Phenotypic, functional, and quantitative characterization of canine peripheral blood monocyte-derived macrophages

The yield as well as phenotypic and functional parameters of canine peripheral blood monocyte-derived macrophages were analyzed. The cells that remained adherent to Teflon after 10 days of culture had high phagocytic activity when inoculated with Leishmania chagasi. Flow cytometric analysis demonstrated that more than 80% of cultured cells were positive for the monocyte/macrophage marker CD14.

Distinct cytokine profiles of circulating mononuclear cells stimulated with Staphylococcus aureus enterotoxin A in vitro during early and late episodes of chronic osteomyelitis

We investigated the cytokine profile of peripheral mononuclear cells from chronic osteomyelitis (OST) patients following in vitro stimulation with staphylococcal enterotoxin A (SEA). We demonstrate that stimulation with SEA induced prominent lymphocyte proliferation and high levels of tumour necrosis factor (TNF)-α, interleukin (IL)-4 and IL-10 secretion in both OST and non-infected individuals (NI). Even though stimulation with SEA had no impact on IL-6 production in either patient group, the baseline level of IL-6 production by cells from OST patients was always significantly less than that produced by cells from NI. After classifying the osteomyelitic episodes based on the time after the last reactivation event as "early" (1-4 months) or "late" osteomyelitis (5-12 months), we found that increased levels of TNF-α and IL-4 in combination with decreased levels of IL-6 were observed in the early episodes. By contrast, increased levels of IL-10, IL-2 and IL-6 were hallmarks of late episodes. Our data demonstrate that early osteomyelitic episodes are accompanied by an increased frequency of "high producers" of TNF-α and IL-4, whereas late events are characterised by increased frequencies of "high producers" of IL-10, IL-6 and IL-2. These findings demonstrate the distinct cytokine profiles in chronic osteomyelitis, with a distinct regulation of IL-6 production during early and late episodes.

High-dose cyclophosphamide followed by autologous peripheral blood progenitor cell transplantation improves the salvage treatment for persistent or sensitive relapsed malignant lymphoma

Trials have demonstrated that high-dose escalation followed by autologous transplantation can promote better long-term survival as salvage treatment in malignant lymphomas. The aim of the present nonrandomized clinical trial was to demonstrate the role of high-dose cyclophosphamide (HDCY) in reducing tumor burden and also to determine the effectiveness of HDCY followed by etoposide (VP-16) and methotrexate (MTX) in Hodgkin's disease plus high-dose therapy with peripheral blood progenitor cell (PBPC) transplantation as salvage treatment. From 1998 to 2000, 33 patients with a median age of 33 years (13-65) affected by aggressive non-Hodgkin's lymphoma (NHL) (60.6%) or persistent or relapsed Hodgkin's disease (39.4%) were enrolled and treated using high dose escalation (HDCY + HDVP-16 plus HDMTX in Hodgkin's disease) followed by autologous PBPC transplantation. On an "intention to treat" basis, 33 patients with malignant lymphomas were evaluated. The overall median follow-up was 400 days (40-1233). Thirty-one patients underwent autografting and received a median of 6.19 x 10(6)/kg (1.07-29.3) CD34+ cells. Patients who were chemosensitive to HDCY (N = 22) and patients who were chemoresistant (N = 11) presented an overall survival of 96 and 15%, respectively (P<0.0001). Overall survival was 92% for chemosensitive patients and 0% for patients who were still chemoresistant before transplantation (P<0.0001). Toxicity-related mortality was 12% (four patients), related to HDCY in two cases and to transplant in the other two. HDCY + HDVP-16 plus HDMTX in only Hodgkin's disease followed by autologous PBPC proved to be effective and safe as salvage treatment for chemosensitive patients affected by aggressive NHL and Hodgkin's disease, with acceptable mortality rates related to sequential treatment.

Age-related changes of the multidrug resistance P-glycoprotein function in normal human peripheral blood T lymphocytes

The multidrug resistance P-glycoprotein is a transmembrane efflux pump expressed by lymphocytes and is involved in their cytolytic activity. In the present study, we investigated the age-related changes of P-glycoprotein function in normal peripheral blood lymphocytes. Blood samples from 90 normal volunteers (age range, 0 to 86 years) were analyzed. P-glycoprotein function was assessed by the flow cytometric rhodamine 123 assay. P-glycoprotein function was highest in cord blood and progressively declined with age in peripheral blood T CD4+ and CD8+ cells. In contrast, P-glycoprotein function did not vary with age in CD19+ B or CD16+CD56+ natural killer cells. These data suggest that the decline in P-glycoprotein function in T CD4+ and CD8+ lymphocytes as a function of age may contribute to the decrease in T cell cytolytic activity with aging.

In vitro activity of hypnophilin from Lentinus strigosus: a potential prototype for Chagas disease and leishmaniasis chemotherapy

Hypnophilin and panepoxydone, terpenoids isolated from Lentinus strigosus, have significant inhibitory activity onTrypanosoma cruzi trypanothione reductase (TR). Although they have similar TR inhibitory activity at 10 μg/mL (40.3 μM and 47.6 μM for hypnophilin and panepoxydone, respectively; ~100%), hypnophilin has a slightly greater inhibitory activity (~71%) on T. cruzi amastigote (AMA) growth in vitro as well as on in vitro phytohemagglutinin (PHA)-induced peripheral blood mononuclear (PBMC) proliferation (~70%) compared to panepoxydone (69% AMA inhibition and 91% PBMC inhibition). Hypnophilin and panepoxydone at 1.25 μg/mL had 67% inhibitory activity onLeishmania (Leishmania) amazonensis amastigote-like (AMA-like) growth in vitro. The panepoxydone activity was accompanied by a significant inhibitory effect on PHA-induced PBMC proliferation, suggesting a cytotoxic action. Moreover, incubation of human PBMC with panepoxydone reduced the percentage of CD16+ and CD14+ cells and down-regulated CD19+, CD4+ and CD8+ cells, while hypnophilin did not alter any of the phenotypes analyzed. These data indicate that hypnophilin may be considered to be a prototype for the design of drugs for the chemotherapy of diseases caused by Trypanosomatidae.

Evaluation of Toll-like, chemokine, and integrin receptors on monocytes and neutrophils from peripheral blood of septic patients and their correlation with clinical outcomes

Recognition of pathogens is performed by specific receptors in cells of the innate immune system, which may undergo modulation during the continuum of clinical manifestations of sepsis. Monocytes and neutrophils play a key role in host defense by sensing and destroying microorganisms. This study aimed to evaluate the expression of CD14 receptors on monocytes; CD66b and CXCR2 receptors on neutrophils; and TLR2, TLR4, TLR5, TLR9, and CD11b receptors on both cell types of septic patients. Seventy-seven septic patients (SP) and 40 healthy volunteers (HV) were included in the study, and blood samples were collected on day zero (D0) and after 7 days of therapy (D7). Evaluation of the cellular receptors was carried out by flow cytometry. Expression of CD14 on monocytes and of CD11b and CXCR2 on neutrophils from SP was lower than that from HV. Conversely, expression of TLR5 on monocytes and neutrophils was higher in SP compared with HV. Expression of TLR2 on the surface of neutrophils and that of TLR5 on monocytes and neutrophils of SP was lower at D7 than at D0. In addition, SP who survived showed reduced expression of TLR2 and TLR4 on the surface of neutrophils at D7 compared to D0. Expression of CXCR2 for surviving patients was higher at follow-up compared to baseline. We conclude that expression of recognition and cell signaling receptors is differentially regulated between SP and HV depending on the receptor being evaluated.

Intranasal sensitization with Blomia Tropicalis antigens induces allergic responses in mice characterized by elevated antigen-specific and non-specific serum ige and peripheral blood eosinophil counts

In order to evaluate the potential allergenicity of Blomia tropicalis (Bt) antigen, IgE production of both specific and non-specific for Bt antigen was monitored in BALB/c mice after exposure to the antigen by nasal route. It was evidenced that B. tropicalis contains a functional allergen in its components. The allergenic components, however, when administered intranasally without any adjuvant, did not function to induce IgE response within a short period. On the other hand, intranasal inoculation of Bt antigens augmented serum IgE responses in mice pretreated by a subcutaneous priming injection of the same antigens. Inoculation of Bt antigen without subcutaneous priming injections induced IgE antibody production only when the antigen was continuously administered for a long period of over 24 weeks. Even when the priming injection was absent, the Bt antigen inoculated with cholera toxin (CT) as a mucosal adjuvant also significantly augmented the Bt antigen-specific IgE responses depending on the dose of CT co-administered. The present study also demonstrated that Bt antigen/CT-inoculated mice showed increased non-specific serum IgE level and peripheral blood eosinophil rates without noticeable elevations of the total leukocyte counts. The immunoblot analysis demonstrated 5 main antigenic components reactive to IgE antibodies induced. These components at about 44-64 kDa position were considered to be an important candidate antigen for diagnosis of the mite-related allergy.

Detection of Bartonella henselae DNA in clinical samples including peripheral blood of immune competent and immune compromised patients by three nested amplifications

Bacteria of the genus Bartonella are emerging pathogens detected in lymph node biopsies and aspirates probably caused by increased concentration of bacteria. Twenty-three samples of 18 patients with clinical, laboratory and/or epidemiological data suggesting bartonellosis were subjected to three nested amplifications targeting a fragment of the 60-kDa heat shock protein (HSP), the internal transcribed spacer 16S-23S rRNA (ITS) and the cell division (FtsZ) of Bartonella henselae, in order to improve detection in clinical samples. In the first amplification 01, 04 and 05 samples, were positive by HSP (4.3%), FtsZ (17.4%) and ITS (21.7%), respectively. After the second round six positive samples were identified by nested-HSP (26%), eight by nested-ITS (34.8%) and 18 by nested-FtsZ (78.2%), corresponding to 10 peripheral blood samples, five lymph node biopsies, two skin biopsies and one lymph node aspirate. The nested-FtsZ was more sensitive than nested-HSP and nested-ITS (p < 0.0001), enabling the detection of Bartonella henselae DNA in 15 of 18 patients (83.3%). In this study, three nested-PCR that should be specific for Bartonella henselae amplification were developed, but only the nested-FtsZ did not amplify DNA from Bartonella quintana. We conclude that nested amplifications increased detection of B. henselae DNA, and that the nested-FtsZ was the most sensitive and the only specific to B. henselae in different biological samples. As all samples detected by nested-HSP and nested-ITS, were also by nested-FtsZ, we infer that in our series infections were caused by Bartonella henselae. The high number of positive blood samples draws attention to the use of this biological material in the investigation of bartonellosis, regardless of the immune status of patients. This fact is important in the case of critically ill patients and young children to avoid more invasive procedures such as lymph nodes biopsies and aspirates.

Kinetics of IFN-gamma, TNF-alpha, IL-10 and IL-4 production by mononuclear cells stimulated with gp43 peptides, in patients cured of paracoccidioidomycosis

We analyzed the kinetics of cytokine production by mononuclear cells from 17 patients who had been treated for paracoccidioidomycosis, using the stimulus of gp43 peptide groups (43kDa glycoprotein of Paracoccidioides brasiliensis) at 0.1 and 1µM, gp43 (1µg/ml) and crude Paracoccidioides brasiliensis antigen (PbAg; 75µg/ml). IFN-gamma production was a maximum at 144 hours in relation to the G2 and G8 peptide groups at 1µM and was greatest at 144 hours when stimulated by gp43 and by PbAg. The maximum TNF-alpha production was at 144 hours for the G2 group (0.1µM) and for gp43. IL-10 production was highest after 48 and 72 hours for G7 and G6 at 1µM, respectively. We also suggest the best time for analysis of IL4 production. These results may contribute towards future studies with gp43 peptides and encourage further investigations with the aim of understanding the influence of these peptides on the production of inflammatory and regulatory cytokines.

Basic Surface Properties of Mononuclear Cells from Didelphis marsupialis

The electrostatic surface charge and surface tension of mononuclear cells/monocytes obtained from young and adult marsupials (Didelphis marsupialis) were investigated by using cationized ferritin and colloidal iron hydroxyde, whole cell electrophoresis, and measurements of contact angles. Anionic sites were found distributed throughout the entire investigated cell surfaces. The results revealed that the anionic character of the cells is given by electrostatic charges corresponding to -18.8 mV (cells from young animals) and -29.3 mV (cells from adult animals). The surface electrostatic charge decreased from 10 to 65.2% after treatment of the cells with each one of trypsin, neuraminidase and phospholipase C. The hydrophobic nature of the mononuclear cell surfaces studied by using the contact angle method revealed that both young and adult cells possess cell surfaces of high hidrofilicity since the angles formed with drops of saline water were 42.5°and 40.8°, respectively. Treatment of the cells with trypsin or neuraminidase rendered their surfaces more hydrophobic, suggesting that sialic acid-containing glycoproteins are responsible for most of the hydrophilicity observed in the mononuclear cell surfaces from D. marsupialis.

Cytokine profile associated with human chronic schistosomiasis mansoni

This study objective was to evaluate the cytokines associated with early events of hepatic fibrosis in schistosomiasis mansoni. Hepatic fibrosis was classified by ultrasonography in 94 patients. Immunological evaluation was performed by measurement of secreted cytokines (interleukin IL-5, IL-10, IL-13, interferon-gamma, tumor necrosis factor-alpha and transforming growth factors-beta) in peripherl blood mononuclear cells stimulated by Schistosoma mansoni antigens. Significantly, higher levels of IL-5, IL-10 and IL-13 were found in supernatants of SEA-stimulated PBMC from subjects with degree III hepatic fibrosis as compared to patients with degree I or II fibrosis, Significant increases in IL-5 and IL-13 levels were also observed in some of the subjects who remained untreated for one year following initial assessment and developed more serious fibrosis during this period. The data suggests a role for type 2 cytokines in early stages of hepatic fibrosis in human schistosomiasis mansoni.

The effects of fluconazole and cytokines on human mononuclear cells

Candida infections are common infections and fluconazole is one of the most frequently administered antifungal agents in their treatment. The resistance developed against antifungal agents has necessitated the improvement of new treatments. This study focuses on the investigation of the effect of fluconazole and cytokines such as interferon-gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha), granulocyte-macrophage colony-stimulating factor (GM-CSF) on chemokine production and anticandidal activity of human monocytes. In the study it was observed that GM-CSF caused an increase in candidacidal activity of monocytes. Anticandidal activity of GM-CSF + IFN-gamma combination was not found to be more effective than GM-CSF or IFN-gamma alone. The presence of cytokine and fluconazole caused an increase in the levels of CCL3 and CCL4 chemokines. Accordingly, it was considered that chemokines could contribute to the efficacy of fluconazole in C. albicans infections. Besides, in order to strengthen the immune system some cytokines might be used in addition to antifungal agents for the treatment.

Polymerase chain reaction of peripheral blood as a tool for the diagnosis of visceral leishmaniasis in children

The diagnosis of visceral leishmaniasis (VL) generally requires the use of invasive tests for the collection of infected tissue (aspirates of bone marrow, spleen, liver or lymph nodes). This difficulty has led to the search for safer and less painful techniques to confirm the occurrence of the disease in children. Polymerase chain reaction (PCR) is a method that is advantageous in that it allows the use of peripheral blood samples for diagnosis. This paper reports the utilisation of PCR on peripheral blood samples to diagnose VL in 45 children in Mato Grosso do Sul, Brazil. This technique is compared with methods carried out using tissue collected by invasive procedures, including direct microscopy, culture and detection of Leishmania DNA by PCR in bone marrow aspirates. The results show that PCR of peripheral blood provides great sensitivity (95.6%) that is similar to that from the PCR of bone marrow aspirates (91.1%) and higher than that achieved with microscopy (80%) or culture (26.7%) methods. PCR of peripheral blood proved to be a suitable tool for the diagnosis of VL in children because it is highly sensitive and safe, with tissue collection being less invasive than in traditional tests.

Peripheral blood fibrocytes: new information to explain the dynamics of Leishmania infection

Fibrocytes are important for understanding the progression of many diseases because they are present in areas where pathogenic lesions are generated. However, the morphology of fibrocytes and their interactions with parasites are poorly understood. In this study, we examined the morphology of peripheral blood fibrocytes and their interactions with Leishmania (L.) amazonensis . Through ultrastructural analysis, we describe the details of fibrocyte morphology and how fibrocytes rapidly internaliseLeishmania promastigotes. The parasites differentiated into amastigotes after 2 h in phagolysosomes and the infection was completely resolved after 72 h. Early in the infection, we found increased nitric oxide production and large lysosomes with electron-dense material. These factors may regulate the proliferation and death of the parasites. Because fibrocytes are present at the infection site and are directly involved in developing cutaneous leishmaniasis, they are targets for effective, non-toxic cell-based therapies that control and treat leishmaniasis.

Protocol for collection and separation of bone marrow mononuclear cells in Chlorocebus aethiops

Abstract: Chlorocebus aethiops is a species of non-human primate frequently used in biomedical research. Some research involves this species as an experimental model for various diseases and possible treatment with stem cells. The bone marrow is one of the main sources of these cells and provides easy access. The aim of this study was to standardize the protocol of collection and separation of bone marrow in C. aethiops. Ten animals were submitted to puncture of bone marrow with access to the iliac crest and cell separation by density gradient. The bone marrow of C. aethiops had an average of 97% viability. From the results achieved, we can conclude that C. aethiops is an excellent model to obtain and isolate mononuclear cells from bone marrow, fostering several studies in the field of cell therapy.