Peritoneal Mononuclear Cells Research Articles

Changes in MCP-1, HGF, and IGF-1 expression in endometrial stromal cells, PBMCs, and PFMCs of endometriotic women following 1,25(OH)2D3 treatment.

1,25(OH)2D3 has anti-inflammatory and growth inhibitory effects. Our study explored the effect of 1,25(OH)2D3 treatment on the expression of monocyte chemotactic protein-1 (MCP-1), hepatocyte growth factor (HGF), and insulin-like growth factor-1 (IGF-1) by peripheral blood mononuclear cells (PBMCs), peritoneal fluid mononuclear cells (PFMCs), endometrial stromal cells (ESCs), and its effect on the proliferation of PBMCs and PFMCs of patients with endometriosis compared with controls. PBMCs, PFMCs, and ESCs were obtained from 10 endometriosis patients and 10 non-endometriotic individuals. After treating cells with 0.1μM of 1,25(OH)2D3 for 6, 24, and 48 h, the gene and protein expression of mentioned factors were evaluated by real-time PCR and ELISA methods, respectively. 1,25(OH)2D3 treatment significantly reduced the protein expression of MCP-1, HGF, and IGF-1 in PBMCs and PFMCs of endometriotic patients at 48 h (p< 0.05-<0.01). Also, this treatment significantly reduced MCP-1, HGF, and IGF-1 gene and/or protein expression in EESCs and EuESCs at 24 and 48 h (p< 0.05-<0.01). 1,25(OH)2D3 treatment also reduced the proliferation of PBMCs and PFMCs of endometriotic patients compared with controls (p < 0.01). 1,25(OH)2D3 can be considered as a potentially effective agent in the prevention and treatment of endometriosis along with other therapies.

P38: The immunoregulatory effect of 1,25‐Dihydroxy Vitamin D in peritoneal mononuclear cells of endometriosis patients

P38: The immunoregulatory effect of 1,25‐Dihydroxy Vitamin D in peritoneal mononuclear cells of endometriosis patients

Glucosylceramides From Lomentospora prolificans Induce a Differential Production of Cytokines and Increases the Microbicidal Activity of Macrophages.

Lomentospora prolificans is an emerging opportunistic fungus with a high resistance to antifungal agents and it can cause localized infections in immunocompetent patients and disseminated infections with a high mortality rate in immunosuppressed patients. Glucosylceramides (GlcCer) are synthetized in the majority of known fungal pathogens. They are bioactive molecules presenting different functions, such as involvement in fungal growth and morphological transitions in several fungi. The elucidation of the primary structure of the fungal surface glycoconjugates could contribute for the understanding of the mechanisms of pathogenicity. In this work, GlcCer species were isolated from mycelium and conidia forms of L. prolificans and their chemical structures were elucidated by mass spectrometry (ESI-MS). GlcCer purified from both forms presented a major species at m/z 750 that corresponds to N-2-hydroxyhexadecanoyl-1-β-D-glucopyranosyl-9-methyl-4,8-sphingadienine. Monoclonal antibodies against GlcCer could recognize L. prolificans GlcCer species from mycelium and conidia, suggesting a conserved epitope in fungal GlcCer. In addition, in vivo assays showed that purified GlcCer species from both forms was able to induce a high secretion of pro-inflammatory cytokines by splenocytes. GlcCer species also promote the recruitment of polymorphonuclear, eosinophils, small peritoneal macrophage (SPM) and mononuclear cells to the peritoneal cavity. GlcCer species were also able to induce the oxidative burst by peritoneal macrophages with NO and superoxide radicals production, and to increase the killing of L. prolificans conidia by peritoneal macrophages. These results indicate that GlcCer species from L. prolificans are a potent immune response activator.

Two Distinct Subsets Are Identified from the Peritoneal Myeloid Mononuclear Cells Expressing both CD11c and CD115

To this date, the criteria to distinguish peritoneal macrophages and dendritic cells (DCs) are not clear. Here we delineate the subsets of myeloid mononuclear cells in the mouse peritoneal cavity. Considering phenotypical, functional, and ontogenic features, peritoneal myeloid mononuclear cells are divided into 5 subsets: large peritoneal macrophages (LPMs), small peritoneal macrophages (SPMs), DCs, and 2 MHCII+CD11c+CD115+ subpopulations (i.e., MHCII+CD11c+CD115+CD14−CD206− and MHCII+CD11c+CD115+CD14+CD206+). Among them, 2 subsets of competent Ag presenting cells are demonstrated with distinct functional characteristics, one being DCs and the other being MHCII+CD11c+CD115+CD14−CD206− cells. DCs are able to promote fully activated T cells and superior in expanding cytokine producing inflammatory T cells, whereas MHCII+CD11c+CD115+CD14−CD206− cells generate partially activated T cells and possess a greater ability to induce Treg under TGF-β and retinoic acid conditions. While the development of DCs and MHCII+CD11c+CD115+CD14−CD206− cells are responsive to the treatment of FLT3 ligand and GM-CSF, the number of LPMs, SPMs, and MHCII+CD11c+CD115+CD14+CD206+ cells are only influenced by the injection of GM-CSF. In addition, the analysis of gene expression profiles among MHCII+ peritoneal myeloid mononuclear cells reveals that MHCII+CD11c+CD115+CD14+CD206+ cells share high similarity with SPMs, whereas MHCII+CD11c+CD115+CD14−CD206− cells are related to peritoneal DC2s. Collectively, our study identifies 2 distinct subpopulations of MHCII+CD11c+CD115+ cells, 1) MHCII+CD11c+CD115+CD14−CD206− cells closely related to peritoneal DC2s and 2) MHCII+CD11c+CD115+CD14+CD206+ cells to SPMs.

Anti-inflammatory activities of Aedes aegypti cecropins and their protection against murine endotoxin shock

BackgroundMosquitoes are armed with physiologically active compounds to suppress the host immunity including host inflammatory reaction. However, the specific anti-inflammatory components in mosquitoes remain unknown.ResultsBy searching for the immunomodulatory molecules from the mosquito Aedes aegypti (Diptera: Culicidae) at NCBI for anti-inflammatory function, five cecropins (for short in this study: AeaeCec1, 2, 3, 4 and 5) were selected. AeaeCec1-5 efficiently inhibited the expression of inducible nitric oxide synthase (iNOS), nitrite, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) in lipopolysaccharide (LPS)-stimulated mouse peritoneal macrophages and human peripheral blood mononuclear cells (PBMCs) with low toxicity to mammalian cells. Among the five analogues, AeaeCec5 had the strongest anti-inflammatory activity, and generated an additive effect with other AeaeCec peptides. In a mouse model of endotoxin shock, AeaeCec1-5 effectively reduced TNF-α, IL-1β and IL-6 expression in lungs, serum and peritoneal lavage and correspondingly reduced lung damage and edema, with AeaeCec5 showing the best protection. In mice infected with Escherichia coli or Pseudomonas aeruginosa, administration of AeaeCec5 reduced the production of TNF-α, IL-1β and IL-6 and correspondingly reduced lung tissue damage. These effects of Ae. aegypti AeaeCec1-5 were attributed to an efficient inhibition of the activation of mitogen-activated protein kinases (MAPKs) and transcriptional nuclear factor-κB (NF-κB) signaling pathways, as well as partial neutralization of LPS.ConclusionsThe current work characterized the specific anti-inflammatory agents in Ae. aegypti and provided AeaeCec5 as a potent anti-endotoxin peptide that could serve as the basis for the development of anti-inflammatory therapy.

Intraperitoneal infusion of mesenchymal stem cell attenuates severity of collagen antibody induced arthritis.

It is unclear how systemic administration of mesenchymal stem cells (MSCs) controls local inflammation. The aim of this study was to evaluate the therapeutic effects of human MSCs on inflammatory arthritis and to identify the underlying mechanisms. Mice with collagen antibody-induced arthritis (CAIA) received two intraperitoneal injections of human bone marrow-derived MSCs. The clinical and histological features of injected CAIA were then compared with those of non-injected mice. The effect of MSCs on induction of regulatory T cells was examined both in vitro and in vivo. We also examined multiple cytokines secreted by peritoneal mononuclear cells, along with migration of MSCs in the presence of stromal cell-derived factor-1 alpha (SDF-1α) and/or regulated on activation, normal T cell expressed and secreted (RANTES). Sections of CAIA mouse joints and spleen were stained for human anti-nuclear antibodies (ANAs) to confirm migration of injected human MSCs. The results showed that MSCs alleviated the clinical and histological signs of synovitis in CAIA mice. Peritoneal lavage cells from mice treated with MSCs expressed higher levels of SDF-1α and RANTES than those from mice not treated with MSCs. MSC migration was more prevalent in the presence of SDF-1α and/or RANTES. MSCs induced CD4+ T cells to differentiate into regulatory T cells in vitro, and expression of FOXP3 mRNA was upregulated in the forepaws of MSC-treated CAIA mice. Synovial and splenic tissues from CAIA mice receiving human MSCs were positive for human ANA, suggesting recruitment of MSCs. Taken together, these results suggest that MSCs migrate into inflamed tissues and directly induce the differentiation of CD4+ T cells into regulatory T cells, which then suppress inflammation. Thus, systemic administration of MSCs may be a therapeutic option for rheumatoid arthritis.

PAI-1 in granulosa cells is suppressed directly by statin and indirectly by suppressing TGF-β and TNF-α in mononuclear cells by insulin-sensitizing drugs.

Plasminogen activator inhibitor-1 (PAI-1) is elevated in women with polycystic ovary syndrome (PCOS), but the regulation in granulosa cells (GCs) is unclear. PAI-1 expression in PCOS ovaries was investigated immunohistologically. PAI-1 expressions in HGrC1, a human GC cell line, were investigated at mRNA and activity levels. The expressions of TGF-β and TNF-α in peritoneal fluid mononuclear cells (PFMCs) were measured with quantitative PCR. Little PAI-1 expression is observed in healthy GCs, whereas GCs of PCOS and atretic follicle exhibit distinct expression in vivo. In vitro study using HGrC1 shows that TGF-β and TNF-α increase PAI-1 mRNA and its activity, and both together exhibit a synergistic effect. The expression of PAI-1 mRNA is suppressed by simvastatin. Moreover, insulin-sensitizing drugs (metformin, pioglitazone, and rosiglitazone) suppress LPS-induced TGF-β and TNF-α mRNA expression in PFMC. Statin and insulin-sensitizing drugs may provide a potential therapy for PCOS via down-regulation of PAI-1 expression in GCs and down-regulation of TGF-β and TNF-α expression in PFMC, respectively.

Cranberry Proanthocyanidins - Protein complexes for macrophage activation.

In this work we characterize the interaction of cranberry (Vaccinium macrocarpon) proanthocyanidins (PAC) with bovine serum albumin (BSA) and hen egg-white lysozyme (HEL) and determine the effects of these complexes on macrophage activation and antigen presentation. We isolated PAC from cranberry and complexed the isolated PAC with BSA and HEL. The properties of the PAC-protein complexes were studied by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS), gel electrophoresis and zeta-potential. The effects of PAC-BSA complexes on macrophage activation were studied in RAW 264.7 macrophage like cells after treatment with lipopolysaccharide (LPS). Fluorescence microscopy was used to study the endocytosis of PAC-BSA complexes. The effects of the PAC complexes on macrophage antigen presentation were studied in an in vitro model of HEL antigen presentation by mouse peritoneal mononuclear cells to a T-cell hybridoma. The mass spectra of the PAC complexes with BSA and HEL differed from the spectra of the proteins alone by the presence of broad shoulders on the singly and doubly charged protein peaks. Complexation with PAC altered the electrophoretic mobility shift assay in native agarose gel and the electrophoretic mobility (ζ-potential) values. These results indicate that the PAC-protein complexes are stable and alter the protein structure without precipitating the protein. Fluorescence microscopy showed that the RAW 264.7 macrophages endocytosed BSA and PAC-BSA complexes in discrete vesicles that surrounded the nucleus. Macrophages treated with increasing amounts of PAC-BSA complexes had significantly reduced COX-2 and iNOS expression in response to treatment with lipopolysaccharide (LPS) in comparison to the controls. The PAC-HEL complexes modulated antigen uptake, processing and presentation in murine peritoneal macrophages. After 4 h of pre-incubation, only trace amounts of IL-2 were detected in the co-cultures treated with HEL alone, whereas the PAC-HEL complex had already reached the maximum IL-2 expression. Cranberry PAC may increase the rate of endocytosis of HEL and subsequent expression of IL-2 by the T-cell hybridomas. These results suggest that PAC-protein complexes modulate aspects of innate and acquired immune responses in macrophages.

Alpha-7 nicotinic acetylcholine receptor (nAChR) agonist inhibits the development of endometriosis by regulating inflammation.

We investigated α-7 nAchR expression in human peritoneal macrophages and examined whether activation of nAchR might be a new therapy for endometriosis. Human peritoneal fluid mononuclear cells (PFMC) were stimulated with lipopolysaccharide (LPS) in the presence of α-7 nAChR agonists. In a murine endometriosis model, α-7 nAChR modulators were administered. Human PFMC expressed α-7 nAChR at the mRNA and protein levels. Activation of α-7 nAChR with its agonists led to significant (P<.01) suppression of LPS-induced interleukin (IL) -1β expression. In a murine endometriosis model, one week after inoculation of endometrium to the peritoneal cavity, α-7 nAChR agonist significantly suppressed the expression of IL-1β mRNA (P<.01), which was negated when α-7 nAChR antagonist was administered simultaneously. α-7 nAChR agonist significantly suppressed the formation of endometriotic lesions, which was reversed with α-7 nAChR antagonist. Activation of nAChR might be a new candidate for treatment of endometriosis.

Regulation of apoptotic pathways during endometriosis: from the molecular basis to the future perspectives

Endometriosis is defined as the presence of endometrial-like endometrial cells, glands and stroma outside the uterus, causing a strong inflammatory-like microenvironment in the affected tissue. This may provoke a breakdown in the peritoneal cavity homeostasis, with the consequent processes of immune alteration, documented by peripheral mononuclear cells recruitment and secretion of inflammatory cytokines in early phases and of angiogenic and fibrogenic cytokines in the late stages of the disease. Considering the pivotal role of interaction between immune and endometriotic cells, in this paper, we aim to shed light about the role of apoptosis pathways in modulating the fine-regulated peritoneal microenvironment during endometriosis. Narrative overview, synthesizing the findings of literature retrieved from searches of computerized databases. In normal conditions, endometriotic cells, refluxed through the fallopian tubes into the peritoneal cavity, should be attacked and removed by phagocytes and NK cells. During endometriosis, the breakdown of peritoneal homeostasis causes the failure of scavenging mechanisms, allowing the survival of endometriotic cells. The consequent so-called "immunoescaping" of endometriotic cells could be due, at least in part, to the reduction of apoptotic-mediated pathways previously described. Considering the large amount of evidence retrieved from in vitro as well as in vivo models, the reduced apoptosis of endometriotic cells together with the increased apoptosis of peritoneal fluid mononuclear cells may address the peritoneal homeostasis to a permissive environment for the progression of the disease.

Cinnamaldehyde modulates LPS-induced systemic inflammatory response syndrome through TRPA1-dependent and independent mechanisms

Cinnamaldehyde is a natural essential oil suggested to possess anti-bacterial and anti-inflammatory properties; and to activate transient receptor potential ankyrin 1 (TRPA1) channels expressed on neuronal and non-neuronal cells. Here, we investigated the immunomodulatory effects of cinnamaldehyde in an in vivo model of systemic inflammatory response syndrome (SIRS) induced by lipopolysaccharide. Swiss mice received a single oral treatment with cinnamaldehyde 1h before LPS injection. To investigate whether cinnamaldehyde effects are dependent on TRPA1 activation, animals were treated subcutaneously with the selective TRPA1 antagonist HC-030031 5min prior to cinnamaldehyde administration. Vehicle-treated mice were used as controls. Cinnamaldehyde ameliorated SIRS severity in LPS-injected animals. Diminished numbers of circulating mononuclear cells and increased numbers of peritoneal mononuclear and polymorphonuclear cell numbers were also observed. Cinnamaldehyde augmented the number of peritoneal Ly6Chigh and Ly6Clow monocyte/macrophage cells in LPS-injected mice. Reduced levels of nitric oxide, plasma TNFα and plasma and peritoneal IL-10 were also detected. Additionally, IL-1β levels were increased in the same animals. TRPA1 antagonism by HC-030031 reversed the changes in the number of circulating and peritoneal leukocytes in cinnamaldehyde-treated animals, whilst increasing the levels of peritoneal IL-10 and reducing peritoneal IL-1β. Overall, cinnamaldehyde modulates SIRS through TRPA1-dependent and independent mechanisms.

The history and current status of anti-sperm antibody research

we have studied the implication of bone morphogenetic proteins (BMP) in the ovarian physiology. In the present study,weexhibit ourdata regarding (1) folliculogenesis, (2) ovulation and (3) polycystic ovary syndrome (PCOS), especially from the point of the relationship between ovarian cells and immune-competent cells. Folliculogenesis:Although the formation of an individual capillary network in the theca cell layer is required for ovarian folliculogenesis, the mechanism has not been clarified. We found that theca cell derived BMP-7 stimulated VEGF secretion levels in GC significantly. In endothelial cells, BMP-7 increased the cell number and induced VEGF receptor mRNA. BMP-7 might work to form vasculature around follicles via induction of VEGF expression in GC and increased sensitivity of endothelial cells to VEGF. Ovulation: As granulocyte colony-stimulating factor (G-CSF) is known to be a candidate for a treatment of luteinized unruptured follicle (LUF), neutrophils might be involved in ovulatory process. BMP cytokine is known to regulate ovulation, as BMP-6 null mice exhibit a decrease in the number of ovulatory follicleswithout effect on either themorphology or the dynamics of follicular development. We found that BMP-6 significantly increased growthregulated oncogene (GRO)levels in human GC. On the other hand, BMP-6 suppressed the relative expression of the protease inhibitors, secretory leukocyte peptidase inhibitor (SLPI) in GC. BMP-6 might play a role in ovulation by increasing the accumulation of neutrophils in the ovulatory follicle and suppressing the effect of protease inhibitor. PCOS: Plasminogen activator inhibitor-1 (PAI-1), the main physiological inhibitor of plasminogen activation, is elevated in women with PCOS and has been linked to PCOS in a mouse model of the disease. We have found that TGFand TNFwhich are involved in the etiology of PCOS, increased PAI-1 secretion levels by cultured GC. Insulin sensitizing drugs suppressed TGFand TNFmRNA expression in peritoneal fluid mononuclear cells. Our data suggested that insulin sensitizing agentsmayprovide a potential therapy for PCOS via down regulation of PAI-1 expression indirectly. To understand the etiology of PCOS, the regulation of immune-competent cells should be taken into consideration.

A rapid and non-invasive 2-step algorithm for diagnosing tuberculous peritonitis using a T cell-based assay on peripheral blood and peritoneal fluid mononuclear cells together with peritoneal fluid adenosine deaminase

A recently developed RD-1 gene-based assay for diagnosing tuberculous peritonitis (TBP) has given promising results. We therefore created a clinical algorithm for differentiating TBP from other diagnoses using peripheral blood and peritoneal fluid mononuclear cells (PBMC/PF-MC) along with conventional tests. All adult patients with suspected TBP in whom enzyme-linked immunosorbent spot (ELISPOT) assays were performed both on PBMC and PF-MC were prospectively enrolled over a 6-year period. Confirmed TBP with positive cultures or Mycobacterium tuberculosis PCR, probable TBP with PF changes consistent with TBP, caseating granuloma, and a successful response to anti-TB therapy, as well as possible TBP without exclusion of TBP, were each defined. A total of 74 patients were enrolled. Of these, 45 (61%) (19 confirmed, 16 probable, and 10 possible) were classified as TBP. The other 29 (39%) patients were classified as not TB. The sensitivity and specificity, respectively, of the tested methods for diagnosing TBP were as follows: PBMC ELISPOT (≥6 spots), 84% and 59%; PF-MC ELISPOT (≥6 spots), 87% and 86%; PF-MC/PBMC ratio (≥3), 69% and 97%; and PF-ADA level (≥21 U/L), 82% and 79%. The areas under the ROC curves were as follows: PF-MC ELISPOT, 0.90; PF-MC/PBMC ratio, 0.82; PBMC ELISPOT, 0.80; and PF-ADA, 0.80, respectively. When a 2-step algorithm ('PBMC ELISPOT ≥6 spots or PF-ADA ≥21 U/L' as a rule-out test and 'PF-MC/PBMC ratio ≥3' as a rule-in test) was applied, 67% (30/45) of the patients with TBP were accurately classified without undergoing invasive procedures. A 2-step algorithm using the PBMC/PF-MC ELISPOT assays and PF-ADA appears to be a promising rapid and non-invasive approach for diagnosing TBP.

The effect of thiamine deficiency on inflammation, oxidative stress and cellular migration in an experimental model of sepsis.

BackgroundSepsis is a prevalent condition in critically ill patients and may be associated with thiamine deficiency (TD). The aim of this study was to evaluate the effect of TD on inflammation, oxidative stress and cellular recruitment in a sepsis model.MethodsThe experimental sepsis model, cecal ligation and puncture (CLP), was utilized on mice in comparison with a sham procedure. The following four groups were compared against each other: SHAM with AIN93G complete chow, SHAM with thiamine deficient (TD) chow, CLP with AIN93G complete chow, and CLP with TD chow. Thiamine pyrophosphate (TPP) blood concentrations were determined, and blood and peritoneal fluid were evaluated for differences in TNF-alpha, IL-1, IL-6, KC and MCP-1/CCL2 levels. In addition, the levels of 4-HNE adducts in liver proteins were evaluated by Western Blot.ResultsThe mean TPP blood concentration from the mice fed with the complete chow was 303.3 ± 42.6 nmol/L, and TD occurred within 10 days. TNF-α and MCP-1 concentrations in the peritoneal fluid were significantly greater in the CLP with TD chow group when compared with the other groups. The blood IL-1β level, however, was lower in the CLP with TD chow group. Liver 4-HNE levels were highest in the TD chow groups. Blood mononuclear cell numbers, as well as peritoneal total leukocyte, mononuclear cell and neutrophil numbers were greater in the CLP with TD chow group. Peritoneal bacterial colony forming units (CFU) were significantly lower in the CLP with TD chow group.ConclusionTD was associated with greater bacterial clearance, oxidative stress and inflammatory response changes.

Inhibition of colitis by IL-25 associates with induction of alternatively activated macrophages

Interleukin (IL)-25, a Th2-related factor, inhibits the synthesis of inflammatory cytokines by macrophages and attenuates experimental colitis in mice. The mechanism underlying the counterregulatory effect of IL-25, however, remains unknown. Since Th2-cytokines can abrogate inflammatory pathways by inducing alternatively activated macrophages (AAMs), we evaluated whether AAMs are involved in the IL-25-mediated anticolitic effect. AAM-related markers were evaluated in peritoneal and lamina propria mononuclear cells of mice with or without 2,4,6-trinitrobenzenesulphonic acid (TNBS)-induced colitis treated with IL-25 and/or neutralizing IL-4, IL-13, and transforming growth factor beta 1 (TGF-β1) antibodies. Peritoneal AAMs induced in vivo by injecting mice with IL-25 were transferred to mice with TNBS colitis. Finally, we assessed the in vitro effect of IL-25 on the alternative activation of peritoneal F4/80+ cells. IL-25 enhanced the expression of AAM-related markers in F4/80(+) cells infiltrating the peritoneum and colon of naïve and colitic mice. Peritoneal F4/80(+) cells isolated from IL-25-treated mice reduced the severity of TNBS colitis when injected intraperitoneally to recipient mice. Since IL-25 did not directly induce AAM in vitro and in vivo in mice, IL-25 administration enhanced the expression of IL-4, IL-13, and TGF-β1, which are known to promote AAM differentiation, we finally assessed whether such cytokines were involved in the IL-25-driven AAM induction. Blockade of IL-4, IL-13, and TGF-β1 with neutralizing antibodies in mice did not inhibit the stimulatory effect of IL-25 on AAM gene expression. The IL-25-mediated anticolitic effect is associated with induction of AAMs, a subset of macrophages with antiinflammatory properties.

Rapid diagnosis of tuberculous peritonitis by T cell-based assays on peripheral blood and peritoneal fluid mononuclear cells

The utility of a newly-developed Mycobacterium tuberculosis-specific enzyme-linked immunosorbent spot (ELISPOT) assay for diagnosis of tuberculous peritonitis (TBP) has not been fully assessed. All patients with suspected TBP in a tertiary care hospital in an intermediate TB burden country were prospectively enrolled over a 30-month period. ELISPOT assays were performed on peripheral mononuclear cells (PBMC) and mononuclear cells from peritoneal fluid (PF-MC). Sixty-four patients with suspected TBP were enrolled. Of these, 30 (47%) were classified as having TBP (27 confirmed and 3 probable cases), and 25 (39%) were classified as not having active tuberculosis. The remaining 9 (14%) with possible TBP were excluded from the final analysis. Five (8%) of the total 64 patients gave indeterminate PBMC ELISPOT results and 7 (18%) of 39 patients who underwent PF-MC ELISPOT assay revealed indeterminate PF-MC ELISPOT results. The sensitivity and specificity, respectively, of the tested methods for diagnosing TBP were as follows: PBMC ELISPOT (≥ 6 spots), 86% and 67%; PF-MC ELISPOT (≥ 14 spots), 92% and 86%; PF-MC ELISPOT/PBMC ELISPOT ratio (≥ 2), 75% and 93%; and PF ADA levels (≥ 38 IU/L), 95% and 100%. The areas under the receiver operating characteristics curves were as follows: PF-MC ELISPOT, 0.96; PF ADA, 0.96; PBMC ELISPOT, 0.88; and PF-MC ELISPOT/PBMC ELISPOT ratio, 0.87, respectively. Although the ELISPOT assay does not outperform PF ADA, the ELISPOT assay using PBMC and PF-MC is a useful adjunct for diagnosing TBP, especially for a rule-in test when PF/MC/PBMC ELISPOT ratio (≥ 2) is used. However, the relatively high proportion of indeterminate results limits test utility, so further studies are needed to develop an optimized assay prototype.

Resolvin D2 is a potent regulator of leukocytes and controls microbial sepsis

A growing body of evidence indicates that resolution of acute inflammation is an active process1,2. Resolvins are a new family of lipid mediators enzymatically generated within resolution networks that possess unique and specific functions to orchestrate catabasis2,3. Resolvin D2 (RvD2) was originally identified in resolving exudates, yet its individual contribution in resolution remained to be elucidated. Here, we established RvD2’s potent stereoselective actions in reducing excessive neutrophil trafficking to inflammatory loci. RvD2 decreased leukocyte:endothelial interactions in vivo by endothelial-dependent nitric oxide production, and direct modulation of leukocyte adhesion receptor expression. In microbial sepsis initiated by cecal ligation and puncture (CLP), RvD2 sharply decreased both local and systemic bacterial burden, excessive cytokine production and neutrophil recruitment, while increasing peritoneal mononuclear cells and macrophage phagocytosis. These multi-level pro-resolving actions of RvD2 translate to increased survival from CLP-induced sepsis and surgery. Together, these results identify RvD2 as a potent endogenous regulator of excessive inflammatory responses that acts via multiple cellular targets to stimulate resolution and preserve immune vigilance.

Diagnosis of abdominal tuberculosis by T-cell-based assays on peripheral blood and peritoneal fluid mononuclear cells

Diagnosing abdominal tuberculosis (TB) remains a challenge. A recently developed RD-1 gene-based assay for diagnosing tuberculosis infection shows promising results. We evaluated the diagnostic usefulness of this assay compared with conventional tests in patients with suspected abdominal TB in clinical practice. All patients with suspected abdominal TB were prospectively enrolled in a tertiary hospital during a 1-year period. In addition to the conventional tests for diagnosing TB, the IFN-gamma-producing T-cell response to ESAT-6 and CFP-10 by ELISPOT assay using peripheral blood mononuclear cells (PBMC) and peritoneal fluid mononuclear cells (PF-MC) were performed. Forty eight patients with suspected abdominal TB were enrolled. Of these patients, 30 (63%) were classified as abdominal TB including 14 TB peritonitis (12 confirmed + 1 probable + 1 possible), 6 abdominal TB lymphadenitis (3 confirmed + 3 probable), 4 hepatic TB (3 confirmed + 1 possible), 2 intestinal TB (1 confirmed + 1 probable), 3 renal TB (1 confirmed + 2 probable), and 1 pancreatic TB (1 confirmed). Eighteen (38%) were classified as not TB. ELISPOT assay using PBMC was performed on samples from all 48 subjects. The sensitivity and specificity of the PBMC ELISPOT assay were 89% (95% CI, 71-98%) and 78% (95% CI, 52-94%), respectively. In the 11 patients in whom PF-MC ELISPOT assay was performed, it was positive in 5 of 6 patients with TB peritonitis, and negative in all 5 patients with not TB. The ELISPOT assay using PBMC and PF-MC is a useful adjunct to the current tests for diagnosing abdominal TB.

Leishmania donovani vs immunity: T-cells sensitized from Leishmania of one donor may modulate their cytokines pattern on re-stimulation with Leishmania from different donor in visceral leishmaniasis

Antimony resistance is frequently encountered during treatment of visceral leishmaniasis (VL) and the differences are well characterized by inadequate IFN-γ dominant type-1 protection mechanisms. The part played by Leishmania parasites derived from antimony treated patients in the outcome of an immune response largely remains to be investigated. In the present study we observed that macrophages of BALB/c mice infected with antimony non-responder (SAG-NR) isolates had a greater amastigote burden than antimony responder (SAG-R) isolates. Later it was observed that antigen from SAG-NR and R L. donovani isolates elicit different cytokine responses in peritoneal blood mononuclear cells (PBMCs) from patients with VL. The production of IFN-γ by T-cells in VL patients increased in response to Leishmania derived from responder patients but this response within same T-cells was lower when sensitized from Leishmania from a non-responder VL patient. On the other hand, IL-4 and IL-10 expression was increased when primed with parasites from non-responder VL source. Such a differential pattern of cytokine expression by the same T-cell population produced to Leishmania from different donors, needs further exploration.

Enhanced immune response induced by BSA loaded in hydroxyethylstarch microparticles

Microparticles and nanoparticles represent promising carriers for the in vivo delivery of peptides, proteins or deoxyribonucleic acid (DNA). In this study, new hydroxyethylstarch (HES) microparticles were obtained by interfacial cross-linking with terephtaloyl chloride. These microparticles exhibit the characteristics required to improve antigen release and presentation to antigen presentating cells compared to free antigens. The adjuvant activity of HES microparticles as vaccine carrier was investigated in mice using bovine serum albumin (BSA) as model antigen. We showed HES microparticles were phagocyted by peritoneal mononuclear cells. The immunization with BSA-microparticles induced antibody synthesis that was predominantly immunoglobulin G1 (IgG1). Aluminium hydroxide remained more efficient to induce IgG synthesis. The analysis of the cytokine profile from spleen cells revealed that BSA-microparticles induced the secretion of both interferon-γ (IFN-γ) and interleukin-4 (IL-4). However, the immune responses induced by BSA-microparticles were qualitatively and quantitatively affected by the route of injection. Taken together, these results demonstrate that HES microparticles induce a mixed T helper 1/T helper 2 response against BSA and may be a suitable delivery and presentation system in the field of vaccine development.