Chemical constituents from Pithecellobium clypearia and their effects on T lymphocytes proliferation

Aim To investigate the chemical constituents from the twigs and leaves of Pithecellobium clypearia Benth and their immunomodulatory effects. Methods The constituents were separated and purified by various chromatographic methods and their structures were identified on the basis of spectral analysis. The immufiomodulatory effects of all the compounds were examined by a Con A-induced T lymphocytes proliferation assay. Results Eight compounds were isolated and identified as （-）- epigallocatechin （1）, （-）-5, 7, 3′, 4′, 5′-pentahydroxyflavan （2）, （-）-epigallocatechin-7-gallate （3）, （-）-5, 3′, 4′, 5′-tetrahydroxyfiavan- 7-gallate （4）, quercitin-3-O-α-L-rhamnpyranoside （5）, myricitin-3-O-α-L-rhamnpyranoside （6）, gallic acid （7）, and ethyl gallate （8）, respectively. Conclusion Compounds 3 and 8 were isolated from this genus for the first time, and compound 1 was isolated from this species for the first time. Compound 3 exhibited a strong inhibition on the T lymphocytes proliferation induced by Con A with an IC50 of 4.4 μmol·L^-1.

Ambiguous nucleus regulates the proliferation and percentage of T lymphocytes in peripheral blood

The aim of this study was to examine the immunomodulatory role of the unilateral ambiguous nucleus （Amb）. We performed electrical stimulation of the unilateral Amb, electrical stimulation of the left parietal cortex and the lateral hypothalamus following unilateral Arab lesion, as well as microinjection of acetylcholine chloride and hemicholine-3 into the unilateral Amb, and electrical stimulation of the unilateral Amb after injection of atropine, mecamylamine, propranolol, and phentolamine. Results showed that the number and proliferation of peripheral blood T lymphocytes were increased after electrical stimulation of the unilateral Arab. The cholinergic neurons in the Amb released choline substances to alter cellular immunity, thus confirming that the Amb mediates the neuro-immunomodulatory process.

Costimulation of resting B lymphocytes alters the IL-4-activated IRS2 signaling pathway in a STAT6 independent manner: implications for cell survival and proliferation

IL-4 is an important B cell survival and growth factor. IL-4 induced the tyrosine phosphorylation of IRS2 in resting B lymphocytes and in LPS- or CD40L-activated blasts. Phosphorylated IRS2 coprecipitated with the p85 subunit of PI 3’ kinase in both resting and activated cells. By contrast, association of phosphorylated IRS2 with GRB2 was not detected in resting B cells after IL-4 treatment although both proteins were expressed. However, IL-4 induced association of IRS2 with GRB2 in B cell blasts. The pattern of IL-4- induced recruitment of p85 and GRB2 to IRS2 observed in B cells derived from STAT6 null mice was identical to that observed for normal mice. While IL-4 alone does not induce activation of MEK, a MEKI inhibitor suppressed the IL-4-induced proliferative response of LPS-activated B cell blasts. These results demonstrate that costimulation of splenic B cells alters IL-4-induced signal transduction independent of STAT6 leading to proliferation. Furthermore, proliferation induced by IL-4 in LPS-activated blasts is dependent upon the MAP kinase pathway.

Lamprey buccal gland secretory protein-2 (BGSP-2) inhibits human T lymphocyte proliferation

Lamprey is a representative of the agnathans, the most ancient class of vertebrates. Parasitic lampreys secrete anticoagulant from their buccal glands and prevent blood coagulation of host fishes. We identified a buccal gland secretory protein-2 （BGSP-2） from a buccal gland cDNA library of Larnpetrajaponica. The full-length BGSP-2 gene was cloned and the recombinant BGSP-2 protein was generated. The role of BGSP-2 on lymphocyte proliferation was studied by examining its effects on human T lymphocytes. We found that lamprey BGSP-2 was able to effectively block the proliferation of T cells in vitro by inducing G1/S cell cycle arrest. Furthermore, it inhibited the proliferation of hmnan T lymphocytes stimulated by phytohemagglutinin （PHA） at a minimum concentration of 0.1μg/ml. Our data suggest that lamprey BGSP-2 is able to block the mitosis of human T lymphocytes at the G1/S point, and has the potential of anti-proliferative effect on PHA-activated T lymphocytes.

Lymphocytes in patients with psoriasis promote proliferation of ker-atinocytes

Objective: To analyze the effect of lymphocytes on proliferation of keratinocytes in patients with psoriasis. Methods: Lymphocytes in lesion and peripheral blood were isolated and amplified, then cultured together with normal keratinocytes. By MTT method, the living cells were quantified in the mixed culture. Results: Compared with normal controls, lymphocytes from lesion and peripheral blood of psoriasis both promote the proliferation of keratinocytes (P<0. 01 and P<0. 05 respectively). The concentrations of IL-2 and IFN-7 in the mixture of lesion lymphocytes and keratinocytes were significantly higher than that of controls. Tripterygium glycosides inhibited this promotion. Conclusion: Lymphocytes in patients with psoriasis (mainly Th1 cell) play an important role in proliferation of keratinocytes. This psoriasis cell model is useful for studies on signal transduction in psoriasis.

Study on Lymphocyte Activation and Proliferation Induced by Anti-CD3 McAb

T cell activation and proliferation via CD3-TCR complex were investigated by lymphocyte DNA synthesis in vitro.Several interfering factors were also discussed.The result indicated that lymphocyte activation and proliferation are calciumdependent.A rise of cytoplasmic free Ca2+ quickly following activation with CD3 McAb is mainly due to intracellular mobilization of Ca2+,while lymphocyte proliferation needs both intracellular mobilization of Ca2+ as well as influx of extracellular Ca2+, It was confirmed that CTX sensitive G protein plays a role in regulating T cell proliferation by pretreatment with CTX suppressing lymphocyte H-TdR incorporation obviously.PLC and PKC inhibitor neomycin and P.S.S could also decrease T cell proliferation.

Immunopotentiality of Ayurvedic polyherbal formulations “Saribadi” and “Anantamul Salsa” with augmentation of IgM production and lymphocytes proliferation:A preliminary study

Objective:To assess the immunopotentiality of Ayurvedic polyherbal preparations,"Saribadi" and "Anantamul Salsa".Methods: Freshly prepared BALB/c mice splenocytes were cultured with "Saribadi" or"Anantamul Salsa" treatment [doses of 0.25%, 0.50%, 0.75%, 1.00%, 1.50%, 2.00%,3.00% and 4.00%(v/v)] at 37C for 5 days. The immunoglobulin M(IgM) production and lymphocytes proliferation were determined by ELISA and MTT methods, respectively.Endotoxin contamination was assessed by treating the preparations with polymyxin B.Results: The doses of "Saribadi" [0.25%, 0.50%, 0.75% and 1.00%(v/v)] significantly increased IgM productions(0.966, 0.728, 0.695 and 0.615 mg/m L vs. control 0.265 mg/m L)and lymphocytes proliferation [absorbance 0.311, 0.394, 0.372 and 0.334 optical density(OD) vs. control 0.162 OD]. Similarly, the doses of "Anantamul Salsa" [0.50%, 0.75%,1.00% and 1.50%(v/v)] promoted IgM productions(0.933, 0.919, 0.917 and 0.892 mg/m L vs. control 0.502 mg/m L) and the doses of "Anantamul Salsa" [0.50%, 0.75%, 1.00%,1.50%, 2.00%, and 3.00%(v/v)] stimulated lymphocytes proliferation(absorbance 0.395,0.326, 0.440, 0.398, 0.452 and 0.355 OD vs. control 0.199 OD). The activity of "Saribadi"and "Anantamul Salsa" was not retarded by the treatment of preparations with polymyxin B.Conclusions: Immunomodulatory activity of "Saribadi" and "Anantamul Salsa" was unveiled for the first time. "Saribadi" and "Anantamul Salsa" possess immunostimulating potential acting through the induction of lymphocyte proliferation and IgM production.These preparations may be useful in strengthening immune responses. However, further cellular and in vivo studies are required.

Effects of Soman on Lymphocyte Proliferation in Mice

Experiments to investisate the effects of soman on lymphocyte proliferation werecarried out on balb/c mice,which were injected subcutaneously with soman in the doseof 154μg/kg (0.8 LD50),96μg/kg (0.5 LD50) and 38.4μg/kg (0.2 LD50) respectively.Itwas found that from the 1st to the 7th day after poisoning with 154μg/kg soman,B-lymphocyte proliferation was severely inhibited （P【0.05）,and it returned to the controllevel on the 10th day.Con A-stimulated T-cell proliferation showed a diphasic change,increasing at first and then markedly decreasing thereafter,after soman poisoning.The re-sults imply that the mitogenic response of both B- and T- cells and the primary increaseof T- cell response are closely related to the dosage of soman but not with the changes ofwhole blood cholinesterase activity.

Isolation of Sturgeon Peripheral Blood Lymphocytes and the Optimal Proliferation Response Conditions

In this study, sterlet （ Acipenser ruthenus ） was chosen as the model species of sturgeon, different solutions were used to isolate the sturgeon peripheral blood lymphocytes and study their optimal proliferate response condition. Phytohemagglutinin （PHA）, concanavalin A （ConA）and lipopolysaccharide （LPS） were used as lymphocyte proliferation mitogen, respectively. According to L 25 （5^6） five-factor five-level orthogonal experimental design, the conditions for sturgeons proliferation response of peripheral blood lymphocytes were optimized using enhanced cell counting Kit-8 （enhanced CCK-8 or WST-8）. Five factors were selected to explore the optimal response conditions, including culture time, culture temperature, cell concentration, fetal bovine serum （FBS） concentration, and mitogen concentration. The results showed that 70% Percoll （1.092 g/ml） used as the sturgeon lymphocyte separation solution had the best separating effect. The optimal proliferation conditions were as follows： 3.625×10 6 initial cells, 20 μg/ml PHA or 50 μg/ml ConA or 10 μg/ml LPS as mitogen, 10%-20% FBS, the temperature at 20-25 ℃, and the culture time of 2 d.

IMMUNOMODULATION OF SYNTHESIZEDPOLYMERS CONTAINING PHOSPHORUSIN THE BACKBONE──EFFECT ON THE PROLIFERATION OF LYMPHOCYTES

The immunomodulation of several charged synthetic polymers containing phosphorus in the backbone was studied in vitro through examining their inhibition or promotion effect on the proliferation of both T and B lymphocytes. It is found that polymers based on long chain alkyl ester of tyrosine exhibit immunomodulative activity. Negatively charged polymers show stimulative activiactivity on LPS-induced B lymphocytes proliferation. Positively charged polymers exhibit inhibitory activity on both Con A-induced T lymphocytes and LPS-induced B lymphocytes proliferation.

Effects of low dose oxymatrine on mouse lymphocyte proliferation stimulated by Con A

Objective To investigate the effects of low dose Oxymatrine(OMT)on mouse lymphocyte proliferation stimulated by Con A making use of fluorescence dyestuff CFDA-SE.Methods CFDA-SE staining and flow cytometry were used to detect the fluorescence intensity of lymphocytes after stimulated by polyclonal stimulators Con A and OMT.Then,related software was used to analyze the effects of OMT on mouse lymphocyte proliferation.Results After cultured for 48 h,CFSE fluorescence could be detected by cytometer,filial generation peaks did not appear in control group,which indicated that lymphocytes did not proliferate.Three peaks were obviously detected in Con A group which indicated that Lymphocytes divided after 48 h stimulated by Con A compared with the halving of the fluorescence intensity of control group.In groups with Con A and OMT treated,Primary generation peaks are all lower while filial generation peaks are significantly higher than groups with Con A treated only.This indicated OMT obviously promote lymphocyte proliferation.After cultured for 72 h,the fluorescence intensity changes between all groups are consistent with those of cultured for 48 h.Analyzed with CELLQuest software,it is shown that OMT could promote lymphocyte proliferation in 16,8,4 and 2μg/mL respectively.Conclusions 1)CFDA-SEdyeing and flow cytometer were both reliable tools to analyze lymphocyte proliferation;2)lower dosage of OMTcould promote the proliferation of lymphocyte as a immunopotentiator.

Function of IgD on lymphocyte activation and effect of hIgD-Fc-Ig fusion protein on human PBMC proliferation

OBJECTIVE This study aimed to investigate the influence of IgD on T/B cell activation and construct h IgD-Fc-Igfusion protein to competitive inhibition IgD binding with IgDR.METHODS T/B cells were sorted by magnetic cell sorting.The differences of m IgD and IgD-R level between different T/B cell subtypes were detected by FCM.Serum IgD level was detected by ELISA.Human IgD-Fc-IgG1-Fc sequence was amplified by cross-PCR and then subcloned into PET28 a(+) empty vector.After prokaryotic expression through escherichia coli,we obtained the h IgD-Fc-Igfusion protein by affinity chromatograph.Western blot was used to identify the h IgD-Fc-Igfusion protein.Human peripheral blood monouclear cells(PBMC) and fibroblast like synoviocytes(FLS) proliferation were detected using a cell counting kit-8(CCK-8).RESULTS The percentage of CD3^+/CD4^+,CD3^+/IgD^+,CD3^+/CD4^+/IgD^+,CD3^+/IgD-R+and CD3^+/CD4^+/IgD-R+cells increased significantly in RA patients comparing to healthy people.IgD can stimulate PBMC proliferation.IgD(1,3,10,30 μg·mL^(-1)) stimulate PBMC proliferation significantly after 24 h.We obtained stable and active h IgD-Fc-Igfusion protein.The h IgD-Fc-Igfusion protein showed no effect on PBMC proliferation.But it could downregulate human IgD protein promoting proliferation effects in human PBMC.CONCLUSION This result suggests that IgD and IgDR play an important role on T/B cell activation in RA patients and the h IgD-Fc-Igfusion protein may competitively inhibit IgD′s function and may play an therapeutic role in autoimmune diseases.

Vitamins C, E, and NADH on in Vitro Lymphocyte Proliferation and Redox Status among Obese Patients

Background: The prevalence of overweight and obesity associated with oxidative stress and immune abnormalities is continuously increasing. Antioxidant supplementations might counteract potential damage caused by ROS to cellular tissues. Objective: To determine the role of vitamins on immune improvement during obesity, we investigated in vitro effects of vitamins C, E, and NADH on mitogen-stimulated proliferation, Th1- and Th2-type cytokine production, and oxidant/antioxidant status of lymphocytes isolated from obese patients. Methods: Peripheral blood lymphocytes were isolated using a density gradient of Histopaque. They were in vitro cultured and stimulated by Con A in the presence or absence of vitamins. Cell proliferation was determined by MTT assay and interleukin-2, interleukin-4 and interferon-γ (INFγ) secretions. Cell oxidant/antioxidant balance was studied by assaying glutathione (GSH), malondialdehyde (MDA), carbonyl protein levels, catalase activity and micronucli frequency. Results: Obesity is associated with enhanced oxidative stress response. Indeed, vitamin C, E and NADH improved significantly lymphocyte proliferation and diminished cellular oxidative stress. Conclusion: Treatments of lymphocytes with vitamins had beneficial effects on lymphocyte proliferation, cytokines secretions and redox status, generating an anti-inflammatory profile and should be considered in therapeutic approaches for normalizing immune cell function in obesity.

Function of IgD on lymphocyte activation and effect of hIgD-Fc-Ig fusion protein on human PBMC proliferation

Aim：Investigate the influence of IgD on T/B cell activation and construct hIgD-Fc-Ig fusion protein to competitive inhibition IgD binding with IgDR. Methods differences of mIgD and IgD-R level between different T/B was detected by ELISA. Human IgD-Fc-IgGI-Fc sequence T/B cells were sorted by magnetic cell sorting. The cell subtypes were detected by FCM. Serum IgD level was amplified by cross-PCR and then subcloned into PET28a（ ＋ ） empty vector. After prokaryotic expression through escherichia？ coli, we obtained the hIgD-Fc-Ig fu- sion protein by affinity chromatograph. Western blot was used to identify the hIgD-Fc-Ig fusion protein. Human pe- ripheral blood monouclear cells （PBMC） and fibroblast like synoviocytes （FLS） proliferation were detected using a cell counting kit-8 （ CCK-8 ）. Results The percentage of CD3 ＋/CD4 ＋ , CD3 ＋/IgD ＋ , CD3 ＋/CD4 ＋/IgD ＋ , CD3 ＋/IgD-R ＋ and CD3 ＋/CD4 ＋/IgD-R ＋ cells increased significantly in RA patients comparing to healthy people. IgD can stimulate PBMC proliferation. IgD （1, 3, 10, 30 μg ＂ ml^-1） stimulate PBMC proliferation significantly after 24 h. We obtained stable and active hlgD-Fc-Ig fusion protein. The hlgD-Fc-Ig fusion protein showed no effect on PBMC proliferation. But it could downregulate human IgD protein promoting proliferation effects in human PBMC. Conclusion This result suggests that IgD and IgDR play an important role on T/B cell activation in RA patients and the hIgD-Fc-Ig fusion protein may competitively inhibit IgD＇s function and may play an therapeutic role in autoimmune diseases.

Effects of“Moxibustion Serum”on Proliferation and Phenotypes of Tumor Infiltrating Lymphocytes

Tumor infiltrating lymphocytes (TIL) were cultured with “moxibustion serum”(MS), and the results were examined by flow cytometry. The results indicated that MS could enhance the proliferation of TIL,accelerate it to reach the exponential growth phase, and assist recombinant interleukin 2 (rIL-2) to enhance successively the percentage of CD3^+ positive cells, maintain the number of CD4^+ positive T cells, promote greatly the percentage of CD8^+ positive T cells among TILs, and reverse the CD4^+/CD8^+ ratio. Such cooperative effects rely on relative specificity of acupoints. It is suggested that MS is beneficial to the growth of TIL both in the aspects of proliferation and phenotypes.

EFFECTS OF β-ENDORPHIN ON PHYTOHEMAGGLUTININ-INDUCED LYMPHOCYTE PROLIFERATION AND MOUSE PLAQUE-FORMING CELL RESPONSE VIA AN OPIOID RECEPTOR MECHANISM

The effects of opioid peptides on iminune responses were investigated. It was found that β-endorphin (β-END) can depress proliferative responses to PHA in rat splenocytes but enhance those in mice, and it could also inhibit the plaque-forming cell (PFC) response to sheep red blood cells when mouse splenocytes immunized in vivo were cultured in vitro with the peptide. The peptide antagonist naloxone was able to reverse β-END suppression of the PFC response. The data indicate that β-END suppresses antibody production or secretion via a specific opioidreceptor-mediated mechanism.

Silencing of peroxiredoxin 2 suppresses proliferation and Wnt/β-catenin pathway,and induces senescence in hepatocellular carcinoma

Hepatocellular carcinoma(HCC),a common malignancy worldwide,still lacks effective clinical treatment.The study aimed to investigate the oncogenes that affect the progression of HCC and their possible mechanisms.In our study,we initially confirmed a higher level of PRDX2 in the bile of HCC patients compared to those with choledocholithiasis by 2-DE,LC-MS,and ELISA.Subsequently,we demonstrated the high expression of peroxiredoxin 2(PRDX2)in HCC based on the TCGA database and clinical sample analysis.Furthermore,PRDX2 overexpression enhanced the viability of HCC cells.And PRDX2 silencing induced senescence of HCC cells.In vivo,knockdown of PRDX2 significantly reduced the weight of xenograft tumors.PRDX2 also was found to activate the Wnt/β-catenin pathway by inducingβ-catenin nuclear translocation.Consequently,we proved that silencing PRDX2 could inhibit proliferation and Wnt/β-catenin pathway while promoting senescence in HCC cells.

Effect of Sargassum filipendula Fucoxanthin against HeLa Cell and Lymphocyte Proliferation

This study aims to （1） obtain a pure extract of Sargassumfilipendula fucoxanthin, （2） determine the effect of concentration of fucoxanthin against HeLa cells, and （3） determine the effect of concentration of fucoxanthin against cancer cell lymphocytes. The benefit of this research is to find out that fucoxanthin can be used as an anti-cancer treatment. The study was conducted in September 2013 at the Laboratory of Fishery Product Technology FPIK UB and UB Medical Biomedical Laboratories, Malang. Sampling brown seaweed （Sargassum filipendula） of the District Rural Padike Talango Sumenep Madura. Extracted isolated by column chromatography using silica gel stationary phase and mobile phase hexane：ethyl acetate （6：4, v/v） ± 100 mL, then identified by TLC using the stationary phase, a silica gel F-254 HPLC with ODS stationary phase （C-18） 5 mL with a mobile phase of methanol, acetone and ammonium acetate （IM） （80：10：10, v/v） and a flow rate of 1.0 mL/min pigment solution of 20 mL compared with the standard of Japan. Research results indicate that, spectra maximum wavelength of 450 nm and 446 nm with a minimum of acetone solvent. HPLC has the same retention time = 10.12 standard, namely Sargassum filipendula = 10.11. By giving some of the best concentrations at 100 ppm is lethal HeLa cells ebesar 5236% which means that fucoxanthin effect against HeLa cells have the potential fucoxanthin that can induce cell apoptosis that occurs in HeLa cells, it is possible because of the anti-carcinogenic fucoxanthin mempuyai structure unique. While the cell lymphocytes in the dosage of 100 ppm were dead at 8.788%, the effect of the toxicity of a substance can be observed from how many dead lymphocytes when compared with the state charity by observing level of lymphocyte proliferation.

High-frequency repetitive transcranial magnetic stimulation promotes neural stem cell proliferation after ischemic stroke

Prolife ration of neural stem cells is crucial for promoting neuronal regeneration and repairing cerebral infarction damage.Transcranial magnetic stimulation(TMS)has recently emerged as a tool for inducing endogenous neural stem cell regeneration,but its underlying mechanisms remain unclea r In this study,we found that repetitive TMS effectively promotes the proliferation of oxygen-glucose deprived neural stem cells.Additionally,repetitive TMS reduced the volume of cerebral infa rction in a rat model of ischemic stro ke caused by middle cerebral artery occlusion,im p roved rat cognitive function,and promoted the proliferation of neural stem cells in the ischemic penumbra.RNA-sequencing found that repetitive TMS activated the Wnt signaling pathway in the ischemic penumbra of rats with cerebral ischemia.Furthermore,PCR analysis revealed that repetitive TMS promoted AKT phosphorylation,leading to an increase in mRNA levels of cell cycle-related proteins such as Cdk2 and Cdk4.This effect was also associated with activation of the glycogen synthase kinase 3β/β-catenin signaling pathway,which ultimately promotes the prolife ration of neural stem cells.Subsequently,we validated the effect of repetitive TMS on AKT phosphorylation.We found that repetitive TMS promoted Ca2+influx into neural stem cells by activating the P2 calcium channel/calmodulin pathway,thereby promoting AKT phosphorylation and activating the glycogen synthase kinase 3β/β-catenin pathway.These findings indicate that repetitive TMS can promote the proliferation of endogenous neural stem cells through a Ca2+influx-dependent phosphorylated AKT/glycogen synthase kinase 3β/β-catenin signaling pathway.This study has produced pioneering res ults on the intrinsic mechanism of repetitive TMS to promote neural function recove ry after ischemic stro ke.These results provide a stro ng scientific foundation for the clinical application of repetitive TMS.Moreover,repetitive TMS treatment may not only be an efficient and potential approach to support neurogenesis for further therapeutic applications,but also provide an effective platform for the expansion of neural stem cells.

Correlation of neutrophil to lymphocyte ratio to severity of coronary artery disease and in-hospital clinical outcomes in patients with acute coronary syndrome: A prospective observational study

Objective:To explore correlation of neutrophil-to-lymphocyte ratio(NLR)to severity of coronary artery disease(CAD)and in-hospital clinical outcomes in patients with acute coronary syndrome(ACS).Methods:In this prospective and observational study,we recruited 500 patients with ACS.For all the eligible patients,demographic details were collected,and laboratory parameters were evaluated.The CAD severity was evaluated in terms of the number of involved vessels.The NLR was calculated based on neutrophils and lymphocytes and the correlation of various risk factors and severity and outcome of CAD was performed.Results:77.2%of Patients was male,and 52%of the patients aged between 55-70 years.Based on the type of ACS,396 out of 500 patients had ST-elevation myocardial infarction.An ascending trend in the white blood cell levels and NLR value was noted as the severity of the ACS increased and the highest white blood cell levels and NLR was noted among classⅣpatients.The mean NLR value among the non-survivors were higher compared to the survivors(9.52±5.72 vs.4.76±2.36;P<0.01).Receiver operating curve showed that the cut-off NLR value was 5.76 with a sensitivity of 75.0%and a specificity of 77.3%.Conclusions:The NLR can be used as an independent prognostic marker in ACS.An elevated NLR value serves as a reliable predictor for short-term complications,notably in-hospital mortality.