Low prevalence of Toxoplasma gondii infection among children in a rural community in Fars province, Southern Iran.

The current study aimed to determine the seroprevalence of Toxoplasma gondii infection and associated risk factors in children in a rural community in Fars province in southern Iran. Blood samples were collected from 671 children living in three rural areas, and sera and buffy coats were isolated from each sample. Anti-T. gondii antibodies were detected by ELISA, using a commercial kit. Also, buffy coats of seropositive children were examined by a PCR method, targeting a 529 bp gene of T. gondii. Of 671 children participating in the study, 319 (51.7%) were boys and 298 (48.3%) were girls. The mean age of the children was 9.7 (±10.7) years. Anti-Toxoplasma antibodies were detected in sera of 23 out of 671 children, corresponding to a seroprevalence rate of 3.8%. Gender and level of education had no significant influence on the risk of Toxoplasma infection (p>0.05). Toxoplasma DNA was not detected in buffy coats of any of the seropositive cases. Geographic location, as well as the low age of the participants, may contribute to the low rate of Toxoplasma infection in children of rural areas in southern Iran.

Identification of SP1683 as a pneumococcal protein that is protective against nasopharyngeal colonization.

Serotype-independent protein-based pneumococcal vaccines represent attractive alternatives to capsular polysaccharide-based vaccines. The aim of this study was to identify novel immunogenic proteins from Streptococcus pneumoniae that may be used in protein-based pneumococcal vaccine. An immunoproteomics approach and a humanized severe combined immunodeficient mouse model were used to identify S. pneumoniae proteins that are immunogenic for the human immune system. Among the several proteins identified, SP1683 was selected, recombinantly produced, and infection and colonization murine models were used to evaluate the capacity of SP1683 to elicit protective responses, in comparison to known pneumococcal immunogenic proteins (PhtD and detoxified pneumolysin, dPly). Immunisation with SP1683 elicited a weaker antibody response than immunisation with PhtD and did not provide protection in the model of invasive disease. However, similar to PhtD, it was able to significantly reduce colonization in the mouse model of nasopharyngeal carriage. Treatment with anti-IL17A and anti-IL17F antibodies abolished the protection against colonization elicited by SP1683 or PhtD + dPly, which indicated that the protection afforded in this model was Th17-dependent. In conclusion, intranasal immunization with the pneumococcal protein SP1683 conferred IL17-dependent protection against nasopharyngeal carriage in mice, but systemic immunization did not protect against invasive disease. These results do not support the use of SP1683 as an isolated pneumococcal vaccine antigen. Nevertheless, SP1683 could be used as a first line of defence in formulations combining several proteins.

Buffy coat volume reduction for optimization of leucapheresis harvests produced by the autoMNC program.

BACKGROUND AND OBJECTIVES: Buffy coat (BC) volume reduction was evaluated in leucapheresis (LA) harvests due to the target monocyte yield and the red blood cell (RBC) content. A packed erythrocyte volume (PEV) of 7.5 ml should not be exceeded to avoid RBC debulking with loss of leucocytes (WBCs) and the monocyte fraction during monocyte counterflow elutriation, a next step of monocyte enrichment prior to cell culture. MATERIALS AND METHODS: Two hundred and fifty-three 5-l leucaphereses (autoMNC program) performed in 102 healthy blood donors (24 female and 78 male donors) were retrospectively analysed. Different categories of BC volumes were compared due to the quality of the LA products measured by blood counts and flow cytometry. RESULTS: Collection of maximum BC volume of 10 ml and more each collection cycle (product volume: 169 ± 21 ml) resulted in 1.58 ± 0·41 × 10e9 CD14(+) monocytes and high volume of packed erythrocyte (18.4 ± 8.8 ml). Low BC volume collection below 6 ml each collection cycle produced only 1.07 ± 0.40 × 10e9 CD14(+) monocytes but reduced PEV significantly by 64% (6.7 ± 4.1 ml). CONCLUSION: By reduction of the BC volume, the PEV in LA products could be reduced, which is a precondition for counterflow elutriation of monocytes. A BC volume between 7 and 8 ml per collection cycle should be adjusted to reduce PEV to 7.5 ml without relevant monocyte loss.

Isolation of biologically-active exosomes from human plasma.

Effects of exosomes present in human plasma on immune cells have not been examined in detail. Immunological studies with plasma-derived exosomes require their isolation by procedures involving ultracentrifugation. These procedures were largely developed using supernatants of cultured cells. To test biologic activities of plasma-derived exosomes, methods are necessary that ensure adequate recovery of exosome fractions free of contaminating larger vesicles, cell fragments and protein/nucleic acid aggregates. Here, an optimized method for exosome isolation from human plasma/serum specimens of normal controls (NC) or cancer patients and its advantages and pitfalls are described. To remove undesirable plasma-contaminating components, ultrafiltration of differentially-centrifuged plasma/serum followed by size-exclusion chromatography prior to ultracentrifugation facilitated the removal of contaminants. Plasma or serum was equally acceptable as a source of exosomes based on the recovered protein levels (in µg protein/mL plasma) and TEM image quality. Centrifugation on sucrose density gradients led to large exosome losses. Fresh plasma was the best source of morphologically-intact exosomes, while the use of frozen/thawed plasma decreased exosome purity but not their biologic activity. Treatments of frozen plasma with DNAse, RNAse or hyaluronidase did not improve exosome purity and are not recommended. Cancer patients' plasma consistently yielded more isolated exosomes than did NCs' plasma. Cancer patients' exosomes also mediated higher immune suppression as evidenced by decreased CD69 expression on responder CD4+ T effector cells. Thus, the described procedure yields biologically-active, morphologically-intact exosomes that have reasonably good purity without large protein losses and can be used for immunological, biomarker and other studies.

Centrifugo-Magnetophoretic Purification of CD4+ Cells from Whole Blood Toward Future HIV/AIDS Point-of-Care Applications.

In medical diagnostics, detection of cells exhibiting specific phenotypes constitutes a paramount challenge. Detection technology must ensure efficient isolation of (often rare) targets while eliminating nontarget background cells. Technologies exist for such investigations, but many require high levels of expertise, expense, and multistep protocols. Increasing automation, miniaturization, and availability of such technologies is an aim of microfluidic lab-on-a-chip strategies. To this end, we present an integrated, dual-force cellular separation strategy using centrifugo-magnetophoresis. Whole blood spiked with target cells is incubated with (super-)paramagnetic microparticles that specifically bind phenotypic markers on target cells. Under rotation, all cells sediment into a chamber located opposite a co-rotating magnet. Unbound cells follow the radial vector, but under the additional attraction of the lateral magnetic field, bead-bound target cells are deflected to a designated reservoir. This multiforce separation is continuous and low loss. We demonstrate separation efficiently up to 92% for cells expressing the HIV/AIDS relevant epitope (CD4) from whole blood. Such highly selective separation systems may be deployed for accurate diagnostic cell isolations from biological samples such as blood. Furthermore, this high efficiency is delivered in a cheap and simple device, thus making it an attractive option for future deployment in resource-limited settings.

Inhibiting CXCL12 blocks fibrocyte migration and differentiation and attenuates bronchiolitis obliterans in a murine heterotopic tracheal transplant model.

OBJECTIVES: Fibrocytes are integral in the development of fibroproliferative disease after lung transplantation. Undifferentiated fibrocytes (CD45+anti-collagen 1+CXCR4+) preferentially traffic by way of the CXCR4/CXCL12 axis and differentiate into smooth muscle actin-producing (CD45+CXCR4+α-smooth muscle actin+) cells. We postulated that an antibody directed against CXCL12 would attenuate fibrocyte migration and fibro-obliteration of heterotopic tracheal transplant allografts. METHODS: A total alloantigenic mismatch murine heterotopic tracheal transplant model of obliterative bronchiolitis was used. The mice were treated with either goat-anti-human CXCL12 F(ab')(2) or goat IgG F(ab')(2). Buffy coat, bone marrow, and trachea allografts were collected and analyzed using flow cytometry. Tracheal luminal obliteration was assessed using hematoxylin-eosin and Direct Red 80 collagen stain. RESULTS: Compared with the controls, the anti-CXCL12-treated mice showed a significant decrease in tracheal allograft fibrocyte populations at 7 and 21 days after transplantation. Bone marrow and buffy coat aspirates showed the same trend at 7 days. In the anti-CXCL12-treated mice, there was a 35% decrease in luminal obliteration at 21 days (65% vs 100% obliterated; interquartile range, 38% vs 10%; P = .010) and decreased luminal collagen deposition at 21 and 28 days after transplantation (P = .042 and P = .012, respectively). CONCLUSIONS: Understanding the role of fibrocytes in airway fibrosis after lung transplantation could lead to a paradigm shift in treatment strategy. Anti-CXCL12 antibody afforded protection against infiltrating fibrocytes and reduced the deterioration of the tracheal allografts. Thus, the CXCR4/CXCL12 axis is a novel target for the treatment of fibro-obliteration after lung transplantation, and the quantification of fibrocyte populations could provide clinicians with a biomarker of fibrosis, allowing individualized drug therapy.

The histamine H4 receptor is highly expressed on plasmacytoid dendritic cells in psoriasis and histamine regulates their cytokine production and migration.

Plasmacytoid dendritic cells (pDC) are present in inflammatory skin lesions, in particular, in psoriasis. In such lesions, the inflammatory mediator histamine is also detected in high amounts. We therefore investigated a possible interaction of pDC with histamine, especially via the most recently described histamine H(4) receptor (H(4)R). We detected the expression of the H(4)R on pDC in the blood and in lesional psoriasis skin. Interestingly, compared with healthy controls and patients with atopic dermatitis, pDC from the blood of psoriasis patients expressed the highest levels of the H(4)R, which was even more upregulated on stimulation with IFN-γ and CpG. After activation of the H(2)R and H(4)R on pDC, we observed downregulation of CpG-induced production of tumor necrosis factor α, IFN-α, and CXCL8, but not of the chemokine CXCL10. Histamine-induced downregulation of cytokine production was more pronounced in pDC derived from psoriasis patients. Furthermore, we observed F-actin polymerization and active migration of pDC in response to H(4)R agonist stimulation. Taken together, our results indicate that the H(4)R is highly expressed on pDC in psoriasis and influences cytokine production and migration of pDC. Therefore, the H(4)R alone or in combination with the H(2)R might be a promising therapeutic target in psoriasis.

A combination of exosomes carrying TSA derived from HLA-A2-positive human white buffy coat and polyI:C for use as a subcellular antitumor vaccination.

To improve its antitumor effect, we used human leukocyte antigen -A2 (HLA-A2)-positive human dendritic cell (DC)-derived DEXs (DC-derived exosomes) to support NY-ESO-1 antigen and polyI:C, with the aim of increasing the proliferation of specific cytotoxic T lymphocytes (CTL) in transgenic mice. Mature dendritic cells derived from peripheral blood mononuclear cells (PBMC) were isolated from the blood of healthy adults with positive HLA-2A. Using centrifuge and membrane ultrafiltration, EXO (exosomes) were extracted from the supernatant of DCs secretions. Transgenic C57 mice were immunized with human-derived tumor testis antigen NY-ESO-1/EXO, with or without polyI:C. Mice were sacrificed four weeks after immunization, and spleen cells were isolated and tested for function. The experiments included antigen-specific CTL proliferation, as tested by dimerization and antitumor effects for K562 cells as well as melanoma, tested at different ratios of effected cells:target cells (0:1, 10:1, 50:1, and 100:1). Dimerization experiments indicated that the effect of DEX/TSA (tumor specific antigens) + PolyI:C was 2.36 ± 1.10% and the control was 0.38 ± 0.31%, while the effect of DEX/TSA was 1.97 ± 0.63% and the control was 0.36 ± 0.07%. Antitumor effects by DEX/TSA: PolyI:C for the cell ratios of 0:1, 10:1, 50:1, and 100:1 were 11.14 ± 1.36%, 14.17 ± 0.62%, 15.71 ± 2.48%, and 24.31 ± 2.91%, respectively, for K562 cells. The antitumor effects for DEX/TSA for the cell ratios of 0:1, 10:1, 50:1, and 100:1 were 12.23 ± 2.25%, 13.10 ± 1.57%, 15.27 ± 2.93%, and 19.87 ± 2.72%, respectively, for K562 cells. With ratios of 10:1 and 100:1, the antitumor effects of DEX/TSA + PolyI:C were better than for the DEX/TSA group (P < 0.05). However, higher ratios of effecter cells to target cells increased, and there were no significant improvements in antitumor effect for control cells. Combining PolyI:C with DEX/TSA derived from healthy human blood positive for HLA-A2 is a promising strategy for developing new subcellular antitumor vaccination.