Increase in circulating CD4⁺CD25⁺Foxp3⁺ T cells in patients with Philadelphia-negative chronic myeloproliferative neoplasms during treatment with IFN-α

Recent reports have described complete or major molecular remission in patients with polycythemia vera after long-term treatment with the immunomodulatory agent IFN-α2. Accordingly, there are reasons to believe that the immune system is a key player in eradicating the JAK2 mutated clone in these patients. Foxp3(+) regulatory T cells play a pivotal role in maintaining immune homeostasis and, importantly, preventing immune reactivity to self-antigens; however, their suppressive activity can compromise an effective antitumor immune response, and high frequencies of regulatory T cells in peripheral blood have been reported in both hematologic and solid cancers. We have analyzed the number, phenotype, and function of circulating CD4(+)CD25(+)Foxp3(+) T cells in patients with chronic myeloproliferative neoplasms. Surprisingly, we found a marked expansion of this subset of lymphocytes in patients treated with IFN-α2 (13.0%; 95% confidence interval [CI] 10.8% to 15.2%) compared with healthy donors (6.1%; 95% CI 4.9% to 7.2%), patients with untreated chronic myeloproliferative neoplasms (6.9%; 95% CI 5.8% to 7.4%), or patients treated with hydroxyurea (5.8%; 95% CI 4.3% to 7.4%; P < .0001).     

Dietary gluten triggers concomitant activation of CD4+ and CD8+ αβ T cells and γδ T cells in celiac disease

Celiac disease is an intestinal autoimmune disease driven by dietary gluten and gluten-specific CD4⁺ T-cell responses. In celiac patients on a gluten-free diet, exposure to gluten induces the appearance of gluten-specific CD4⁺ T cells with gut-homing potential in the peripheral blood. Here we show that gluten exposure also induces the appearance of activated, gut-homing CD8⁺ αβ and γδ T cells in the peripheral blood. Single-cell T-cell receptor sequence analysis indicates that both of these cell populations have highly focused T-cell receptor repertoires, indicating that their induction is antigen-driven. These results reveal a previously unappreciated role of antigen in the induction of CD8⁺ αβ and γδ T cells in celiac disease and demonstrate a coordinated response by all three of the major types of T cells. More broadly, these responses may parallel adaptive immune responses to viral pathogens and other systemic autoimmune diseases.     

Characterization of CD4 and CD8 T cells producing IFN-γ in human latent and active tuberculosis

Patients with pulmonary tuberculosis (PTB) frequently have reduced IFN-γ production in response to mycobacterial antigens, compared to individuals with latent Mycobacterium tuberculosis infection (LTBi). However, it is not clear whether this reduced responsiveness is restricted to a particular T cell subset. Herein, PBMCs from 26 PTB patients, 30 household contacts (HHCs) of PTB, and 30 tuberculin positive (TST+) healthy subjects not recently exposed to PTB, were stained with CFSE and stimulated non-specific (PPD) for 120?h, and specific (CFP-10/ESAT-6) and latency (HSpX) mycobacterial antigens for 144?h and the percentage of CD4(+) and CD8(+)IFN-γ(+) T cells responding determined by flow cytometry, in addition to their memory phenotype by the CD45RO and CD27 expression. PTB had decreased frequency of both CD4(+) and CD8(+) precursor cells, as well as decreased number of CD4(+)IFN-γ(+) cells in response to all antigens, whereas CD8(+)IFN-γ(+) cells were decreased in response to PPD and ESAT-6, but not to CFP-10 and HSpX. HHCs exhibited the highest precursor frequencies and IFN-γ responses, irrespective of the antigen employed. The CD4(+)/CD8(+) cell ratios showed that in response to PPD CD4(+) precursor and IFN-γ-producer cells are more frequent than their CD8(+) counterparts, and that PTB have a decreased CD4(+)IFN-γ(+)/CD8(+)IFN-γ(+) ratio in response to PPD, CFP-10, and ESAT-6. CD4(+)IFN-γ(+) and CD8(+)IFN-γ(+) cells exhibited a central memory phenotype (CD45RO(+)CD27(+)), irrespective of the group of subjects and the antigen used for stimulation. In conclusion, PTB patients had a decreased percentage of CD4(+) and CD8(+) precursor cells and CD4(+)IFN-γ(+). HHCs exhibited the highest frequency of CD4(+) and CD8(+) precursors and CD4(+)IFN-γ(+)-producing cells.     

Effects of galantamine on β-amyloid release and beta-site cleaving enzyme 1 expression in differentiated human neuroblastoma SH-SY5Y cells

Galantamine (Gal) is an acetylcholinesterase inhibitor and used to treat the symptoms of Alzheimer's disease (AD). Recent studies show that Gal may affect amyloid precursor protein (APP) metabolism and increase release of secretory APPα (sAPPα). However the effect of Gal on amyloid-β peptide (Aβ) release and β-site cleaving enzyme 1 (BACE1) expression is still unknown. Consequently, we investigated the effect of Gal on the level of Aβ and BACE1. In a differentiated human neuroblastoma cell line (SH-SY5Y), Gal (0.3 μM) was found to significantly decrease Aβ release and BACE1 expression following treatment for 6, 12, and 24 h. Increasing Gal to 0.9 μM or 10 μM had no further effect. The effect of Gal (0.3 μM for 18 h) was maximal on BACE1 expression but not on Aβ secretion. At higher concentration (0.9 μM and 10 μM), Gal had no effect on the level of full-length APP but could still stimulate further decrease in Aβ secretion and release of sAPPα. These observations suggested that 0.3 μM Gal exerts its effect on Aβ production by inhibiting BACE1 expression, while 0.9 μM or 10 μM Gal mainly reduces Aβ production by stimulating the non-amyloidogenic pathway to decrease the amount of APP substrate available for β-secretase cleavage. In addition, α7 nicotinic acetylcholine receptor (α7nAChR) and multiple second messengers (including PKC, MEK, and p38MAPK) were found to be involved in the regulation of Gal-inhibited Aβ release and BACE1 expression.     

Effects of dendritic cells from cord blood CD34^＋ cells on human hepatocarcinoma cell line BEL-7402 in vitro and in SCID mice

AIM: To develop a cancer vaccine of dendritic cells derived from human cord blood CD34+ cells and to investigate its cytotoxicity on human hepatocarcinoma cells in vitro and in sever combined immunodeficiency (SCID) mice. METHODS: Lymphocytes from cord blood or peripheral blood were primed by DCs, which were derived from cord blood and pulsed with whole tumor cell lysates. Nonradiative neutral red uptake assay was adopted to detect the cytotoxicity of primed lymphocytes on human hepatocartinoma cell line BEL-7402 in vitro. The anti-tumor effect of primed lymphocytes in vivo was detected in SCID mice, including therapeutic effect and vaccination effect. RESULTS: The cytotoxicity of DC vaccine primed lymphocytes from cord blood or peripheral blood on human hepatocarcinoma cell line BEL-7402 was significantly higher than that of unprimed lymphocytes in vitro (44.09% vs 14.69%, 47.92% vs 19.44%, P<0.01). There was no significant difference between the cytotoxicity of primed lymphocytes from cord blood and peripheral blood (P>0.05). The tumor growth rate and tumor size were smaller in SCID mice treated or vaccinated with primed lymphocytes than those with unprimed lymphocytes. SCID mice vaccinated with primed lymphocytes had a lower tumor incidence (80% vs 100%, P<0.05) and delayed tumor latent period compared with mice vaccinated with unprimed lymphocytes (11d vs 7 d,P<0.01). CONCLUSION: Vaccine of cord blood derived-DCs has an inhibitory activity on growth of human hepatocarcinoma cells in vitro and in SCID mice. The results also implicate the potential role of cord blood derived-DC vaccine in clinical tumor immunotherapy.     

Analysis for expression and significance of interleukin-17, RORγt in CD4+ T cells from patients with ankylosing spondylitis

Objective To study the expression of IL-17, RORγt in CD4+T cells from patients with ankylosing spondylitis (AS). Methods The specimens of venous blood PBMC were collected from 28 patients with AS and 15 healthy subjects. Intracellular flow cytometry detection of IL-17 was established after isolation of human CD4+ T cells from PBMC. The expression level of IL-17, RORγt mRNA in CD4+ T cells was determined from 28 AS patients and 15 healthy controls by real-time fluorescence quantitative RT-PCR using Anti -CD3/Anti -CD28 as stimulators or not. Analysis of variance and Pearson correlation were selected. Results The isolation of human CD4+ T cells from PBMC was effective and its purity reached 90%. The percentage of intracellular IL-17 in CD4+ T cells from AS pati-ents in the AS active group was higher than that of the AS stable group and healthy control group (P<0.01). The expression level of IL-17, RORγt mRNA in CD4+ T cells was significantly higher in patients with AS than in controls. After stimulated with anti-CD3/ anti-CD28 stimulation, the percentage of IL-17, RORγt mRNA was increased significantly (P<0.01). The percentage of IL-17, RORγt mRNA in the 12 h group was higher than that of the 24 h group, while both of them were higher than those without stimulation (P<0.05). Conclusion There is an abnormal expression of IL-17, RORγt in human CD4+ T cells from AS patients. Our results indicate that the abnormal expression of IL-17 might play a role in the development and progression of AS.     

Analysis for expression and significance of interleukin-17, RORγt in CD4+ T cells from patients with ankylosing spondylitis

Objective To study the expression of IL-17, RORγt in CD4+T cells from patients with ankylosing spondylitis (AS). Methods The specimens of venous blood PBMC were collected from 28 patients with AS and 15 healthy subjects. Intracellular flow cytometry detection of IL-17 was established after isolation of human CD4+ T cells from PBMC. The expression level of IL-17, RORγt mRNA in CD4+ T cells was determined from 28 AS patients and 15 healthy controls by real-time fluorescence quantitative RT-PCR using Anti -CD3/Anti -CD28 as stimulators or not. Analysis of variance and Pearson correlation were selected. Results The isolation of human CD4+ T cells from PBMC was effective and its purity reached 90%. The percentage of intracellular IL-17 in CD4+ T cells from AS pati-ents in the AS active group was higher than that of the AS stable group and healthy control group (P<0.01). The expression level of IL-17, RORγt mRNA in CD4+ T cells was significantly higher in patients with AS than in controls. After stimulated with anti-CD3/ anti-CD28 stimulation, the percentage of IL-17, RORγt mRNA was increased significantly (P<0.01). The percentage of IL-17, RORγt mRNA in the 12 h group was higher than that of the 24 h group, while both of them were higher than those without stimulation (P<0.05). Conclusion There is an abnormal expression of IL-17, RORγt in human CD4+ T cells from AS patients. Our results indicate that the abnormal expression of IL-17 might play a role in the development and progression of AS.     

Increased TTS abrogates IDO-mediated CD4+ T cells suppression in patients with Graves’ disease

Indoleamine 2,3-dioxygenase (IDO)-expression in antigen-presenting cells (APCs) may control autoimmune responses by depleting the available tryptophan, whereas tryptophanyl-tRNA synthetase (TTS) may counteract this effect. The study aims to determine whether abnormal IDO and TTS activities in autoreactive T, B and dendritic cells (DCs) lead to tryptophan metabolism disorder, inducing the immune imbalance in patients with Graves’ disease (GD). The concentrations of serum kynurenine and tryptophan and the mRNA expressions of IDO and TTS were analyzed, and the mixed leukocyte reaction (MLR) was employed to assess the interaction of IDO-expressing DCs and TTS-expressing CD4⁺ T cells. Compared with healthy donors (HD), the ratio of serum kynurenine to tryptophan (P < 0.0001) was increased in GD patients, which was associated with the increased IDO expression in B cells (P < 0.01) and DCs (P < 0.01). GD-derived CD4⁺ T cells enhanced TTS expression (P < 0.01), and its proliferation was not inhibited in the presence of IDO-expressing DCs from the GD patients. In contrast, the proliferation of HD-derived CD4⁺ T cells with low TTS expression was inhibited. Increased TTS expression from CD4⁺ T cells resists IDO-mediated immunosuppression from DCs, which might link to a pathogenic mechanism involved in autoreactive T cells being sustained in vivo in GD patients. Shu Wang and Chaoming Mao contributed equally to this study.     

Effects of dermatan sulfate derivatives on platelet surface P-selectin expression and protein C activity in blood of inflammatory bowel disease patients

AIM:To investigate the effect of dermatan sulfate(DS)derivatives on platelet surface P-selectin expression andblood activated protein C(APC)activity in patients withinflammatory bowel disease(IBD),and to clarity the anti-inflammatory mechanism of D5 derivatives.METHODS:Dermatan sulfate(DS)was sulfated withchlorosulfonic acid to prepare polysulfated dermatan sulfate(PSDS).The major disaccharides of D5 and PSD5 weredetermined by ~1H nuclear magnetic resonance spectroscopy(~1H-NMR)and ~(13)C-NMR.Both DS and PSDS were depolymerizedwith hydrogen peroxide.The fragments were separatedby gel filtration chromatography.The effects of DS derivativeson P-selectin expression were assayed by ELISA method,and blood APC activity was assayed by the syntheticchromogenic substrate method.RESULTS:The major disaccharides of DS and PSDS wereIdoA-1→3-GalNAc-4-SO_3 and IdoA-2SO_3-1→3-GalNAc4,6-diSO_3,respectively.Compared with the adenosine diphosphatestimulated group and IBD control group,DS and its derivativesall had significant inhibitory effects on P-selectin expression(P<0.01),but there was no difference between DS-derivedoligosaccharides(DSOSs)and PSDS-derived oligosaccharides(PSDSOSs).The experiments on APC activity showed thatDS and its derivatives all enhanced APC activity.The mostactive D505 was the one with a relative molecular weight(M_r)of 4 825,which enhanced the APC activity from106.5±11.5% to 181.8 22.3%(P<0.01).With the decreaseof M_1,the activity of DSOSs decreased gradually.The effectof PSDS on APC activity enhancement was more significantthan that of DS,and the APC activity was raised to205.2±22.1%(P<0.01).All the PSDSOSs were more activethan DSOSs on the basis of comparable M.With the decreaseof M_1,the activity of PSDSOSs increased gradually,and the most active PSDSOS was PSDSOS_3 with M_1 of 2 749,whichenhanced the APC activity to 331.2±27.8%(P<0.01),thenthe activity of PSDSOSs decreased gradually.CONCLUSION:DS and its derivatives can significantlyinhibit P-selectin expression on platelet surface,but the effecthas no correlation with DS molecular mass and sulfation.The effect of DS or its derivatives on APC activity atmolecular level involves complex mechanisms that dependon the molecular mass,the degree of sulfation,and theheterogeneous composition of DS.On the same molecularsize,the higher the degree of DS sulfation,the more significantthe effect on enhancing APC activity.     

Effects of Rheum tanguticum polysaccharide on TNBS -induced colitis and CD4^＋T cells in rats

AIM: To study the effects of Rheum tanguticum polysaccharide-1 (RTP-1) on ulcerative colitis in rats induced by 2, 4, 6-trinitrophene sulphonic acid (TNBS) and their possible mechanism.METHODS: RTP1 (200 mg@kg-1, ig) extracted from Rheum tanguticum Maxim. Ex Regel was administrated to rats with colitis induced by TNBS for 5 d, 7 d, 10 d and 14 d,respectively. The effects of RTP1 and dexamethasone (DX,0.2 mg@kg-1, ig) were contrastively investigated. The MPO level and SOD activity were determined by chromatometry.The expansion and protein expression of CD4+T lymphocytes isolated from colon mucosae and mesenteric lymph nodes of colitis rats were performed by immunohistochemical analysis and Western-blot methods.RESULTS: Treatments of RTP1 (200 mg@kg-1, ig) significantly reduced diarrhea, mortality, colon mass, ulcer areas and MPO level in colon mucosae on days 5, 7, 10 and 14 (5.2±1.4,5.4±0.7, 5.2±1.8, P＜0.05.3.4±0.8, P＜0.01. 16.1±12.1,P＜0.01.31.8±8.6, 17.7±5.3, 12.7±4.1, P＜0.05). The effectsof RTP1 were similar to those noted above in DX group, but there were no immunosupressive effects of DX in RTP-1group, such as body mass loss, thymus and spleen atrophy.The decreased number and down-regulated protein levels of CD4+T cells isolated from the colon of colitis rats treated with RTP1 were found.CONCLUSION: RTP1 shows significantly protective effects but lower side effects on rats with colitis induced by TNBS.The mechanism may be due to the resistance to over expansion of CD4.     

HIV disease progression to CD4 count <200 cells/μL and death in Saskatoon,Saskatchewan

OBJECTIVE:

To characterize and identify determinants of HIV disease progression among a predominantly injection drug use (IDU) HIV population in the highly active antiretroviral therapy era.

METHODS:

The present retrospective study was based on 343 HIV patients diagnosed from 2005 to 2010 from two clinics in Saskatoon, Saskatchewan. Disease progression was defined as the time from diagnosis to immunological AIDS (CD4 count <200 cells/μL) and death. Uni- and multivariable Cox proportional hazards models were used.

RESULTS:

Of the 343 patients, 79% had a history of IDU, 77% were hepatitis C virus (HCV) coinfected and 67% were of Aboriginal descent. The one-year and three-year immunological AIDS-free probabilities were 78% and 53%, respectively. The one-year and three-year survival probabilities were 97% and 88%, respectively. Multicollinearity among IDU, HCV and ethnicity was observed and, thus, separate models were built. HCV coinfection (HR 2.9 [95% CI 1.2 to 6.9]) was a significant predictor of progression to immunological AIDS when controlling for baseline CD4 counts, treatment, age at diagnosis and year of diagnosis. For survival, only treatment use was a significant predictor (HR 0.34 [95% CI 0.1 to 0.8]). HCV coinfection was marginally significant (P=0.067).

CONCLUSION:

Baseline CD4 count, HCV coinfection, year of diagnosis and treatment use were significant predictors of disease progression. This highlights the importance of early treatment and the need for targeted interventions for these particularly vulnerable populations to slow disease progression.     

Diurnal variation and effect of insulin on circulating high molecular weight (HMW) adiponectin and NF-κB activity in human endothelial cells

ObjectiveTo study the diurnal variation and the effect of insulin on adiponectin multimers and nuclear factor-kappaB (NF-κB) activity in human endothelial cells.Methods and resultsWe utilized a prolonged insulin–glucose infusion in six healthy human subjects. HMW and total adiponectin levels were higher in the morning and lower at night; NF-κB activities in serum treated human microvascular endothelial cells (HMEC-1) cells were lower in the morning and higher at night. Hyperinsulinemic induction significantly decreased HMW and total adiponectin levels but increased NF-κB activity in serum treated HMEC-1 cells (P < 0.05, P < 0.01). There were no significant changes to MMW and LMW adiponectin levels (P > 0.05).ConclusionCircadian rhythm of HMW adiponectin and NF-κB activity are altered by hyperinsulinemia providing novel insights adiponectin and NF-κB biology, which may be pertinent to insulin resistant states, e.g. obesity and type 2 diabetes mellitus.     

Thymic tissue is not evident on high-resolution computed tomography and [¹⁸F]fluoro-deoxy-glucose positron emission tomography scans of aviraemic HIV patients with poor recovery of CD4⁺ T cells

Some previously immunodeficient HIV patients responding to antiretroviral therapy display poor recovery of CD4? T cells. Evaluation of the contribution of thymic function requires sensitive detection and quantitation of metabolically active thymic tissue. We describe patients with low but detectable thymopoiesis assessed as circulating CD4? naive T cells expressing CD31. High-resolution computed tomography and PET scans found no residual thymic tissue even though metabolic activity was demonstrable by PET in lymph nodes.