway-focused arrays reveal increased matrix metalloproteinase-7 (matrilysin) transcription in trachomatous trichiasis.

Several genes that are associated with protection from or susceptibility to trachomatous trichiasis (TT) have been identified through genetic association studies. Yet there have been few studies in which gene expression profiles were assessed in TT cases and disease-free controls. The purpose was to identify genes that are differentially expressed in the upper tarsal conjunctiva of subjects with TT. Pathway-focused gene arrays were used to screen conjunctival RNA expression of 226 gene transcripts of interest. The screening was followed by validation of differentially expressed genes by qRT-PCR on an independent set of samples. Three different techniques were then used to test for quantitative differences in the recovered conjunctival protein fraction. Focused arrays identified a set of 13 differentially expressed genes. Validation by qRT-PCR confirmed differential expression in four of these genes (COL1A1, COL7A1, MMP7, and TLR6). Increased expression of MMP7 was the only consistent differentially regulated gene in the conjunctival samples of trichiasis subjects. MMP7 was present in isolated conjunctival proteins and in the tissue culture supernatants of peripheral blood lymphocytes after stimulation. There is an imbalance in extracellular matrix turnover with minimal contribution of adaptive immune responses at this stage of trichiasis. There was little evidence of broad differential expression in genes characteristic of polar responses of adaptive T cells or macrophages. The control of the MMP7 response and its activity appears significant in the fibrotic changes observed in TT.

Immune response toPlasmodium vivaxhas a potential to reduce malaria severity

Plasmodium falciparum infection causes transient immunosuppression during the parasitaemic stage. However, the immune response during simultaneous infections with both P. vivax and P. falciparum has been investigated rarely. In particular, it is not clear whether the host's immune response to malaria will be different when infected with a single or mixed malaria species. Phenotypes of T cells from mixed P. vivax-P. falciparum (PV-PF) infection were characterized by flow cytometry, and anti-malarial antibodies in the plasma were determined by an enzyme-linked immunosorbent assay. We found the percentage of CD3+delta2+-T cell receptor (TCR) T cells in the acute-mixed PV-PF infection and single P. vivax infection three times higher than in the single P. falciparum infection. This implied that P. vivax might lead to the host immune response to the production of effector T killer cells. During the parasitaemic stage, the mixed PV-PF infection had the highest number of plasma antibodies against both P. vivax and P. falciparum. Interestingly, plasma from the group of single P. vivax or P. falciparum malaria infections had both anti-P. vivax and anti-P. falciparum antibodies. In addition, antigenic cross-reactivity of P. vivax or P. falciparum resulting in antibodies against both malaria species was shown in the supernatant of lymphocyte cultures cross-stimulated with either antigen of P. vivax or P. falciparum. The role of delta2 +/- TCR T cells and the antibodies against both species during acute mixed malaria infection could have an impact on the immunity to malaria infection.

Immune responses induced by recombinant major outer membrane protein of Chlamydia trachomatis serovar E.

Objective To investigate specific immune responses in mice induced by recombinant major outer membrane protein(rMOMP)of C.trachomatis serovaf E.Methods Thirty-six female BALB/cmice aged 3 to 4 weeks Were divided into three groups.i.e.,adjuvant group vaccinated、with purified rMOMP and Freund's adjutant,solitary group vaccinated with rMOMP only and control group vaccinated with phosphate buffered saline(PBS).All the mice were intramuscularly vaccinated on week 0,2 and 4.Blood samples and vaginal washes were obtained from these mice on week 6,then,mice were challenged with elementary body(EB)of C.trachomatis serovar E at the footpad followed by the observation of delayed hypersensitivity.On week 7.mice were genitally infected with C.trachomatis EB;one week later,blood samples and vaginal washes were obtained again;six weeks later,spleen lymphocytes were isolated from the mice and stimulated bv C.trachomatis or ConA followed by the detection of cell proliferation with MTT assay.In vitro neutralization assay was also performed.ELISA was used to determine the titers of Chlamydia-specific IgO antibody in sera and IgA antibody in vaginal washes,as well as the level of IFN-γ in culture supernatant of lymphocytes and sefa of mice.Vaginal swabs were collected after genital challenge and subjected to C.trachomatis culture.Results The absorbance at 405 ms of Chlamydia-specific IgG antibody and proliferation index of lymphocytes were 0.641±0.059 and 5.085±1.291.respectively,in mice immunized with rMOMP and Frennd's adjuvant.significantly higher than those in mice immunized with rMOMP only(0.424±0.015 and 3.123 ±0.840.both P<0.05).The thickness of right hind footpad increased by 0.324±0.054 mm and 0.272±0.064 mm,respectively,in solitary group and adjuvant group,respectively,with significant difference between the two groups(P<0.05).A significant increase was also observed in the adjuvant group compared with the control group in the above three parameters(all P<0.01).Conclusion The rMOMP of C.trachomatis could efficiently induce Chlamydia-specific humoml and cellular immune responses in mice. Key words: Cldamydia tmchomafis; Bacterial outer membrane proteins; Vaccines; Immunity

Generation of mesenchymal stromal cells in the presence of platelet lysate: a phenotypic and functional comparison of umbilical cord blood- and bone marrow-derived progenitors

Mesenchymal stromal cells are employed in various different clinical settings in order to modulate immune response. However, relatively little is known about the mechanisms responsible for their immunomodulatory effects, which could be influenced by both the cell source and culture conditions. We tested the ability of a 5% platelet lysate-supplemented medium to support isolation and ex vivo expansion of mesenchymal stromal cells from full-term umbilical-cord blood. We also investigated the biological/functional properties of umbilical cord blood mesenchymal stromal cells, in comparison with platelet lysate-expanded bone marrow mesenchymal stromal cells. The success rate of isolation of mesenchymal stromal cells from umbilical cord blood was in the order of 20%. These cells exhibited typical morphology, immunophenotype and differentiation capacity. Although they have a low clonogenic efficiency, umbilical cord blood mesenchymal stromal cells may possess high proliferative potential. The genetic stability of these cells from umbilical cord blood was demonstrated by a normal molecular karyotype; in addition, these cells do not express hTERT and telomerase activity, do express p16(ink4a) protein and do not show anchorage-independent cell growth. Concerning alloantigen-specific immune responses, umbilical cord blood mesenchymal stromal cells were able to: (i) suppress T- and NK-lymphocyte proliferation, (ii) decrease cytotoxic activity and (iii) only slightly increase interleukin-10, while decreasing interferon-gamma secretion, in mixed lymphocyte culture supernatants. While an indoleamine 2,3-dioxygenase-specific inhibitor did not reverse mesenchymal stromal cell-induced suppressive effects, a prostaglandin E(2)-specific inhibitor hampered the suppressive effect of both umbilical cord blood- and bone marrow-mesenchymal stromal cells on alloantigen-induced cytotoxic activity. Mesenchymal stromal cells from both sources expressed HLA-G. Umbilical cord blood- and bone marrow-mesenchymal stromal cells may differ in terms of clonogenic efficiency, proliferative capacity and immunomodulatory properties; these differences may be relevant for clinical applications.

The Th1-promoting effects of dehydroepiandrosterone can provide an explanation for the stronger Th1-immune response of women.

Estrogens foster immunological processes driven by CD4+ Th2 cells and B cells and androgens foster Th1 CD4+ and CD8+ cell activity. Higher levels of IFN-gamma and IL-2 and lower levels of IL-4 and IL-10 are detected in the phytohemagglutinin-stimulated lymphocyte culture supernatants of men compared with women. It is documented that the physiologic levels of estrogens produced during the luteal phase of the menstrual cycle shift the female immune system toward a Th2-type response and that the Th1 cytokines are increased in postmenopausal women. However, the Th1 immune response is also surprisingly stronger in women, hence affording them a better protection against infections. Nickel sensitivity, a Th1 immune reaction, seems to be more common in women even if men wear earrings. Further, not only the Th2 but also the Th1 autoimmune diseases are generally more common in women than men. How do women advance a stronger Th1 response than men? It is suggested that in contrast to the paradigm that estrogens lead to a Th2 bias, estrogens can enhance Th1 cytokine production also. However, the discrepant effects of estrogens are difficult to be reconciled from a molecular viewpoint and hence are not advocated by all authors. This paper provides an explanation: The effects of dehydroepiandrosterone on Th1/Th2 balance seem to be model-specific; in humans dehydroepiandrosterone, represents a pivotal up-regulator of Th1 immune response. Steroid sulphatase is a microsomal enzyme that cleaves the sulphate group of dehydroepiandrosterone sulphate. This enzyme is controlled by an X-linked gene that escapes the Lyon effect of X-inactivation; as a result, women usually have about twice steroid sulphatase in their cells, including macrophages, as have men. Putting all these facts together, it could be concluded that women's macrophages, which contain higher steroid sulphatase levels and enter peripheral lymphoid organs through afferent lymphatic drainage, produce higher levels of dehydroepiandrosterone in these organs; and higher levels of this hormone produce stronger Th1 immune responses.

Mesenchymal Stems Cells and Regulatory T Cells Inhibit Alloantigen Mediated Graft-Versus-Host Reaction but Also Impair Graft-Versus-Tumor Reactions Targeting Tumor Associated Antigens

Graft-versus-host disease (GvHD) is a common and potentially life-threatening complication after allogeneic hematopoietic stem cell transplantation (HSCT). It is mediated by donor immune cells reacting against alloantigens presented by host or donor derived professional antigen presenting cells or expressed on host tissue. In addition, donor derived T-cells contribute to the beneficial graft-versus-tumor (GvT) reaction targeting minor histocompatibility antigens and, as previously demonstrated (Stelljes et al., Blood 2004; 104:1210-6), tumor associated antigens (TAAs). Several publications have shown that regulatory T-cells (Tregs) and mesenchymal stem cells (MSCs) can be used to prevent or treat GvHD. Since both cell populations react in a largely unspecific manner on alloantigen mediated immune responses, it remains a matter of debate whether intervention with these cell populations might interfere with GvT reactions directed against TAAs. In order to investigate the effects of MSCs and Tregs on GvHD and tumor specific GvT reactions, we used a murine parent-into-F1 transplant model. After total body irradiation with 9 Gy F1[BALB/c × C57BL/6] (CB6F1) mice were transplanted with parental bone marrow and splenocytes from C57BL/6 (B6) or BALB/c donors, respectively. Following transplantation, CB6F1 recipients received either three doses of 1×106 MSCs of B6 or BALB/c origin s.c. within the first two weeks or 1×106 Tregs together with the transplant. To evaluate TAA specific GvT effects, transplanted recipients were inoculated with tumor cells sharing the MHC background of the donor strain. GvHD score and tumor size were monitored every other day. Treatment with unstimulated MSCs of B6 origin had only marginal effects on GvHD intensity. In contrast, mice treated with unstimulated MSCs of BALB/c origin or BL/6 MSCs stimulated in vitro with supernatant of allogeneic mixed lymphocyte cultures, showed no signs of GvHD like weight loss or skin damage. TAA specific GvT reactions were significantly impaired by MSCs of either origin resulting in markedly increased tumor growth. Analysis of in vivo primed T cells 25 days after transplantation by IFN-γ ELIspot assay revealed significantly reduced TAA specific T-cells in recipients treated with MSCs as compared to transplanted tumor bearing control animals. In our experimental setting, Tregs also reduced GvHD compared with transplanted controls. Again, specific immune responses targeting TAAs were also abolished. In conclusion, cellular immune modulation using MSCs or Tregs might be effective to modulate GvHD. However, both strategies clearly impair tumor specific GvT reactions in our experimental setting suggesting that these interventions might bear the risk of higher relapse rates after allogeneic HSCT. The different effects on GvHD by stimulated and unstimulated MSCs of B6 or BALB/c origin are being explored.

Suppression of lymphokine-activated killer (LAK) cell induction mediated by interleukin-4 and transforming growth factor-β1: effect of addition of exogenous tumour necrosis factor-alpha and interferon-gamma, and measurement of their endogenous production

Recombinant human interleukin-4 (rhIL-4) and transforming growth factor-beta 1 (TGF-beta 1) suppressed the induction of lymphokine-activated killer (LAK) activity induced by recombinant human interleukin-2 (rhIL-2) in peripheral blood lymphocytes. DNA synthesis and the expression of the p55 alpha chain of the IL-2 receptor (Tac antigen) were also inhibited. The inhibitory effect was greatest when these factors were added during the first 48 h of a 4-day culture, with reduced cytolytic activity against both natural killer (NK) resistant and NK-sensitive tumour cell line targets. The suppressive action of both cytokines was accompanied by a reduction in tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) levels in lymphocyte culture supernatants. Recombinant human IFN-gamma (rhIFN-gamma), but not recombinant human TNF-alpha (rhTNF-alpha) was able to overcome the inhibitory effect of recombinant human interleukin-4 (rhIL-4) on LAK induction and DNA synthesis but not Tac antigen expression. However, cytotoxicity induced by rhIFN-gamma alone was also suppressed by rhIL-4 and TGF-beta 1, inferring that rhIFN-gamma-mediated abrogation of rhIL4 suppression was not simply a direct IL-2-independent effect on cytotoxicity. In addition, rhIL-4 did not increase TGF-beta production from rhIL-2-activated peripheral blood mononuclear cells, suggesting that rhIL-4 did not mediate reduction of rhIL-2 responses through the induction of TGF-beta release.

Expression of α-AR Subtypes in T Lymphocytes and Role of the α-ARs in Mediating Modulation of T Cell Function

Objectives: Previous work in our laboratory has shown that α-adrenoreceptors (α-ARs) and β-ARs exist on lymphocytes from functional profile, and that the receptors mediate the regulation of lymphocyte function by catecholamines. In the present study, we directly examined the expression of α-AR subtypes, α₁-AR and α₂-AR mRNAs, in T lymphocytes and explored the roles of the α-AR subtypes and intracellular signal transduction mechanisms linked to the receptors in mediating the modulation of T lymphocyte function. Methods: T lymphocytes from mesenteric lymph nodes of rats were purified by using a nylon wool column. Reverse transcription polymerase chain reaction was used to detect the expression of α₁-AR and α₂-AR mRNAs in the freshly isolated T cells and the mitogen concanavalin A (Con A)-activated lymphocytes. Colorimetric methylthiazoletetrazolium assay was employed to measure lymphocyte proliferation induced by Con A. Interferon-γ (IFN-γ) and interleukin-4 (IL-4) levels in the Con A-stimulated lymphocyte culture supernatants were examined by enzyme-linked immunosorbent assay. Results: T cells expressed both α₁-AR and α₂-AR mRNAs. The expression of both α₁-AR and α₂-AR mRNAs was significantly higher in the Con A-activated lymphocytes than in the resting lymphocytes. Phenylephrine, a selective α₁-AR agonist, had no evident effect on lymphocyte proliferation nor on IFN-γ and IL-4 production induced by Con A. However, the selective α₂-AR agonist clonidine attenuated Con A-induced lymphocyte proliferation as well as IFN-γ and IL-4 production. The inhibited lymphocyte proliferation and IFN-γ and IL-4 production by clonidine were blocked by yohimbine, an α₂-AR antagonist. Either phospholipase C inhibitor U-73122 or protein kinase C inhibitor chelerythrine partially prevented the suppressive effect of clonidine on Con A-stimulated lymphocyte proliferation and IL-4 production. Conclusions: T lymphocytes express both α₁-ARs and α₂-ARs, but only the α₂-ARs participate in the suppressive modulation of lymphocyte proliferation and cytokine production in vitro. The inhibitory effect of α₂-AR stimulation on lymphocyte function is partially mediated via the phospholipase C-protein kinase C pathway.

Pattern of Macrophage Activation in Yersinia‐Resistant and Yersinia‐Susceptible Strains of Mice

Th1 cells, in cooperation with activated macrophages, are required to overcome Yersinia enterocolitica infection in mice. The pathway macrophages utilize to metabolize arginine can alter the outcome of inflammation in different ways. The objective of this study was to verify the pattern of macrophages activation in Y. enterocolitica infection of BALB/c (Yersinia-susceptible) and C57BL/6 (Yersinia-resistant) mice. Both strains of mice were infected with Y. enterocolitica O:8 WA 2707. Peritoneal macrophages and spleen cells were obtained on the 1st, 3rd and 5th day post-infection. The iNOS and the arginase activities were assayed in supernatants of macrophage cultures, by measuring their NO/citrulline and ornithine products, respectively. TGFbeta-1 production was also assayed. The Th1 and Th2 responses were evaluated in supernatants of lymphocyte cultures, by IFN-gamma and IL-4 production. Our results showed that in the early phase of Y. enterocolitica infection (1st and 3rd day), the macrophages from C57BL/6 mice produced higher levels of NO/citrulline and lower levels of ornithine than macrophages from BALB/c mice. The infection with Y. enterocolitica leads to an increase in the TGF-beta1 and IL-4 production by BALB/c mice and to an increase in the IFN-gamma levels produced by C57BL/6 mice. These results suggest that Y. enterocolitica infection leads to the modulation of M1 macrophages in C57Bl/6 mice, and M2 macrophages in BALB/c mice. The predominant macrophage population (M1 or M2) at the 1st and 3rd day of infection thus seems to be important in determining Y. enterocolitica susceptibility or resistance.

TNF‐α from monocyte of patients with pre‐eclampsia‐induced apoptosis in human trophoblast cell line

In pre-eclampsia, fetal growth restriction is frequently observed, and the possible involvement of inhibitory substances on trophoblast cell proliferation and differentiation has been suggested. The objective of this study was to investigate the effects humoral factors, such as cytokines, produced in immune cells on proliferation of an immortalized trophoblastic cell line (TCL) that we established. Serum and lymphocyte layers were isolated from the blood of normal pregnant and preeclamptic women. The lymphocyte layer was further fractionated into different immune cell populations by the Stem Sep method. Immortalized trophoblastic cells were cultured with the sera diluted. The cytokine concentrations in the supernatants of lymphocyte cultures were compared between normal pregnancy and pre-eclampsia. The number, DNA content and induced apoptosis were examined on the immortalized trophoblastic cells at the end of culture. The sera from preeclamptic women significantly inhibited the immortalized trophoblastic cell proliferation in comparison with those from normal pregnancy. Among the lymphocyte fractions, monocytes significantly inhibited the immortalized trophoblastic cell proliferation. The monocytes from preeclamptic women were found to produce higher levels of tumor necrosis factor-alpha (TNF-alpha) in the culture supernatant than those from normal pregnant women. The coculture with the monocytes from preeclamptic women increased the frequency of TUNEL-positive TCL cells. TNF-alpha inhibited immortalized trophoblastic cell proliferation in a dose-dependent manner and induced apoptosis. The present results suggest that monocytes are activated and that cytokines, such as TNF-alpha, which is produced by monocytes, induce apoptosis and inhibit proliferation of trophoblast cells in pre-eclampsia.

Alloanatigenic Reactions after Hematopoietic Cell Transplantation Induce Genomic Alterations in Epithelial Cells as Shown in Human Studies and in an In Vitro Model.

Previous studies from our group demonstrated frequent genomic alterations measured by microsatellite instability (MSI) in non-neoplastic epithelial tissues of patients who underwent allogeneic hematopoietic cell transplantation (HCT) (Blood 2006; 107:3389–3396). These genomic alterations were found only after allogeneic but not after autologous HCT, and therefore we hypothesized that an “allogeneic” effect is substantially involved in the mutation process. We extended our previous analyses by examining 210 bucall swabs obtained from 70 patients between day (d+) 26 and d+3514 after allogeneic HCT for the presence of MSI. MSI analysis was performed by PCR and denaturing capillary electrophoresis at three tetranucleotide (THO-1, SEE33, D14S120) and three mononucleotide microsatellite (ZP3, BAT26, SRY) loci. MSI was found in the buccal smears of 38% allografted patients (median time of occurence 322 days). In a prospective trial, in which patients were followed from time before transplantation until d+365, 5 out of 14 (35%) patients exhibited MSI after transplantation although all of them showed stable microsatellites before transplantation. We are currently pefroming statistical analyses in order to identify which clinical factors influence the presence of MSI and we will present the data in the meeting. To test the hypothesis that an “alloantigenic” effect is responsible for th induction of MSI, we developed a model system in which keratinocyte (HaCaT) cells were transfected with a palsmid vector which carries a G418 (neo) selectable marker and a microsatellite repeat (CA) that places the sequence for Hygromycin Resistance (HygR) out of frame for protein translation. In this reporter system, DNA slippage mutations can restore the HygR reading frame and become detectable by hygromycin treatment as hygromycin resistant (HygR+) colonies. Pools of stably transfected (neo+) HaCaT cells were treated with supernatant (SN) of major histocompatibility complex nonmatched mixed lymphocyte cultures (MLC) and assayed for HygR+ colonies 48h later. Cells transfected with a control, in-frame hygromycin B gene construct (p12) were used as positive controls. Using this system, we found that HaCaT cells aquire hygromycin resistance after treatment with supernatatant from MLC. Treatment of cells with hydrogen hyperoxid which has been shown in a E. Coli system to induce MSI (PNAS 1998, 95:12468–12473) generated HygR+ colonies at a >80% lower frequency than the SN-MLC treatment. Control p12 transfected cells were grown with high efficiency in the presence of hygromycin B. In summary, our in vivo data confirm our previous results and provide evidence of genomic alterations after allogeneic HCT and our in vitro data are compatible with the hypothesis that an “alloantigenic” factor is the driving force in producing detectable MSI in the allografted patients. Elucidating the ultimate mechanisms underlying the genomic instability following allogeneic HCT may prove to be of major therapeutic value.

Wnt-10b secreted from lymphocytes promotes differentiation of skin epithelial cells

Wnt-10b was originally isolated from lymphoid tissue and is known to be involved in a wide range of biological actions, while recently it was found to be expressed early in the development of hair follicles. However, few studies have been conducted concerning the role of Wnt-10b with the differentiation of skin epithelial cells. To evaluate its role in epithelial differentiation, we purified Wnt-10b from the supernatant of a concanavalin A-stimulated lymphocyte culture using an affinity column and investigated its effects on the differentiation of adult mouse-derived primary skin epithelial cells (MPSEC). MPSEC cultured with Wnt-10b showed morphological changes from cuboidal to spindle-shaped with inhibited proliferation, and also obtained characteristics of the hair shaft and inner root sheath of the hair follicle, represented by red-colored Ayoub Shklar staining, and reactions to AE-13 and AE-15 as seen with immunocytology. Further, RT-PCR analysis demonstrated the expression of mRNA for keratin 1, keratin 2, loricrin, mHa5, and mHb5, in association with a decreased expression of the basal cell marker keratin 5, in Wnt-10b-treated MPSEC. In addition, involvement of the canonical Wnt signal pathway was demonstrated by a TCF reporter (pTOPFLASH) assay. These results suggest that Wnt-10b promotes the differentiation of MPSEC and may play an important role in hair follicle development by promoting differentiation of epithelial cells.