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PubMedDate not supplied

Proinflammatory cytokine production by mitogen-stimulated peripheral blood mononuclear cells (PBMCs) in trauma patients fed immune-enhancing enteral diets.

Widespread metabolic changes associated with injury facilitate the delivery of nutrients to the immune system. The effect of specific nutrients administered by the enteral route on the immune response in trauma victims is not well understood. The purpose of this study was to examine whether the synthesis of proinflammatory cytokines (tumor necrosis factor-alpha [TNF-alpha], interleukin-1 beta [IL-1 beta], and interleukin-6 [IL-6]) by peripheral blood mononuclear cells (PBMCs) are influenced by the nature of the dietary fat in critically injured trauma victims. We measured plasma TNF-alpha, IL-1 beta, and IL-6 and their release stimulated by phytohemagglutinin (PHA) and endotoxin (lipopolysaccharide, LPS) from PBMCs of 13 severely injured (injury severity score = 30 +/- 2) patients once within 48-60 h after injury and then after 7 d of enteral feeding (1.5 g protein[P].kg-1.d-1). Group I (n = 6) received diet A (Crucial) and group II (n = 7) received diet B (Impact). The plasma levels of TNF-alpha and IL-1 beta in trauma patients are not significantly different from those in healthy volunteers, but plasma IL-6 levels are significantly increased (five times) in severely injured patients. Stimulation of TNF-alpha and IL-1 beta secretion by LPS and PHA were significantly higher in patients than in control subjects; in contrast, there was no stimulation of IL-6 because of trauma or nutritional support by either of the diets. Stimulation of IL-1 beta by LPS was normalized by Crucial but was further enhanced by Impact. The higher fat content in Crucial may contribute in part to the apparent immunomodulation. Crucial seems to be a better choice in correcting the nutritional deficiency.

Adult↗
PubMed1999

The effect of simulated microgravity conditions on the TNF-alpha production by human PBMCS.

Our earlier space experiments demonstrated that the interferon production of human lymphocytes in microgravity is 4-8 times higher than those of the synchronous ground controls in vitro (Talas et al. 1983). These data suggested that the microgravity has a significant effect on cells. Since the possibilities to perform space-experiments are very limited and our study raised many interesting questions, we wished to simulate microgravity conditions in our laboratory. For this reason we purchased a Rotary Cell Culture System (RCCS) equipment to study different cell lines and human peripheral blood mononuclear cells (PBMCs) in experimental microgravity conditions. RCCS is a horizontally rotated bubble free culture vessel with membrane diffusion gas exchange. We report here an analysis of TNF-alpha (tumor necrosis factor-alpha) production by human PBMCs (control cultures exposed to simulated microgravity in RCCS). The cells were incubated in the presence or absence of either NDV (Newcastle Disease Virus) or one of the different forms (PHA-M or -P) of Phytohaemagglutinin.

Cell Culture Techniques↗
PubMed2001

Drug resistance testing in PBMCs of HIV infected patients.

HIV infected patients are considered a sort of reservoir having different genetically distinct viral variants (quasispecies), that evolve from the starting virus inoculum. Frequently, during replication, HIV can generate nucleotide differences in the new viral population; such genetic changes may be uninfluential in viral "fitness" (replication capacity) or give the virus some advantages under a selective pressure, due to immune response or drug treatment. The use of potent combination therapy for the treatment of HIV infections has certainly improved the "quality of life" for patients, decreasing the viral load in the plasma (HIV RNA). In our study, we investigated whether detection of drug resistance-related mutations was possible in circulating PBMCs, which represent a sort of genetic archive of viral drug resistances, when the levels of viral RNA were reduced to below 400 or 50 copies/ml, since, generally, plasma samples with more than 1,000 copies/ml of HIV RNA are needed to generate some results. The study was successfully performed sequencing proviral HIV DNA in PBMCs from 32 samples belonging to 25 patients, using a new modified protocol, that showed a good reproduciblity and very interesting data, also in patients with low or without circulating HIV RNA levels.

Anti-HIV Agents↗
PubMed2002

Effect of simulated microgravity on the production of IL-12 by PBMCs.

During space flight immunity is altered. This phenomenon is partly due to the microgravity condition itself. Our earlier space experiments (INTERFERON) indicated that microgravity has a significant effect at the cellular level. In our subsequent terrestrial studies we applied the Rotating Cell Culture System (RCCS) developed by NASA to mimick microgravity on ground. Previously we reported that human peripheral blood mononuclear cells (PBMCS) respond to simulated microgravity conditions with elevated tumor necrosis factor-alpha (TNF-alpha) production. We extended our investigations to the production of interleukin (IL)-12 under modelled microgravity conditions by separated PBMCs. In simulated microgravity we found significantly elevated level of secreted IL-12 compared to static, standard tissue culture conditions. Following a maximum of TNF-alpha production at 24 hours, the peak of IL-12 production was observed at 48 hours after the start of the experiment. Our results suggest that simulated microgravity favors the establishment of a Th1 type cytokine response.

Journal Article↗
PubMed2007

[Subpopulations and cytokine expression of naïve and memory T cells in normal human PBMCs].

AIM: To determine subpopulations and cytokine expression of naïve and memory T cells by polychromatic flow cytometry(PFC). METHODS: PBMCs were separated from normal human peripheral blood and stimulated by superantigen (SEB). After incubation for 5 hours, the cells were fixed, permeablized and stained with multiplecolor-labeled mAbs for cell surface and intracellular cytokines. Then they were detected by polychromatic flow cytometry and the data were analyzed by Flow Jo software. RESULTS: Based on the expression of CD45RO molecules, CD4(+) and CD8(+) T cells were classified as naïve and memory cells, which were further devided into several subsets according to the expression of CD62L and CCR7. When PBMCs were stimulated by SEB for 5 hours, CD45RO(+) and CD45RO(-) CD4(+) or CD8(+) T cells expressed IL-2, IFN-gamma and TNF-alpha. Further analysis indicated that CD62L(lo) CCR7(lo) and CCR7(+) T cells but not CD62L(hi) and CD62L(hi) CCR7(+) T cells expressed cytokines. In addition, the percentage of cytokine expression in CD62L(lo) CCR7(lo) subsets was markedly higher than that in CCR7(+) subsets. CONCLUSION: It is not scientific to identify naïve and memory cells in human T cells only based on the expression of CD45RO. Accurate determination of naïve, effector and memory cell populations can be achieved not only based on the expression of CD45RO but also according to the expression of CD62L.

CD4-Positive T-Lymphocytes↗
PubMed2002

The effect of X4 and R5 HIV-1 on C, C-C, and C-X-C chemokines during the early stages of infection in human PBMCs.

To better define a mechanism underlying the increase in expression of certain proinflammatory chemokines during HIV-1 infection, we analyzed the effect of X4 HIV-1 infection on C, C-C, and C-X-C chemokine mRNA levels. We demonstrate that X4 HIV-1 infection augments the expression of RANTES, IP-10, MCP-1, and Ltn in peripheral blood mononuclear cells (PBMCs). R5 HIV-1 also induces an increase in both IP-10 and MCP-1 production. Binding of UV-inactivated HIV-1 elevates MCP-1, RANTES, MIP-1alpha, MIP-1beta, and IL-8 expression, but fails to alter the production of IP-10, suggesting that the induction of IP-10 is dependent on downstream events following viral internalization. Indeed, recombinant gp120 alone was able to stimulate an eightfold increase in MCP-1 expression, but was unable to induce any detectable increase in IP-10 protein. HIV-induced modulation of chemokine expression suggests a mechanism by which HIV-infected monocytes and T cells might recruit target cells to sites of active viral replication, thus potentially aiding in the spread of the virus.

Chemokine CCL2↗
PubMed1994

Detection of CTL activity in PBMCS taken from HIV-ENV immunized individuals after in vitro viral infection.

The cellular immune response to the AIDS virus in healthy individuals immunized with HIV-1 antigens has not yet been entirely understood. Unlike HIV-1 infected patients where direct measurements of anti HIV-1 CTL activities can be readily performed with fresh peripheral blood mononuclear cells, uninfected volunteers immunized against HIV-1 antigens have fewer circulating CTL necessitating an in vitro activation in order to amplify the cytotoxic signal and make it measurable. This study presents experiments where specific CTLs are successfully obtained simply by in vitro infection of PBMCs from HIV-1 Envelope immunized individuals.

Acquired Immunodeficiency Syndrome↗
PubMed2006

IL-2 increased RANTES production and CD25 expression in cultured PBMCs only from antiretroviral treated HIV-1+ patients with detectable viral loads.

In order to better understand the possible beneficial effects of intermittent IL-2 treatment as complement of antiretroviral therapy in HIV-1+ patients, we have measured the levels of RANTES in the supernatants and the CD25 expression in cultured PBMCs obtained from HIV-1+ individuals in presence of IL-2. The results showed a significant increases in RANTES production and in the expression of CD25+ in the cultures with IL-2 of PBMC obtained from HIV-1+ patients with a detectable viral load in comparison with both, HIV-1+ patients with no detectable viral loads and with healthy individuals. These results suggest that therapeutic IL-2 administered in addition to highly active anti-retroviral therapy (HAART) may contribute to increase the effect of this therapy by rising both RANTES production and CD25 expression only in HIV-1+ patients with detectable viral loads.

Antigens, CD↗
PubMed2003

The HIV-1 chimeric protein CR3 expressed by poxviral vectors induces a diverse CD8+ T cell response in mice and is antigenic for PBMCs from HIV+ patients.

Recombinant avipoxvirus vectors are attractive for vaccination against human immunodeficiency virus type 1 (HIV-1), where induction of a cytotoxic CD8(+) T cell (CTL) response seems to be an important component of protective immunity. We expressed the chimeric protein CR3, composed by CTL epitopes rich regions from, RT, Gag and Nef and conserved Th cell epitopes from gp120, gp41 and Vpr of HIV-1 in a fowlpox virus (FWPV) vector (FPCR3), and used this vector to induce HIV-specific CTL responses in mice. Mice immunised twice intraperitoneally with FPCR3, developed a CD8(+) T cell response measured as production of IFN-gamma by splenocytes in response to stimulation with P815 cells infected with recombinant vaccinia viruses (rVV) expressing CR3, Gag and Nef. The number of IFN-gamma secreting cells was markedly higher when a P815 cell line constitutively expressing CR3 was used as target cells for Enzyme-linked-immunospot (ELISPOT). CR3 epitopes were also specifically recognised by human PBMCs from three HIV(+) patients with different haplotypes. These results confirm the potential of FWPV vectors expressing these novel HIV-1 chimeric proteins to induce a simultaneous CD8(+) T cell response against conserved viral targets and early expressed regulatory proteins.

Animals↗
PubMed1997

Simian immunodeficiency viruses containing mutations in the long terminal repeat NF-kappa B or Sp1 binding sites replicate efficiently in T cells and PHA-stimulated PBMCs.

The long terminal repeats (LTRs) of primate lentiviruses contain conserved binding sites for the NF-kappa B and Sp1 cellular transcription factors. In order to study the role that these sites play in simian immunodeficiency virus (SIV) replication, we have introduced mutations that disrupt either the NF-kappa B or Sp1 binding sites in the LTR of an infectious molecular clone of SIVmac239. An additional mutation also disrupted the SF3 transcription factor binding site that overlaps the NF-kappa B site. Viruses containing point mutations or deletions of the NF-kappa B, SF3, or Sp1 binding sites retained the ability to replicate efficiently in the CEMx174 and MT4 cell lines, as well as in PHA-stimulated primary rhesus macaque peripheral blood mononuclear cells (PBMCs). Efficient replication of SIVs mutated in either NF-kappa B or Sp1 binding sites suggests that the SIV LTR promoter contains multiple functionally redundant elements capable of supporting sufficient transcription to allow productive viral replication.

Animals↗
PubMed2004

The effects of calpain inhibition on IkB alpha degradation after activation of PBMCs: identification of the calpain cleavage sites.

Human peripheral blood mononuclear cells (PBMCs) were activated using anti-CD3/CD28 (HIT3A/CD28.2) resulting in degradation of IkB alpha, an inhibitor of NFkB, relative to unactivated cells. Degradation of IkB alpha began by 30 min and proceeded for at least 5 h. Calpeptin, a calpain inhibitor, inhibited IkB alpha degradation in a time- and dose-dependent manner. Furthermore, calpain inhibition increased IkB alpha levels compared to nonactivated controls. Recombinant IkB alpha was incubated with purified porcine m-calpain in the presence of 0.1% Triton X-100, and the degradation products were monitored by SDS-PAGE and sequenced. Most of the degradation products were peptides derived from calpain, but one was derived from IkB alpha cleaved between amino acids 50 and 51 (glutamine and glutamic acid). The liberated fragment included the entire signal response domain (SRD), a region containing key serine and threonine residues necessary for phosphorylation by the IKKinase complex and sites required for ubiquitination. The results suggest that calpain plays an important role in IkB alpha degradation, a crucial event in T cell activation.

Amino Acid Sequence↗
PubMed2000

PBMCs from both atopic asthmatic and nonatopic children show a TH2 cytokine response to house dust mite allergen.

BACKGROUND: The hypothesis that in atopic diseases the T-helper response is skewed toward a T(H)2-type cytokine response was based on studies with mitogen stimulation, T-cell clones, or both. OBJECTIVE: Using primary cultures, we investigated (1) whether atopic asthmatic patients have a T(H)2 response and nonatopic subjects have a T(H)1 response to allergen and (2) whether atopic patients have a decreased ability to mount T(H)1 immune responses to mycobacterial antigens. METHODS: The responses of PBMCs to allergen (house dust mite [HDM]) or purified protein derivative of Mycobacterium tuberculosis (PPD) stimulation from 10 severely and 14 moderately asthmatic patients (all allergic to HDM) were compared with those of 17 nonatopic healthy black (Xhosa) children. RESULTS: HDM-stimulated proliferation, IL-5 release, and the IL-5/IFN-gamma ratio were significantly increased in subjects with atopic asthma, whereas IFN-gamma release was not significantly different. IL-4 levels were below the level of detection. PPD-stimulated proliferation, IL-5 release, IFN-gamma release, and the IL-5/IFN-gamma ratio were not significantly different among the groups. Each group had a significantly higher IL-5/IFN-gamma ratio in response to HDM than to PPD (a T(H)1 stimulus). CONCLUSION: Our study, which used primary cultures to investigate the hypothesis that nonatopic subjects have a T(H)1 response to allergens, indicates that HDM stimulates a T(H)2 cytokine response in both atopic and nonatopic subjects but that the response is enhanced in atopic patients. Our results with PPD suggest that normal and atopic asthmatic subjects can have a T(H)1 cytokine response to mycobacteria, but there is a subgroup of atopic subjects that have a T(H)2 response.

Allergens↗
PubMed2001

Immunostimulatory sequence DNA linked to the Amb a 1 allergen promotes T(H)1 cytokine expression while downregulating T(H)2 cytokine expression in PBMCs from human patients with ragweed allergy.

BACKGROUND: Recent studies have demonstrated that bacterially derived immunostimulatory sequences (ISSs) of DNA can activate the mammalian innate immune system and promote the development of T(H)1 cells. Promotion of T(H)1 immunity by means of immunotherapy in allergic patients has led to the alleviation of symptoms that result from allergen-specific T(H)2 responses. OBJECTIVE: Our purpose was to investigate whether the T(H)1-enhancing properties of ISSs could be used to alter the T(H)2-dominated immune response of allergic PBMCs in vitro. METHODS: Ragweed protein-linked ISS (PLI) was generated from a specific, highly active 22-base ISS and Amb a 1, the immunodominant allergen in ragweed pollen, to combine the T(H)1-enhancing properties of ISSs with allergen selectivity, and its activity was investigated in PBMC cultures from subjects with ragweed allergy. RESULTS: PLI was markedly successful at reversing the dominant allergen-induced T(H)2 profile while greatly enhancing IFN-gamma production. Delivering ISSs in a linked form proved to be much more effective at modulating the resulting cytokine profile than delivering free ISSs in a mixture with unlinked Amb a 1. PLI also demonstrated cytokine-modulating properties, even when used to stimulate cells that had already been primed for 6 days with Amb a 1. The antigen specificity of the action of PLI was confirmed by the observations that PLI enhances Amb a 1--specific T-cell proliferation. CONCLUSION: These data indicate that delivery of ISSs within an antigen-specific context exhibits potent cytokine-modulating activity and, combined with its reduced allergenicity, makes this molecule a strong candidate for use in improved immunotherapy applications.

Adjuvants, Immunologic↗
PubMed2001

The immunomodulatory drugs cyclosporin A, mycophenolate mofetil, and sirolimus (rapamycin) inhibit allergen-induced proliferation and IL-5 production by PBMCs from atopic asthmatic patients.

We have used an optimized, physiologically relevant in vitro assay system to show that in a concentration-dependent fashion the immunomodulatory drugs cyclosporin A, mycophenolate mofetil, and sirolimus (rapamycin), as well as the glucocorticoid dexamethasone, inhibit allergen-driven T-cell proliferation and IL-5 production in PBMCs from allergen-sensitized atopic asthmatic individuals at physiologic concentrations. This effect of cyclosporin A might at least partially account for its established clinical efficacy in sparing systemic glucocorticoid therapy while improving lung function in chronic, severe, glucocorticoid-dependent asthma. The data are also compatible with the hypothesis that the newer immunomodulatory drugs mycophenolate mofetil and sirolimus exert similar effects, perhaps with a more favorable benefit/risk ratio.

Asthma↗
PubMed2002

Evidence for aberrant regulation of the p21Ras pathway in PBMCs of patients with chronic idiopathic urticaria.

BACKGROUND: Recent data suggest that a subpopulation of patients with chronic urticaria has an autoimmune disorder. Aberrant expression and regulation of the p21Ras pathway has been reported in lymphoid cells in a variety of systemic autoimmune diseases but not in chronic idiopathic urticaria (CIU). OBJECTIVES: The aim of this study was to examine the expression, regulation, and function of the p21Ras pathway in patients with CIU. METHODS: Twenty-four patients with CIU and 14 control subjects were enrolled. All patients and 9 control subjects were intradermally injected with autologous serum. PBMCs were isolated, and the p21Ras and its regulatory proteins were studied. RESULTS: We found increased expression of the p21ras proto-oncogene in patients with CIU. This was associated with a low expression of the p21Ras stimulatory element human son of sevenless (hSOS1) but normally expressed p21Ras inhibitory element p120GTPase-activating protein. The basal nonstimulated membrane/cytoplasmic ratio of hSOS1, which indicates the p21Ras pathway activity, was higher in patients compared with that seen in control subjects. Moreover, after stimulation, both patients and control subjects decreased their hSOS1 membrane/cytoplasmic ratio. The magnitude of this decrease was much higher in patients than in control subjects: 14- and approximately 2-fold, respectively. The basal and stimulated activities of the p21Ras downstream key regulatory enzyme mitogen-activated protein kinase were comparable in patients and control subjects, as was their in vitro mitogen-stimulated lymphocyte proliferation. CONCLUSION: Our data demonstrate for the first time an aberrant signaling through the p21Ras pathway in lymphocytes of patients with CIU. This finding further supports the autoimmune basis of this disease.

Adult↗
PubMed2005

CD8+ T cells specific for a potential HLA-A*0201 epitope from Chlamydophila pneumoniae are present in the PBMCs from infected patients.

Infection with the common pathogen Chlamydophila pneumoniae (Cpn, previously Chlamydia pneumoniae) has a high prevalence in patients suffering from arteriosclerosis and may trigger or contribute to heart disease. In mice, CD8-positive T cells are critical for the eradication of the infection and the development of immune memory against Cpn. Although several H2-class I epitopes have been described, no HLA-class I-associated peptides from Cpn are known. In order to define HLA-A*0201 epitopes from Cpn, we focused on the bacterial heat shock proteins (HSP) 60 and 70 which are known to be recognized by the immune system. Using epitope prediction, peptide binding studies and peptide-specific CTLs from HLA-A2 transgenic mice, we could define a potential HSP-70-derived epitope. The study of PBMCs from Cpn-infected individuals using fluorescent MHC tetramers revealed that some patients have CD8(+) T cells capable of recognizing the Cpn HSP-70 HLA-A*0201 epitope. Our studies pave the way to the immunomonitoring of the anti-Cpn CTL immune response present in patients suffering from different diseases potentially linked to Cpn or anti-Cpn immunity.

Animals↗
PubMed2003

Identification of a sequence from the genome of porcine circovirus type 2 with an inhibitory effect on IFN-alpha production by porcine PBMCs.

Porcine circovirus type 2 (PCV-2) has been identified as the causal agent of postweaning multisystemic wasting syndrome and has been associated with several other disease syndromes in pigs. To date, however, little is known regarding the mechanism(s) underlying the pathogenesis of PCV-2-induced diseases and the interaction of the virus with the host immune system. In the present study, oligodeoxynucleotides (ODNs), with central CpG motifs selected from the genome of PCV-2, were demonstrated to modulate the immune response of porcine PBMCs. Four of the five ODNs tested were demonstrated to act in a stimulatory manner via induction of IFN-alpha production, whereas only one of the five ODNs showed inhibitory activity. Also, this inhibitory ODN was demonstrated to completely inhibit IFN-alpha production induced by the other stimulatory ODNs and showed a variable degree of inhibitory action on other known inducers of IFN-alpha. Although no single common characteristic among resistant or susceptible inducers could be identified, the presence of immune modulatory sequences in the genome of PCV-2 may represent an underlying mechanism of the pathogenesis of PCV-2-associated diseases.

Animals↗
PubMed2005

Comparison of apoptosis and mortality measurements in peripheral blood mononuclear cells (PBMCs) using multiple methods.

Death through apoptosis is the main process by which aged cells that have lost their function are eliminated. Apoptotic cells are usually detected microscopically by changes in their morphology. However, determination of early apoptotic events is important for in vitro (and ex vivo) studies. The main objective of the present study is to find the most sensitive method for apoptosis detection in human peripheral blood mononuclear cells (PBMCs) by comparing six different methods following five different means of immunological stimulation at 3 and 5 days. Each of six apoptosis quantification methods, except the trypan blue exclusion test, is a combination of two stains, one for the specific detection of apoptotic cells and the other for the unspecific detection of dead cells. Values for apoptosis and mortality were compared with a reference method. The choice of apoptosis detection method is more important following 3 days of stimulation than after 5 days of stimulation (P=2x10(-6) versus P=1x10(-2)). In contrast, we find mortality measurements following the different means of stimulation highly significant at both 3 and 5 days (F2.28=7.9, P=1.4x10(-6) at 3 days and F2.28=8.5, P=4.5x10(-7) at 5 days). Variation as a result of the combination of specific PBMC stimulation and the method used to detect apoptosis is reduced considerably with time (F1.58+3.7, P+3x10(-7) at 3 days to F=1.58=0.97, P=0.5 at 5 days). Based on Tukey's test, YO-PRO-1 is the most sensitive stain for apoptosis and, when combined with 7-AAD, provides an accurate measure of apoptosis and mortality. In conclusion, we propose YO-PRO-1/7-AAD as a new combination and low-cost alternative for the sensitive detection of early apoptosis.

Antibodies↗