phospholipase C (PC-PLC), protects mice from lethal shock induced either by TNF-␣, LPS, or staphylococcal enterotoxin B (SEB) (14). Surprisingly, D609 did not reduce the SEB-in- duced IL-1, TNF-␣, or IFN-␥ levels in these animals (14). The proposed mechanism of action of D609 is via blockade of the cytotoxic action of TNF-␣. In another study, D609 improved the survival of mice with LPS-induced shock (20), accompa- nied by a reduction in the levels of IL-1 and IL-6, but not TNF-␣, in serum. In the present study, the modulatory effect of D609 on various cytokines and chemokines induced by both bacterial LPS and superantigens in human peripheralbloodmononuclearcells (PBMC) was further examined in vitro.
We examined the progressive and irreversible loss of antigen-specific lymphoproliferative responses in peripheralbloodmononuclearcells (PBMC) obtained from blood exposed for prolonged periods to EDTA as an anticoagulant. The responses of these lymphocytes to interleukin-2 or to concanavalin A were, however, unaffected. The observed loss was not due to depletion of metal ions by EDTA, since the addition of several divalent cations to whole blood during storage in EDTA or to lymphocytes from EDTA-stored blood during antigen stimulation in vitro did not alleviate the defect. Reconstitution of antigen-specific T-cell lines or Percoll-purified T cells with adherent antigen-presenting cells in antigen stimulation assays revealed that the presenting cells and not the effector T-cells were the targets of EDTA-mediated damage. The anticoagulant heparin helped to circumvent this problem. Surprisingly, EGTA, another metal ion chelator, could successfully replace EDTA, with a marginal loss in antigen-specific responses. Lymphoproliferative responses to antigens of Japanese encephalitis virus (JEV) and Mycobacterium tuberculosis were both significantly preserved in EGTA. JEV antigen-specific responses of PBMC obtained from the blood of convalescent JEV patients and stored in EGTA for as long as 24 h (n ⴝ 20) were comparable to those of fresh PBMC (n ⴝ 10), while PBMC from blood stored in EDTA (n ⴝ 17) for 16 h or longer failed to respond. We recommend that EGTA be used as the anticoagulant of choice for applications that require the lymphocyte proliferation assay, especially when on-site testing facilities are not available.
Loss of anti-human immunodeficiency virus type 1 (HIV-1) memory cytotoxic T-lymphocyte (CTLm) re- sponses is associated with disease progression in HIV-1 infection. In this study, nonspecific stimulation of peripheralbloodmononuclearcells (PBMC) from HIV-1-infected homosexual men with anti-CD3 monoclonal antibody (MAb) was compared with antigen-specific stimulation with inactivated, autologous B lymphoblastoid cells (B-LCL) infected with a vaccinia virus vector encoding HIV-1 IIIb Gag, Pol, and Env (VV-GPE) for activation of HIV-1-specific CTLm responses in a bulk lysis assay and by precursor frequency analysis. The results show that VV-GPE-infected B-LCL stimulated on average 10-fold greater anti-HIV-1 CTLm activity, as detected in the bulk lysis assay, and 55-fold-greater CTLm precursor frequencies specific for the three HIV-1 structural proteins than did stimulation with anti-CD3 MAb. This effect was noted with both freshly donated and frozen-thawed PBMC. The lysis was mediated by CD8 1 T cells and was restricted by the major histo- compatibility class I complex. These data indicate that antigen-specific stimulation with VV-GPE-infected B-LCL is a highly efficient method for detection of anti-HIV-1 CTLm responses that is applicable to noncurrent prospective studies with frozen PBMC.
Drug-resistant mutants of human immunodeficiency virus type 1 (HIV-1) recede below the limit of detection of most assays applied to plasma when selective pressure is altered due to changes in antiretroviral treatment (ART). Viral variants with different mutations are selected by the new ART when replication is not suppressed or wild-type variants with greater replication fitness outgrow mutants following the cessation of ART. Mutants selected by past ART appear to persist in reservoirs even when not detected in the plasma, and when conferring cross-resistance they can compromise the efficacy of novel ART. Oligonucleotide ligation assay (OLA) of virus in plasma and peripheralbloodmononuclearcells (PBMC) was compared to consensus sequence dideoxynucleotide chain terminator sequencing for detection of 91 drug resistance mutations that had receded below the limit of detection by sequencing of plasma. OLA of plasma virus detected 27.5% (95% confidence interval [CI], 19 to 39%) of mutant genotypes; consensus sequencing of the PBMC amplicon from the same specimen detected 23.1% (95% CI, 14 to 34%); and OLA of PBMC detected 53.8% (95% CI, 44 to 64%). These data suggest that concentrations of drug-resistant mutants were greater in PBMC than in plasma after changes in ART and indicate that the OLA was more sensitive than consensus sequencing in detecting low levels of select drug-resistant mutants.
In the present study, we have demonstrated that all three plants A. squamosa, D. metel, and M. piperita extracts have cytotoxic effects on cultured lymphocytes from human. Among three extracts, we found that A. squamosa showed the ability to inhibit cell survival. It is confirmed by three different methods: trypan blue dye exclusion assay, morphological observation of cells by three staining methods. Previous studies showed that the hot water extract of A. squamosa leaf has significant hypoglycemic and thus anti-diabetic activity in experimental animals  and seeds of plant have showed remarkable anti-microbial and cytotoxic activities , whereas ethanolic extract of leaf and stem has reported anticancer . The Annona sp. is a promising source of potential compounds exhibiting cytotoxic activities against PBMC and our investigation showed good result on PBMC. Further research is needed to identify active anitproliferative drug from aqueous extract of A. squamosa. In our study, D. metel leaf aqueous extract showed moderately cytotoxic effect on lymphocytes. This is in agreement with Soumen Roy’s work on evaluation of in vitro cytotoxic and antioxidant activity of D. metel and Cynodon dactylon extracts . The cytotoxicity of Labiatae family including the Mentha genus has been reported. Moreover, the Mentha preparations have been used for therapy of human cervical cancer . The cytotoxic effect of essential oil of M. pulegium on ovarian adenocarcinoma (SK-OV-3), human malignant cervix carcinoma (Hela) and human lung carcinoma (A549) cell lines has been shown by Shirazi et al. In this study methanolic extract at a concentration of 1000 μg/ml did not show any cytotoxic effect . On the other hand, in our study, there was gradual increase in cytotoxicity from 50- 150 mg/mL, which is 50 times greater than that in Shirazi’s study in terms of concentration used. Furthermore, they used clonogenic and neutral red (NR) assays for assessment of cytotoxicity, while we studied the effect of aqueous extract of M. piperita and used trypan blue dye exclusion for evaluation of its cytotoxicity.
Despite some indications from animal experiments , it is currently unknown, whether systemic inflam- masome activation is associated with human ageing. Our data present clear evidence for this hypothesis by demon- strating, that gene expression of AIM2, NLRP3, ASC, CASP1, CASP5, and IL1B in PBMC of vascular patients in- creases with age. Future studies on inflammasome gene ex- pression of PBMC in healthy people of different ages will be necessary to demonstrate whether this phenomenon ap- plies to ageing in general, although the definition of “healthy” might be difficult in individuals above 70 years.
Suspensions of peripheralbloodmononuclearcells (PBMC), monocytes, T or B lymphocytes, platelets or granulocytes, and cell-depleted supernatant fluids of these suspensions inhibited activation of Hageman factor (HF, Factor XII) by ellagic acid, a property not shared by erythrocytes. PBMC also inhibited HF activation by glass or
Figure 1 Peripheralbloodmononuclearcells (PBMC) adherence after no treatment (unTx), ethanol (EtOH Tx) treatment, EtOH and feline immunodeficiency virus (FIV) treatment (EtOH&FIV Tx) or FIV treatment (FIV Tx) of endothelial cells, astrocytes, and/or microglia. Astrocytes and/or microglia in the lower chamber of an in vitro cell culture insert system were treated or sham treated for 24 hours, and washed. Then inserts with confluent brain endothelial cells (BECs) were added to the well and cocultured with PBMCs for 24 hours. In the absence of astrocytes and microglia, BeCs received the treatment or control medium instead, and then were cocultured with PBMCs. error bars reflect standard error of the mean. Ethanol treatment showed dramatic, significant increases in mean adherence compared to untreated cells with all configurations of cells as did ethanol and FIV treatment (*P 0.05). The ethanol or ethanol and FIV treatment was significantly increased over FIV treatment within most configurations of cells ( † P 0.05). For the treatment group receiving ethanol and FIV (dark gray bar) among the different configurations, those including microglia were significantly decreased
Molecular docking study was performed with the Hex molecular modelling package version 8.0. 28 . Docking study of the synthesized compounds 4-15 were evaluated against PeripheralBloodMononuclearCells (PBMCs) (PDB ID: 3FLY). In the present study, an effort was made to evaluate their anti-cancer behaviour, we have selected PeripheralBloodMononuclearCells (PDB ID: 3FLY) to obtained docking scores (binding interaction energy). The results were tabulated in Table-4 and graphically presented in figure-2. The synthesized molecules 4-15 binds with various amino acid receptor (PDB ID: 3FLY) in the active pocket sites and given a molecular interaction energy (E- total value) at -219.91 to -265.00 (Kcal/mol). The compounds 6, 11, 12, 13 and 15 showed higher binding energy as compared with the compounds 4, 5, 7, 8, 9, 10 and 14. The estimated binding affinity of molecules 4-15 with the complex hydrogen network and other interactions with amino acids were MET78, LEU74, ILE84, ILE166, ASN155, LYS152, ASN155, ASP150, ASN155, LEU167, ASN155, GLY170, HIS148, SER208, TYR188, ILE212, SER208, LYS152 and APS150, which were presented in active sites of PBMCs respectively. It explained the role of hydrogen bond formation and other interactions for effective enzyme binding.
study and seen consecutively at the Department of Pediatrics and Respiratory Disease, Homeostasis and Cell Dysfunction Unit Research 99/UR/08-40, Abderrahman Mami Hospital (Ariana, Tunisia). Thirty children without asthma served as healthy controls. The Abderrahman Mami Hospital Ethics Committee approved the project, and the parents and children gave their informed consent to be enrolled in the study. Children with mild and moderate asthma were treated with regular inhaled glucocorticoids (ICS), but variable daily doses were required to control the symptoms (at the time of evaluation daily ICS dose ranged 100–1000 mg/day). Nine children with exacerbation were excluded from the study. Table 1 describes the characteristics of the asthmatic patients in the study: 60.5% had mild asthma and 39.5% had moderate asthma. The study analyzed TBX21, GATA-3, RORC, FOXP3, and EBI3 mRNA transcript in peripheralbloodmononuclearcells (PBMC) from young asthmatic patients compared to PBMC from healthy children (mean age ± SD: 14 ± 3.8 years; range: 4–15 years).
3. Banchereau J. Cells and Cytokines in Lung Inflammation. Mediators Inflamm. 1994; 3(1): 61-99. 4. Aliverdilo M MDM, Eftekhari Z, Paryan Mahdi. Evaluation of Lung Surfactant on Gene Expression of MCP-1 in PeripheralBloodMononuclearCells in Rabbit Model. Journal Of Animal Biology 2017;9 (4):67-77. 5. Frerking I, Günther A, Seeger W, Pison U. Pulmonary surfactant: functions, abnormalities and therapeutic options. Intensive care med. 2001; 27(11): 1699-717. 6. Alcorn JL. Innate Immunity and Pulmonary Inflammation: A Balance Between Protection and Disease. Translational Inflammation: Elsevier; 2019;153- 75.
In humans, memory T-cell subsets can be phenotypic- ally classified by CCR7 expression in combination with that of CD45RA . It was shown that the effector CD45RA + CD28 − CD8+ T-cell subset is expanded among peripheralblood lymphocytes in patients positive for the Human papillomavirus (HPV+) and with CC . Likewise, cytokines represent part of the complex pattern of the im- mune response, which can contribute to the development of cancer, as well as to combating it . In this study, we characterized the percentage of CD8+ T cells with the phenotype CD28–, CD16+/56+, and effector memory subsets in the peripheralbloodmononuclearcells (PBMC) of controls and patients with a diagnosis of human papil- lomavirus infection without neoplastic changes (HPV-I), as well as patients with CIN-I; we genotyped HPV in both groups. In addition, we measured plasma levels of Tumor necrosis factor alpha (TNF-α), Interferon gamma (IFN-γ), and Interleukin-6 (IL-6), IL-17, IL-10, IL-2, and IL-4.
The human immunodeficiency virus type 1-specific Vpu protein is a small integral membrane phosphopro- tein that induces degradation of the virus receptor CD4 in the endoplasmic reticulum and, independently, increases the release of progeny virions from infected cells. To address the importance of Vpu for virus replication in primary human cells such as peripheralbloodmononuclearcells (PBMC) and monocyte-derived macrophages (MDM), we used three different sets of monocyte-tropic molecular clones of human immunode- ficiency virus type 1: a primary isolate, AD8 1 , and two chimeric variants of the T-cell-tropic isolate NL4-3 carrying the env determinants of either AD8 1 or SF162 monocyte-tropic primary isolates. Isogenic variants of these chimeric viruses were constructed to express either wild-type Vpu or various mutants of Vpu. The effects of these mutations in the vpu gene on virus particle secretion from infected MDM or PBMC were assessed by determination of the release of virion-associated reverse transcriptase into culture supernatants, Western blot (immunoblot) analysis of pelleted virions, and steady-state or pulse-chase metabolic labeling. Wild-type Vpu increased virus release four- to sixfold in MDM and two- to threefold in PBMC, while nonphosphorylated Vpu and a C-terminal truncation mutant of Vpu were partially active on virus release in primary cells. These results demonstrate that Vpu regulates virus release in primary lymphocyte and macrophage cultures in a similar manner and to a similar extent to those previously observed in HeLa cells or CD4 1 T-cell lines. Thus, our findings provide evidence that Vpu functions in a variety of human cells, both primary cells and continuous cell lines, and mutations in Vpu affect its biological activity independent of the cell type and virus isolate used.
To elucidate the pathological roles of staphylococcal enterotoxin C (SEC) in bovine staphylococcal mastitis, a histopathological analysis of SEC-inoculated mammary glands was performed. SEC-inoculated mammary glands exhibited interstitial inflammation, and the leukocytes that migrated into the gland were predominantly polymorphonuclear neutrophils (PMN). In the gland cistern tissues dissected from SEC-inoculated mammary glands, epithelial cellular degeneration was observed. We also investigated the physiological effects of SEC on PMN in vitro. PMN migration was induced by culture supernatant of SEC-stimulated peripheralbloodmononuclearcells (S-PBMC sup) but not by that of nonstimulated PBMC (N-PBMC sup). The concentration of interleukin-8 was significantly (P < 0.05) higher in S-PBMC sup than N-PBMC sup, and a significantly (P < 0.05) higher mRNA expression of growth-regulated oncogenes was detected in SEC-stimulated PBMC than in nonstimulated PBMC. Milk PMN collected from SEC-inoculated mammary glands produced more than 2 times the amount of superoxide at 1 day postinoculation (dpi) than at 0 dpi in the presence of phorbol 12-myristate 13-acetate (PMA). PMN cultured with S-PBMC sup for 24 h also produced significantly (P < 0.05) larger amounts of superoxide than those cultured with N-PBMC sup in the presence of PMA. Moreover, S-PBMC sup induced the long-time survival of PMN. These results indicate that SEC induces the activation of PMN via the stimulation of mononuclearcells.
There is good evidence that C. pneumoniae circulates in the bloodstream as a cell-associated infection. We  and others  have demonstrated that the plasma fractions of PBMC-positive patients were uniformly negative for C. pneumoniae DNA. However, it remains unclear which cells within the peripheralbloodmononuclear cell layer con- tain C. pneumoniae. Peripheralbloodmononuclearcells contain monocytes, dendritic cells, and lymphocytes, and may be contaminated with polymorphonuclear cells or platelets. Using CD14 monoclonal selection, Maass et al  demonstrated a prevalence of 27.0% C. pneumoniae DNA positivity among patients presenting to hospital with acute coronary syndromes. It is unknown whether this prevalence was higher than in whole blood or buffy coat alone. Kaul et al  used adherence to select CD14 cells and found that the CD3+ lymphocytes fraction was positive more often (10 of 28 patients, 35.7%) than the "adherent" fraction of CD14+ cells (3 of 28, 10.7%). Fur- ther work is required to determine which cells are infect- ed, and whether cell enrichment would improve detection compared with mononuclear cell preparations.
The antigen-specific immune unresponsiveness seen in bancroftian filariasis was studied by examining lymphokine production in peripheralbloodmononuclearcells (PBMC) or PBMC subpopulations from 10 patients with asymptomatic microfilaremia, 13 patients with elephantiasis and 6 normal North Americans. In each group of patients, the kinetics of the lymphokine response and the response to mitogens and nonparasite antigens did not differ significantly. In marked contrast, when antigen-induced lymphokine production was
Two novel simian immunodeficiency virus (SIV) strains from wild-caught red-capped mangabeys (Cercocebus torquatus torquatus) from Nigeria were characterized. Sequence analysis of the fully sequenced SIV strain rcmNG411 (SIVrcmNG411) and gag and pol sequence of SIVrcmNG409 revealed that they were genetically most closely related to the recently characterized SIVrcm from Gabon (SIVrcmGB1). Thus, red-capped mangabeys from distant geographic locations harbor a common lineage of SIV. SIVrcmNG411 carried a vpx gene in addition to vpr, suggesting a common evolutionary ancestor with SIVsm (from sooty mangabeys). However, SIVrcm was only marginally closer to SIVsm in that region than to any of the other lentiviruses. SIVrcm showed the highest similarity in pol with SIVdrl, isolated from a drill, a primate that is phylogenetically distinct from mangabey monkeys, and clustered with other primate lentiviruses (primarily SIVcpz [from chimpanzees] and SIVagmSab [from African green monkeys]) discordantly in different regions of the genome, suggesting a history of recombination. Despite the genetic relationship to SIVcpz in the pol gene, SIVrcmNG411 did not replicate in chimpanzee peripheralbloodmononuclearcells (PBMC), although two other viruses unrelated to SIVcpz, SIVmndGB1 (from mandrills) and SIVlhoest (from L’Hoest monkeys), were able to grow in chimpanzee PBMC. The CCR5 24-bp deletion previously described in red-capped mangabeys from Gabon was also observed in Nigerian red-capped mangabeys, and SIVrcmNG411, like SIVrcmGB1, used CCR2B and STRL33 as coreceptors for virus entry. SIVrcm, SIVsm, SIVmndGB1, and all four SIVlhoest isolates but not SIVsun (from sun-tailed monkeys) replicated efficiently in human PBMC, suggesting that the ability to infect the human host can vary within one lineage.
The Quantitative real-time PCR was performed using SYBR ® Premix Ex Taq™ II (Takara Co., Ltd.), with specific primers (Sigma-Aldrich; Table 2) based on the provided guideline. The IL-4, GATA3, IL-17, RORC and GAPDH gene expression was performed using StepOnePlus™ Real-Time PCR Systems (Applied Biosystems, Foster City, CA, USA). The relative expressions of target gene mRNA, compared to the endogenous control, (GAPDH mRNA) were evaluated using the ΔCT method, with reference to each amplification plot (fluorescence signal vs cycle number). The mean difference (ΔCT) between the Table 2. Primer Sequences for the effects of β-D Mannuronic Acid on IL-4, GATA3, IL-17 and RORC gene expression in the peripheralbloodmononuclearcells (PBMC) of patients with inflammatory bowel diseases (IBD)
We characterized changes of gene expression in peripheralbloodmononuclearcells (PBMC) isolated at different times from rhesus macaques that were uninfected, infected with the virulent strain LCMV-WE, or infected with the nonvirulent strain LCMV-Armstrong (LCMV-Arm). Although arenavi- ruses replicate well in periarterial spaces, liver, spleen, and peripheral lymphoid organs soon after inoculation (46, 65), they cannot infect most PBMC (primarily lymphocytes and immature monocytes), and hence viremia does not appear until day 4 in this model. Our goal was to identify molecular events in blood that could (i) serve as markers for disease progression and (ii) discriminate between virulent and non- virulent infection. We characterized two major stages, previre- mic and viremic, and compared samples to uninfected samples or samples of blood from monkeys infected with LCMV that did not have disease. This analysis of the primate transcrip- tome after in vivo LCMV infection reveals critical molecular events that can be related to disease progression.