Mitogen-stimulated Lymphocytes Research Articles

Evaluation of the quality of life-enhancing effect of allogeneic feline adipose mesenchymal stem cells in cats with osteoarthritis: A pilot study

Osteoarthritis (OA) is a progressive degenerative disease in older cats, and often leads to decreased quality of life (QOL). Mesenchymal stem cells (MSCs) have been used in novel therapies for inflammatory diseases. We aimed to evaluate quantitatively allogeneic adipose-derived MSC (ADSC) therapy in cats with naturally occurring OA, based on QOL assessment resources. To characterize the in vitro properties of ADSCs, we estimated ADSCs from four healthy cats with respect to morphology, differentiation potential, and immunomodulatory potential. Six cats with OA were administered a single intravenous injection of allogeneic ADSCs. Based on the feline musculoskeletal pain index (FMPI), the outcome measure was QOL. The cultured cells were adherent, exhibited a spindle shape without becoming flattened or large, and maintained doubling time until passage 5. After induction, the cells had osteogenic, adipogenic, and chondrogenic phenotypes. These cells expressed CD44 and CD90 and lacked expression of CD14 and CD45, had significantly suppressed the production of interferon -ɤ released from mitogen-stimulated lymphocytes (P < 0.05). The FMPI of all cats with OA significantly increased one month after ADSC therapy (P < 0.05). No adverse effects associated with ADSC administration were observed during follow-up in any of the cats. In conclusion, ADSC therapy with immunomodulatory potential could have beneficial effects on the QOL in cats with OA. Further research is necessary to carry out larger studies of the effectiveness of ADSC therapy.

Prenatal blood metals, per- and polyfluoroalkyl substances and antigen- or mitogen-stimulated cord blood lymphocyte proliferation and cytokine secretion

Prenatal blood metals, per- and polyfluoroalkyl substances and antigen- or mitogen-stimulated cord blood lymphocyte proliferation and cytokine secretion

Prenatal blood metals, per- and polyfluoroalkyl substances and antigen- or mitogen-stimulated cord blood lymphocyte proliferation and cytokine secretion

BackgroundEvidence suggests that prenatal per- and polyfluoroalkyl substances (PFAS) and metals, two classes of chemicals found ubiquitously in human populations, influence immune system development and response. ObjectiveWe evaluated whether first trimester blood PFAS and metals were associated with antigen- or mitogen-stimulated cord blood lymphocyte proliferation and cytokine secretion. MethodsWe measured six PFAS, as well as six nonessential and four essential metals, in first trimester blood from participants in the longitudinal pre-birth Project Viva cohort, recruited between 1999 and 2000 in eastern Massachusetts. We measured antigen- or mitogen-stimulated cord blood mononuclear cell proliferation responses (n = 269–314) and cytokine secretion (n = 217–302). We used covariate-adjusted least absolute shrinkage and selection operator (LASSO) for variable selection and multivariable regression to estimate associations with the immune markers. ResultsEach ng/mL of MeFOSAA was associated with a 3.6% (1.4, 5.8) higher lymphocyte proliferation response after stimulation with egg antigen, as well as 0.8 (0.7, 1.0) reduced odds of having IFN-γ detected in response to dust mite. Each ng/g increment of cesium was associated with 27.8% (−45.1, −4.9) lower IL-10 levels in response to dust mite. Each ng/g increment of mercury was associated with 12.0% (1.3, 23.8) higher IL-13 levels in response to mitogen PHA. Each ng/g increment of selenium and zinc was associated with 0.2% (0.01, 0.4) and 0.01% (0.002, 0.02) higher TNF-α in response to mitogen PHA, respectively. ConclusionsPrenatal metals and PFAS influence cord blood lymphocyte proliferation and cytokine secretion in ways that may increase risk for atopic disease in childhood.

Antitubercular and immunomodulatory activities of Eugenia astringens n-hexane fraction

BackgroundIn Brazil, the Eugenia genus is traditionally used to treat several types of human diseases, including diabetes, gastric ulcers and rheumatoid arthritis. Previous studies demonstrated that medicinal properties of Eugenia astringens Cambess (Myrtaceae), popularly known as “baguaçu" in Brazil, were associated with anti-inflammatory and antimicrobial activities exhibited by the plant extracts. Anti-tubercular effects of E. astringens have not been elucidated yet. Aim of the studyTo study anti-tubercular and immunomodulatory potential of the E. astringens leaves extracts (syn. E. umbelliflora O. Berg), as well as the chemical profiles of active fractions. MethodsThe plant material was collected in the Parque Nacional da Restinga de Jurubatiba (Rio de Janeiro) and the leaves were dried and extracted with ethanol. The extracts were partitioned, to yield n-hexane (Hex), dichloromethane (DCM), ethyl acetate (EtOAc) and n-butanol (BuOH) fractions. The crude extract and fractions were evaluated for antitubercular effects in broth cultures of M. tuberculosis strains and assessed for cytotoxicity in mammalian cell cultures by the MTT method. The Hex fraction, exhibiting low cytotoxicity and stronger inhibitory effect on mycobacterial growth relative to other fractions, was selected for the further studies. Immunomodulatory effects of the Hex fraction were evaluated in the RAW 264.7 macrophages, stimulated by LPS to produce inflammatory mediators, such as TNF-α, IL-6 and nitric oxide. Additionally, the effects on mitogen-stimulated human lymphocyte functions were estimated by measuring cell proliferation and IFN-γ production. ResultsThe E. astringens Hex fraction inhibited growth of mycobacterial strains both in the bacterial culture and infected macrophages, demonstrating strong anti-tubercular potential. In addition, this fraction suppressed secretion of inflammatory cytokines and reduced production of nitric oxide through inhibition of iNOS expression in the LPS-stimulated macrophages. In the mitogen-stimulated lymphocyte assay, Hex fraction inhibited cell proliferation but did not alter IFN-γ production. GC–MS analyses showed that the Hex fraction is composed by caryophyllene-type, eudesmane-type, cadinene-type, and aromadendrene-type sesquiterpenes; and 28 compounds were identified, including spathulenol, previously described for antitubercular acitivity. It is possible that the overall biological efficiency of the Hex fraction depends on the cumulative and/or synergistic effect of these sesquiterpenes. ConclusionThe Hex fraction of E. astringens leaves showed strong antitubercular and immunomodulatory activities, demonstrating pharmacological potential of the identified compounds for the development of new anti-TB drugs.

Duck Interleukin-22: Identification and Expression Analysis in Riemerella anatipestifer Infection.

Riemerella anatipestifer is one of the most devastating pathogens affecting the global duck farms. Infection is involved in secretion of proinflammatory cytokines, including interleukin- (IL-) 17A. During the immune response to infection, IL-22 and IL-17A are often produced concurrently and at high levels in inflamed tissues. Little is known about duck IL-22 (duIL-22) during R. anatipestifer infection. We describe the characterization of duIL-22 and its mRNA expression analysis in splenic lymphocytes and macrophages treated with heat-killed R. anatipestifer and in the spleens and livers of R. anatipestifer-infected ducks. Full-length cDNA of duIL-22 encoded 197 amino acids. The deduced amino acid sequence of duIL-22 shared a 30.4–40.5% similarity with piscine counterparts, 57.4–60.1% with mammalian homologs, and 93.4% similarity to the chicken. Duck IL-22 mRNA expression level was relatively high in the skin of normal ducks. It was increased in mitogen-stimulated splenic lymphocytes and in killed R. anatipestifer-activated splenic lymphocytes and macrophages. Compared with healthy ducks, IL-22 transcript expression was significantly upregulated in the livers and spleens on days 1 and 4 postinfection, but not on day 7. IL-17A was significantly increased in the spleens only on day 4 postinfection and in the livers at all time points. When splenic lymphocytes were stimulated with heat-killed R. anatipestifer, CD4+ cells predominantly produced IL-22 while IL-17A was expressed both by CD4+ and CD4− cells. These results suggested that IL-22 and IL-17A are likely expressed in different cell types during R. anatipestifer infection.

Long-Term Trypsin Treatment Promotes Stem Cell Potency of Canine Adipose-Derived Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) isolated from adipose tissue (adipose-derived stem cells [ADSCs]) are considered one of the most promising cell types for applications in regenerative medicine. However, the regenerative potency of ADSCs may vary because of heterogeneity. Long-term trypsin treatment (LTT) is known to significantly concentrate multilineage-differentiating stress-enduring (Muse) cells from human MSCs. In this study, we aimed to generate cells with high stem cell potency from canine ADSCs using LTT. After 16 h of treatment with trypsin, surviving ADSCs (LTT-tolerant cells) had significantly enhanced expression of stage-specific embryonic antigen (SSEA)-1, a mouse embryonic stem cell marker, and fucosyltransferase 9, one of several fucosyltransferases for SSEA-1 biosynthesis. However, LTT-tolerant cells did not enhance the expression of SSEA-3, a known human Muse cell marker. LTT-tolerant cells, however, showed significantly higher self-renewal capacity in the colony-forming unit fibroblast assay than ADSCs. In addition, the LTT-tolerant cells formed cell clusters similar to embryoid bodies and expressed undifferentiated markers. Moreover, these cells differentiated into cells of all three germ layers and showed significantly higher levels of α 2-6 sialic acid (Sia)-specific lectins, known as differentiation potential markers of human MSCs, than ADSCs. LTT-tolerant cells had a normal karyotype and had low telomerase activity, showing little carcinogenetic potency. LTT-tolerant cells also showed significantly increased activity of transmigration in the presence of chemoattractants and had increased expression of migration-related genes compared with ADSCs. In addition, LTT-tolerant cells had stronger suppressive activity against mitogen-stimulated lymphocyte proliferation than ADSCs. Overall, these results indicated that the LTT-tolerant cells in canine ADSCs have similar properties as human Muse cells (although one of the undifferentiated markers is different) and are expected to be a promising tool for regenerative therapy in dogs.

Pluripotency and immunomodulatory signatures of canine induced pluripotent stem cell-derived mesenchymal stromal cells are similar to harvested mesenchymal stromal cells

With a view towards harnessing the therapeutic potential of canine mesenchymal stromal cells (cMSCs) as modulators of inflammation and the immune response, and to avoid the issues of the variable quality and quantity of harvested cMSCs, we examined the immunomodulatory properties of cMSCs derived from canine induced pluripotent stem cells (ciMSCs), and compared them to cMSCs harvested from adipose tissue (cAT-MSC) and bone marrow (cBM-MSC). A combination of deep sequencing and quantitative RT-PCR of the ciMSC transcriptome confirmed that ciMSCs express more genes in common with cBM-MSCs and cAT-MSCs than with the ciPSCs from which they were derived. Both ciMSCs and harvested cMSCs express a range of pluripotency factors in common with the ciPSCs including NANOG, POU5F1 (OCT-4), SOX-2, KLF-4, LIN-28A, MYC, LIF, LIFR, and TERT. However, ESRRB and PRDM-14, both factors associated with naïve, rather than primed, pluripotency were expressed only in the ciPSCs. CXCR-4, which is essential for the homing of MSCs to sites of inflammation, is also detectable in ciMSCs, cAT- and cBM-MSCs, but not ciPSCs. ciMSCs constitutively express the immunomodulatory factors iNOS, GAL-9, TGF-β1, PTGER-2α and VEGF, and the pro-inflammatory mediators COX-2, IL-1β and IL-8. When stimulated with the canine pro-inflammatory cytokines tumor necrosis factor-α (cTNF-α), interferon-γ (cIFN-γ), or a combination of both, ciMSCs upregulated their expression of IDO, iNOS, GAL-9, HGF, TGF-β1, PTGER-2α, VEGF, COX-2, IL-1β and IL-8. When co-cultured with mitogen-stimulated lymphocytes, ciMSCs downregulated their expression of iNOS, HGF, TGF-β1 and PTGER-2α, while increasing their expression of COX-2, IDO and IL-1β. Taken together, these findings suggest that ciMSCs possess similar immunomodulatory capabilities as harvested cMSCs and support further investigation into their potential use for the management of canine immune-mediated and inflammatory disorders.

M230 EVALUATION OF IMMUNE DEFICIENCY IN A 72 YEAR OLD PATIENT WHO LIVED BY CHERNOBYL

This is a 72-year-old Russian female with history of multiple cancers and recently with recurrent infection. Patient was referred by infectious disease due to recurrent lung infection and cellulitis of unknown etiology. History was negative for recurrent childhood infections or family members with recurrent infection. This case was done by telemedicine, so no exam was able to be done. In reviewing the medical records, the chest CT showed some non-specific lesions and possible bronchiectasis. Her labs showed IGG1 subtype deficiency. In the history. I asked where she immigrated from in Russia and she said she lived near Chernobyl in 1986 during the nuclear reactor melt down. Our research revealed that long term immune system changes have been shown to occur following high dose radiation exposure including antibody deficiency and reduction of lymphocyte function. We did further labs including Lymphocyte Mitogen Stimulation with multiple agents since low response was reported as a sequelae of high dose radiation.

Cytogenetically-based biodosimetry after high doses of radiation.

Dosimetry is an important tool for triage and treatment planning following any radiation exposure accident, and biological dosimetry, which estimates exposure dose using a biological parameter, is a practical means of determining the specific dose an individual receives. The cytokinesis-blocked micronucleus assay (CBMN) is an established biodosimetric tool to measure chromosomal damage in mitogen-stimulated human lymphocytes. The CBMN method is especially valuable for biodosimetry in triage situations thanks to simplicity in scoring and adaptability to high-throughput automated sample processing systems. While this technique produces dose-response data which fit very well to a linear-quadratic model for exposures to low linear energy transfer (LET) radiation and for doses up for 5 Gy, limitations to the accuracy of this method arise at larger doses. Accuracy at higher doses is limited by the number of cells reaching mitosis. Whereas it would be expected that the yield of micronuclei increases with the dose, in many experiments it has been shown to actually decrease when normalized over the total number of cells. This variation from a monotonically increasing dose response poses a limitation for retrospective dose reconstruction. In this study we modified the standard CBMN assay to increase its accuracy following exposures to higher doses of photons or a mixed neutron–photon beam. The assay is modified either through inhibitions of the G2/M and spindle checkpoints with the addition of caffeine and/or ZM447439 (an Aurora kinase inhibitor), respectively to the blood cultures at select times during the assay. Our results showed that caffeine addition improved assay performance for photon up to 10 Gy. This was achieved by extending the assay time from the typical 70 h to just 74 h. Compared to micronuclei yields without inhibitors, addition of caffeine and ZM447439 resulted in improved accuracy in the detection of micronuclei yields up to 10 Gy from photons and 4 Gy of mixed neutrons-photons. When the dose-effect curves were fitted to take into account the turnover phenomenon observed at higher doses, best fitting was achieved when the combination of both inhibitors was used. These techniques permit reliable dose reconstruction after high doses of radiation with a method that can be adapted to high-throughput automated sample processing systems.

Level of Cyclin D1 protein in peripheral blood lymphocytes of Chornobyl clean-up workers in remote period after radiation exposure

Aim: to assess changes of products of Cyclin D1 protein in lymphocytes of peripheral blood of Chernobyl clean-up workers as remote results of the action of ionizing irradiation. Methods: there were examined 120 Chernobyl clean-up workers in the remote period after radiation exposure and 45 persons of the control group. For assessing mitogen-induced levels of Cyclin D1, the micro-method of cultivating erythrocytes of whole blood was used. The quantitative assessment of the spontaneous and mitogen-induced levels of Cyclin D1 in lymphocytes of peripheral blood (PB) was realized using the reagents FITC Mouse Anti-Human Cyclin D1 Antibody Set (BD, USA) by the method of flow cytometry.Research results: there was determined the dose-dependent increase of the spontaneous level of Cyclin D1 in PB lymphocytes of Chornobyl clean-up workers. High values of the parameter were established in the subgroup of Chornobyl clean-up workers, irradiated in the diapason of doses as 500-1000 mSv. Maximal values of Cyclin D1 level in PB lymphocytes were observed in Chornobyl clean-up workers at exacerbation of the bronchial-pulmonary pathology, bronchial asthma in anamnesis and in reconvalescents of acute radiation sickness with the radiation doses D ≥ 500 mSv. After mitogen stimulation of lymphocytes, there was noted the decrease of Cyclin D1 level in the group of Chornobyl clean-up workers and the increase in persons of the control group.Conclusions: the research revealed the differences in products of Cyclin D1 in PB lymphocytes of Chornobyl clean-up workers and persons of the control group. The revealed changes of the spontaneous and mitogen-induced levels of Cyclin D1 in PB lymphocytes of Chornobyl clean-up workers with the somatic pathology reflect disorders in regulation processes of proliferation and cellular cycle. The obtained data add ideas about mechanisms of the radiation-induced disorder of the cellular cycle that may be a manifestation of genome instability and become a trigger factor of carcinogenesis in the remote period after radiation exposure

Tumor Cell Death via Apoptosis and Improvement of Activated Lymphocyte Cytokine Secretion by Extracts from Euphorbia Hebecarpa and Euphorbia Petiolata.

Background:Immunomodulatory materials from natural herbs and the characterization of their immune enhancement effects may have tremendous potential as cancer treatment. The aim of the present study was to investigate the apoptosis-inducing activities of Euphorbia hebecarpa Boiss and Euphorbia petiolata Banks & Sol. plant extracts and their effects on cytokine secretion by lymphocytes. Materials and Methods:We assessed the apoptosis-inducing effect of the plants’ hexane extracts on previously determined sensitive cell lines (HeLa for E. hebecarpa and K562 for E. petiolata) by flow cytometry and measurement of caspase 3 activation. The apoptosis-related gene expressions were examined by real-time PCR. The effects of the extracts on lymphocyte proliferation and cytokine secretion were examined.Results:Flow cytometry analysis showed that the inhibitory effect of the extracts on tumor cell growth was due to cell apoptosis. The plant extracts at the 100 µg/ml dose induced apoptosis in HeLa (98.5 ± 0.1%) and K562 (57.7 ± 1.9%) cells. The extracts increased caspase 3 activation (≈2-fold>control). Real-time PCR showed Fas and Bax gene upregulation and Bcl-2 downregulation, which resulted in an increased Bax/Bcl-2 expression ratio. The extracts increased lymphocyte proliferation and increased levels of IFN-γ production in the presence and absence of mitogen (p < 0.05). They significantly increased IL-4 and decreased IL-10 secretion by mitogen-stimulated lymphocytes. E. hebecarpa also increased IL-17 release. Conclusion:These results have shown that both extracts possess antitumor activity by inducing apoptosis, possibly through both intrinsic and extrinsic pathways. In addition, they induced secretion of different T helper subset related cytokines that are effective in the immune response against cancer.

SUN-253 Effects of Growth Hormone Stimulation on the Immunologic Cellular Landscape in Pediatric Patients

Background: Multiple complex interactions exist between growth hormone (GH)-Insulin like growth factor-1 axis and the immune system. We and others have demonstrated GH receptors on the cell surface of peripheral blood mononuclear cells, human IM-9 cultured lymphocytes, thymus, spleen and lymph nodes. Administration of GH to GH deficient (GHD) children has shown a transient decrease in percent B cells and T cell percentages, interleukin-2 receptor levels and lymphocyte mitogenic stimulation response. Data about acute effects of GH on the immune system are lacking. Aim: The aim of this study was to evaluate the acute effect of endogenous GH on the cellular landscape of the immune system. Methods: A prospective study was conducted in pediatric patients being evaluated for short stature who underwent a standard 3-hour growth hormone stimulation test using arginine and glucagon. Exclusion criteria included genetic syndromes, renal failure, recent immunosuppressant or steroid use and small for gestational age. Blood samples for immunologic markers, that included complete blood count (CBC) and time of flight mass flow cytometry (CyTOF), were collected at the beginning (T0) and end (T3) of the test. Differences in patients by time point (T0 and T3) and by GH response to stimulation (growth hormone sufficient {GHS} versus GHD) were calculated using a two-way ANOVA test with repeated measures over time, considering the interaction between time point and GH status. Results: Fifty-four patients (39 boys, 15 girls) aged 5-18 years, were enrolled in the study. Twenty-two participants had peak GH level <10 ng/ml (GHD). Both GHD and GHS groups were not statistically different in age, gender, pubertal status, height standard deviations (SDs), weight SDs, BMI and IGF-1 SD values, but did differ in growth velocity. Absolute lymphocyte count, lymphocyte percentage, absolute eosinophil count, absolute monocyte count and platelet count demonstrated significant (p<0.05) decrease from T0 to T3. Consistent with CBC analysis, CyTOF analysis showed a decrease (p<0.05) from T0 to T3 in percentages of B cells, monocytes, plasmacytoid dendritic cells, T helper cells and a subset of cytotoxic T cells with low CD57 expression. These results are consistent with previous reports of chronic GH administration. Total white blood cell (WBC) count, absolute neutrophil count and neutrophil percentage demonstrated increase (P<0.05) from T0 to T3, as did granulocyte percentage by CyTOF. Overall, no significant differences were found between CBC or CyTOF measurements and GH status at either time point (T0 and T3). Conclusions: The study provides the first high-resolution map of acute changes in the immune system with GH stimulation. CBC and CyTOF analyses showed significant changes, consistent with previous studies during chronic GH administration. No significant differences were observed between the GHD and GHS groups.

Comparative analysis and characterization of soluble factors and exosomes from cultured adipose tissue and bone marrow mesenchymal stem cells in canine species

The two main sources of mesenchymal stem cell (MSCs) in the canine species are bone marrow (cBM-MSCs) and adipose tissue (cAd-MSCs). The secretion of multitude bioactive molecules, included under the concept of secretome and found in the cultured medium, play a predominant role in the mechanism of action of these cells on tissue regeneration. Although certain features of its characterization are well documented, their secretory profiles remain unknown. We described and compared, for the first time, the secretory profile and exosomes characterization in standard monolayer culture of MSCs from both sources of the same donor as well as its immunomodulatory potential. We found that despite the similarity in surface immunophenotyping and trilineage differentiation, there are several differences in terms of proliferation rate and secretory profile. cAd-MSCs have advantages in proliferative capacity, whereas cBM-MSCs showed a significantly higher secretory production of some soluble factors (IL-10, IL-2, IL-6, IL-8, IL-12p40, IFN-γ, VEGF-A, NGF-β, TGF-β, NO and PGE2) and exosomes under the same standard culture conditions. Proteomics analysis confirm that cBM-MSCs exosomes have a greater number of characterized proteins involved in metabolic processes and in the regulation of biological processes compared to cAd-MSCs. On the other hand, secretome from both sources demonstrate similar immunomodulatory capacity when tested in mitogen stimulated lymphocyte reaction, but not in their exosomes at the dose used. Considering that the use of secretome open as a new therapeutic strategy for different diseases, without the need to implant cells, those biological differences should be considered, when choosing the MSCs source, for either cellular implantation or direct use of secretome for a specific clinical application.

Hemozoin-induced activation of human monocytes toward M2-like phenotype is partially reversed by antimalarial drugs-chloroquine and artemisinin.

Plasmodium falciparum malaria is the most severe form of malaria with several complications. The malaria pigment‐hemozoin (Hz) is associated with severe anemia, cytokine dysfunction, and immunosuppression, thus making it an interesting target for developing new strategies for antimalarial therapy. Monocytes (MO) in circulation actively ingest Hz released by Plasmodium parasites and secrete pro‐ and anti‐inflammatory cytokines. M1 and M2 types represent the two major forms of MO/macrophages (MQ) with distinct phenotypes and opposing functions. Imbalance in the polarization of these types is reported in many infectious diseases. Though the association of Hz with immunosuppression is well documented, its role in activation of MO in context of M1/M2 phenotypes remains to be addressed. We report here that natural Hz drives human MO toward M2‐like phenotype as evidenced by the expression of M2 signature markers. Hz‐fed MO showed elevated transcript and secreted level of IL‐10, CCL17, CCL1, expression of mannose‐binding lectin receptor (CD206), and arginase activity. Hz attenuated HLA‐DR expression, nitric oxide, and reactive oxygen species production, which are the features of M1 phenotype. Our data also implicate the involvement of p38 MAPK, PI3K/AKT, and NF‐κB signaling pathways in skewing of Hz‐fed MO toward M2‐like type and suppression of mitogen‐stimulated lymphocyte proliferation. Importantly, antimalarial drugs—chloroquine and artemisinin—partially reversed activation of Hz‐induced MO toward M2‐like phenotype. Considering the limitations in the current therapeutic options for malaria, we propose that these drugs may be re‐examined for their potential as immunomodulators and candidates for adjunctive treatment in malaria.

Copper nanoparticles induce early fibrotic changes in the liver via TGF-β/Smad signaling and cause immunosuppressive effects in rats

Copper nanoparticles (Cu NPs) have various uses, including as additives in polymers/plastics, lubricants for metallic coating, and biomedical applications. We investigated the role of transforming growth factor (TGF)-β1 signaling in hepatic damage caused by Cu NPs and explored the effects of a 28-day repeated oral administration to Cu NPs on the immune response. The exposure to Cu NPs caused a dose-dependent increase in Cu levels in the liver and spleen. Cu NPs caused hepatic damage and markedly increased oxidative stress in liver tissues. Cu NPs induced activation of TGF-β1/Smad signaling by induction of vascular endothelial growth factor and matrix metalloproteinase-9. Exposure to Cu NPs also induced activation of Smad-independent pathways, phosphorylation of mitogen-activated protein kinases (MAPKs) and Akt/FoxO3. Consistent with the activation of TGF-β1/Smad-dependent and -independent pathways, Cu NPs markedly increased the deposition and induction of extracellular matrix components, α-smooth muscle actin, and collagens in liver tissues. In addition, repeated exposure to Cu NPs suppressed the proliferation of mitogenically stimulated T- or B-lymphocytes and decreased CD3+ (particularly, CD3+CD4+CD8−) and CD45+ population, followed by decreased levels of immunoglobulins and Th1/Th2 type cytokines. Collectively, Cu NPs caused hepatic damage and induced pro-fibrotic changes, which were closely related to the activation of oxidative stress-mediated TGF-β1/Smad-dependent and -independent pathways (MAPKs and Akt/FoxO3). We confirmed the immunosuppressive effect of Cu NPs via the inhibition of mitogen-stimulated spleen-derived lymphocyte proliferation and suppression of B- or T-lymphocyte-mediated immune responses.

Sea Buckthorn Pomace Supplementation in the Diet of Growing Pigs—Effects on Fatty Acid Metabolism, HPA Activity and Immune Status

There is evidence that sea buckthorn, as a source of n-3 polyunsaturated fatty acids (n-3 PUFA), possesses health-enhancing properties and may modulate neuroendocrine and immune functions. In the present study, we investigated the effect of sea buckthorn pomace (SBP) supplementation in the diet of growing German Landrace pigs on fatty acids in the blood and hypothalamus, peripheral immune parameters and mRNA expression of corticotropin-releasing hormone (CRH), mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) in the hypothalamus and spleen. Pigs were fed diets supplemented with 12% of dried SBP or 0% SBP (control group) over an intervention period of eight weeks. The fatty acid profiles in blood plasma were significantly affected by SBP supplementation only for C18:2n-6 and n-6/n-3 PUFA ratio compared with the control group. SBP supplementation did not significantly affect the fatty acid concentrations in the hypothalamus. Furthermore, there were no significant differences in mRNA expression of CRH, MR and GR in the hypothalamus or of GR mRNA expression in the spleen. Concerning the immune status, the plasma IgG levels tended to be higher in SBP pigs, whereas the leukocyte distribution, mitogen-stimulated lymphocyte proliferation, and serum IgM levels remained unchanged. In conclusion, the SBP supplementation of the diet only caused moderate effects on fatty acid metabolism, but no significant effects on hypothalamic–pituitary–adrenal (HPA) activity and immunity in growing pigs. It seems that a beneficial effect of dietary n-3 PUFA on health and welfare is more likely to be expected during stressful situations.

Реакція бластної трансформації лімфоцитів крові нелінійних самиць щурів, їх приплоду на ранніх етапах постембріонального розвитку на фоні впливу сольового екстракту Hirudoverbena

Реакція бластної трансформації лімфоцитів крові нелінійних самиць щурів, їх приплоду на ранніх етапах постембріонального розвитку на фоні впливу сольового екстракту Hirudoverbena

Enhanced CD8+ cytolytic T cell responses in the peripheral circulation of patients with sarcoidosis and non-Löfgren's disease.

The role of CD4+ T cells in the immunopathogenesis of pulmonary sarcoidosis is well-established, while less is known about the phenotype and function of CD8+ cytolytic T cells (CTLs). CD8+ CTLs were explored in peripheral blood and bronchoalveolar lavage (BAL) samples obtained from up to 25 patients with sarcoidosis and 25 healthy controls. The proportion of CTLs was assessed by the expression of cytolytic effector molecules perforin, granzyme B and granulysin in CD8+ T cells, using flow cytometry. Cytolytic function in blood lymphocytes was assessed using a standard 51Cr-release assay. Patients with Löfgren´s syndrome (LS) and an acute disease onset, were compared to non-LS patients with an insidious onset. Higher proportions of peripheral CD8+ CTLs expressing perforin and granzyme B were observed in sarcoidosis compared to healthy controls. Blood CTLs from non-LS patients had significantly higher expression of perforin, granzyme B and granulysin compared to matched BAL, while LS patients maintained lower levels of effector molecules in both compartments. Mitogen-stimulated peripheral lymphocytes from sarcoidosis patients, particularly from the non-LS group, showed a higher target cell lysis compared to controls. These results demonstrated enhanced peripheral CD8+ CTL responses in sarcoidosis, especially in non-LS patients who have an increased risk of chronic disease. Further comprehensive clinical studies are warranted to increase our understanding of CD8+ CTL responses in sarcoidosis.

Effect of levamisole on alkaline phosphatase activity and immunoglobulin secretion of lipopolysaccharide-stimulated murine splenic lymphocytes

Background: Alkaline phosphatases (APase) are a group of enzymes whose activity increases above normal levels in conditions of diseases such as cancer. In the present study, the role of APase using mitogen-stimulated murine lymphocytes was investigated. Methods: U266B1 cells and RPMI 8226 cultures were kept at 37°C in a humidified incubator with 5% CO 2. The cultures were pulsed with 0.5 μCi of 3 H-thymidine and were harvested onto glass fiber filter using Skatron automatic cell harvester. The dried filters were transferred into toluene-based scintillation cocktail, and the radioactivity was measured using Beckman scintillation counter. APase activity was determined by p-nitrophenol phosphate hydrolysis. The intracellular immunoglobulin E (IgE) content was quantified by western blot assay. U266 B1 and RPMI 8226 were cultured at 0.25 × 10 6 /ml with and without 1 mM levamisole for 48 h, and 15 ml of the culture supernatant was collected and lyophilized. Electrophoresis was carried out on 30 μl of the dialyzed samples. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed and the gels were silver stained. Results: APase may be involved in the constitutive proliferation as well as in Ig secretion of myeloma cells. It was observed that murine splenic lymphocytes showed an increase in proliferative response concomitant with an increase in the APase activity and Ig secretion upon mitogenic stimulation (0.5–2.5 mM, P > 0.05). Conclusion: Levamisole significantly inhibited the APase activity when added to the lipopolysaccharide-stimulated cells at the initiation of the culture. Significance of the present study is discussed in the light of existing literature.

Characterization and Immunomodulatory Effects of Canine Adipose Tissue- and Bone Marrow-Derived Mesenchymal Stromal Cells.

BackgroundMesenchymal stromal cells (MSC) hold promise for both cell replacement and immune modulation strategies owing to their progenitor and non-progenitor functions, respectively. Characterization of MSC from different sources is an important and necessary step before clinical use of these cells is widely adopted. Little is known about the biology and function of canine MSC compared to their mouse or human counterparts. This knowledge-gap impedes development of canine evidence-based MSC technologies.Hypothesis and ObjectivesWe hypothesized that canine adipose tissue (AT) and bone marrow (BM) MSC (derived from the same dogs) will have similar differentiation and immune modulatory profiles. Our objectives were to evaluate progenitor and non-progenitor functions as well as other characteristics of AT- and BM-MSC including 1) proliferation rate, 2) cell surface marker expression, 3) DNA methylation levels, 4) potential for trilineage differentiation towards osteogenic, adipogenic, and chondrogenic cell fates, and 5) immunomodulatory potency in vitro.Results1) AT-MSC proliferated at more than double the rate of BM-MSC (population doubling times in days) for passage (P) 2, AT: 1.69, BM: 3.81; P3, AT: 1.80, BM: 4.06; P4, AT: 2.37, BM: 5.34; P5, AT: 3.20, BM: 7.21). 2) Canine MSC, regardless of source, strongly expressed cell surface markers MHC I, CD29, CD44, and CD90, and were negative for MHC II and CD45. They also showed moderate expression of CD8 and CD73 and mild expression of CD14. Minor differences were found in expression of CD4 and CD34. 3) Global DNA methylation levels were significantly lower in BM-MSC compared to AT-MSC. 4) Little difference was found between AT- and BM-MSC in their potential for adipogenesis and osteogenesis. Chondrogenesis was poor to absent for both sources in spite of adding varying levels of bone-morphogenic protein to our standard transforming growth factor (TGF-β3)-based induction medium. 5) Immunomodulatory capacity was equal regardless of cell source when tested in mitogen-stimulated lymphocyte reactions. Priming of MSC with pro-inflammatory factors interferon-gamma and/or tumour necrosis factor did not increase the lymphocyte suppressive properties of the MSC compared to untreated MSC.Conclusions/SignificanceNo significant differences were found between AT- and BM-MSC with regard to their immunophenotype, progenitor, and non-progenitor functions. Both MSC populations showed strong adipogenic and osteogenic potential and poor chondrogenic potential. Both significantly suppressed stimulated peripheral blood mononuclear cells. The most significant differences found were the higher isolation success and proliferation rate of AT-MSC, which could be realized as notable benefits of their use over BM-MSC.