Th2-skewed T cells correlate with B cell response to α-Gal and tick antigens in α-Gal syndrome.

Tick bites have been shown to transmit a novel form of severe food allergy, the galactose-α-1,3-galactose (α-Gal) syndrome (AGS). Cellular responses to α-Gal in patients with AGS have, to date, not been thoroughly scrutinized. Therefore, we investigated T and B cell proliferation, activation, and cytokine profiles in response to tick protein extract (TE) and α-Gal-free TE in patients with AGS and in healthy controls.

A Novel Sandwich ELISA for Tartrate-Resistant Acid Phosphatase 5a and 5b Protein Reveals that Both Isoforms are Secreted by Differentiating Osteoclasts and Correlate to the Type I Collagen Degradation Marker CTX-I In Vivo and In Vitro.

Tartrate-resistant acid phosphatase type 5 (TRAP) exists as two isoforms, 5a and 5b. 5b is a marker of osteoclast number and 5a of chronic inflammation; however, its association with bone resorption is unknown. In this study, a double-TRAP 5a/5b sandwich ELISA measuring 5a and 5b protein in the same sample was developed.

Intensive weight gain therapy in patients with anorexia nervosa results in improved serum tartrate-resistant acid phosphatase (TRAP) 5a and 5b isoform protein levels.

Aim: Tartrate-resistant acid phosphatase (TRAP) exists as isoforms 5a and 5b. TRAP 5a is a biomarker of chronic inflammation and influences adipose tissue and 5b associates with bone metabolism/pathologies. The aim was to investigate the association of serum TRAP 5a/5b isoforms with fat and bone markers and anthropometric parameters in patients with anorexia nervosa (AN) during weight gain therapy.

A novel adjuvant G3 induces both Th1 and Th2 related immune responses in mice after immunization with a trivalent inactivated split-virion influenza vaccine.

A preferred adjuvant should promote both Th1 and Th2 responses. However, most adjuvants in common use are biased towards a Th2-driven response. Therefore, the ability of a novel saponin-based adjuvant G3 to inducing balanced Th1 and Th2 responses in BALB/c mice immunized with a split trivalent seasonal influenza vaccine was evaluated in comparison to that of the adjuvant Al(OH).

The adipokine tartrate-resistant acid phosphatase 5a in serum correlates to adipose tissue expansion in obesity.

Purpose: Tartrate-resistant acid phosphatase (TRAP) exists as two isoforms, 5a and 5b. TRAP 5a is elevated in adipose tissue of obese women, interacts with pre-adipocytes and is linked to insulin-sensitive hyperplastic obesity when overexpressed in mice. The aim of this study was to investigate the relation between serum TRAP 5a, adiposity indices and metabolic syndrome risk markers in lean and obese women, using a newly developed TRAP 5a-specific ELISA.

Activation of Neutrophils via IP3 Pathway Following Exposure to Demodex-Associated Bacterial Proteins.

Rosacea is a chronic inflammatory condition that predominantly affects the skin of the face. Sera from rosacea patients display elevated reactivity to proteins from a bacterium (Bacillus oleronius) originally isolated from a Demodex mite from a rosacea patient suggesting a possible role for bacteria in the induction and persistence of this condition. This work investigated the ability of B.

Increased Number of Plasma B Cells Producing Autoantibodies Against Aβ42 Protofibrils in Alzheimer's Disease.

The Alzheimer's disease (AD)-related peptide amyloid-β (Aβ) has a propensity to aggregate into various assemblies including toxic soluble Aβ protofibrils. Several studies have reported the existence of anti-Aβ antibodies in humans. However, it is still debated whether levels of anti-Aβ antibodies are altered in AD patients compared to healthy individuals.

ELISpot and ELISA analyses of human IL-21-secreting cells: Impact of blocking IL-21 interaction with cellular receptors.

Interleukin (IL)-21 is crucial for the regulation of lymphocytes and is implicated in autoimmune and other diseases. The relevance of being able to measure human IL-21 prompted us to develop ELISA and ELISpot assays for analysis of IL-21 levels and IL-21-producing cells, respectively. Monoclonal antibodies (mAbs) to IL-21 were made and ELISA and ELISpot assays were developed.

ApoD mediates binding of HDL to LDL and to growing T24 carcinoma.

Apolipoprotein (Apo) D is an important protein produced in many parts of the body. It is necessary for the development and repair of the brain and protection from oxidative stress. The purpose of this study was to investigate the extent to which apoD interacts with lipoproteins in human plasma.

Caveolae-mediated endocytosis of the glucosaminoglycan-interacting adipokine tartrate resistant acid phosphatase 5a in adipocyte progenitor lineage cells.

Adipogenesis depends on growth factors controlling proliferation/differentiation of mesenchymal stem cells (MSCs). Membrane binding and endocytosis of growth factors are often coupled to receptor activation and downstream signaling leading to specific cellular responses. The novel adipokine tartrate-resistant acid phosphatase (TRAP) 5a exhibits a growth factor-like effect on MSCs and pre-adipocytes and induces hyperplastic obesity in vivo.

ApoE production in human monocytes and its regulation by inflammatory cytokines.

The apoE production by tissue macrophages is crucial for the prevention of atherosclerosis and the aim of this study was to further elucidate how this apolipoprotein is regulated by cytokines present during inflammation. Here we studied apoE production in peripheral blood mononuclear cells (PBMC) and analysis was made with a newly developed apoE ELISpot assay. In PBMC, apoE secretion was restricted to monocytes with classical (CD14(++)CD16(-)) and intermediate (CD14(+)CD16(+)) monocytes being the main producers.

Clinical validation of IFNγ/IL-10 and IFNγ/IL-2 FluoroSpot assays for the detection of Tr1 T cells and influenza vaccine monitoring in humans.

The type of T cell polarization and simultaneous production of multiple cytokines have been correlated with vaccine efficacy. ELISpot is a T cell detection technique optimized for the measurement of a secreted cytokine at the single cell level. The FluoroSpot assay differs from ELISpot by the use of multiple fluorescent-labeled anticytokine detection antibodies, allowing optimal measurement of multiple cytokines.

Adjuvant properties of mesoporous silica particles tune the development of effector T cells.

Alum is the most frequently used adjuvant today, primarily inducing Th2 responses. However, Th1-type responses are often desirable within immune therapy, and therefore the development of new adjuvants is greatly needed. Mesoporous silica particles with a highly ordered pore structure have properties that make them very interesting for future controlled drug delivery systems, such as controllable particle and pore size; they also have the ability to induce minor immune modulatory effects, as previously demonstrated on human-monocyte-derived dendritic cells (MDDCs).

Heavy-chain complementarity-determining regions determine conformation selectivity of anti-aβ antibodies.

Background/aims: Amyloid-β (Aβ) protofibrils are neurotoxic soluble intermediates in the Aβ aggregation process eventually forming senile plaques in Alzheimer's disease. This Aβ species is a potential biomarker for Alzheimer's disease and also a promising target for immunotherapy. In this study, we investigated the characteristics of conformation-dependent Aβ antibodies specific for Aβ protofibrils.

Interference from heterophilic antibodies in amyloid-β oligomer ELISAs.

Amyloid-β (Aβ) oligomers of different sizes and forms have recently been the focus formany Alzheimer's disease (AD) researchers. Various immunoassays have been used to detect low concentrations of these elusive Aβ species in different forms of human samples using little or no sample dilutions. However, the possibility that positive results may be caused by interference from heterophilic antibodies (HA) is often overlooked.

Selective expansion of HIV-1 envelope glycoprotein-specific B cell subsets recognizing distinct structural elements following immunization.

The HIV-1 envelope glycoprotein (Env) functional spike has evolved multiple immune evasion strategies, and only a few broadly neutralizing determinants on the assembled spike are accessible to Abs. Serological studies, based upon Ab binding and neutralization activity in vitro, suggest that vaccination with current Env-based immunogens predominantly elicits Abs that bind nonneutralizing or strain-restricted neutralizing epitopes. However, the fractional specificities of the polyclonal mixture of Abs present in serum, especially those directed to conformational Env epitopes, are often difficult to determine.

Efficient internalization of mesoporous silica particles of different sizes by primary human macrophages without impairment of macrophage clearance of apoptotic or antibody-opsonized target cells.

Macrophage recognition and ingestion of apoptotic cell corpses, a process referred to as programmed cell clearance, is of considerable importance for the maintenance of tissue homeostasis and in the resolution of inflammation. Moreover, macrophages are the first line of defense against microorganisms and other foreign materials including particles. However, there is sparse information on the mode of uptake of engineered nanomaterials by primary macrophages.

ELISpot analysis of LPS-stimulated leukocytes: human granulocytes selectively secrete IL-8, MIP-1beta and TNF-alpha.

Granulocytes and monocytes/macrophages represent key effector cells of the innate immune system. While human monocytes have been recognized as capable of secreting a broad spectrum of cytokines, the situation has been less clear in granulocytes with studies often showing conflicting results. In this study, lipopolysaccharide (LPS)-induced cytokine secretion from polymorphonuclear cells (PMN) and peripheral blood mononuclear cells (PBMC) was analyzed at the single cell level with the enzyme-linked immunospot (ELISpot) assay.

Sensitive ELISA detection of amyloid-beta protofibrils in biological samples.

Amyloid-beta (Abeta) protofibrils are known intermediates of the in vitro Abeta aggregation process and the protofibrillogenic Arctic mutation (APPE693G) provides clinical support for a pathogenic role of Abeta protofibrils in Alzheimer's disease (AD). To verify their in vivo relevance and to establish a quantitative Abeta protofibril immunoassay, Abeta conformation dependent monoclonal antibodies were generated. One of these antibodies, mAb158 (IgG2a), was used in a sandwich ELISA to specifically detect picomolar concentrations of Abeta protofibrils without interference from Abeta monomers or the amyloid precursor protein (APP).

A new multi-clade DNA prime/recombinant MVA boost vaccine induces broad and high levels of HIV-1-specific CD8(+) T-cell and humoral responses in mice.

The results presented here are from the preclinical evaluation in BALB/c mice of a DNA prime/modified vaccinia virus Ankara (MVA) boost multi-gene multi-subtype human immunodeficiency virus-1 (HIV-1) vaccine intended for use in humans. The plasmid DNA vaccine was delivered intradermally using a Biojector, and the MVA was delivered intramuscularly by needle. This combination of recombinant DNA and MVA proved to induce extraordinarily strong cellular responses, with more than 80% of the CD8(+) T cells specific for HIV-1 antigens.

Direct exosome stimulation of peripheral human T cells detected by ELISPOT.

Exosomes from APC are nano-vesicles that can induce antigen-specific T cell responses and are presently explored as therapeutic tools in different clinical settings. Investigations of the capacity of exosomes to stimulate T cells in vitro have mostly been performed on T cell hybridomas, clones or lines. Whether exosomes can stimulate T cells directly or need the presence of dendritic cells (DC) is debated.

Detection of macaque perforin expression and release by flow cytometry, immunohistochemistry, ELISA, and ELISpot.

Simian immunodeficiency virus (SIV)-infection in macaques provides an important animal model for human immunodeficiency virus-1 (HIV-1) infection. The involvement of perforin (PFN), released by cytotoxic cells to mediate killing of virus-infected cells, has been difficult to assess in this experimental model due to a lack of reagents. We therefore evaluated monoclonal antibodies (mAbs) Pf-80, Pf-164 and Pf-344, previously raised against human PFN, for cross-reactivity with macaque PFN.

Detection of human perforin by ELISpot and ELISA: ex vivo identification of virus-specific cells.

The perforin (PFN) protein is essential for the elimination of target cells by cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. The study of cells releasing PFN has been hampered by a lack of sensitive methods. We therefore produced PFN-reactive monoclonal antibodies (mAb) and developed capture enzyme-linked immunosorbent (ELISA) and enzyme-linked immunospot (ELISpot) assays.

cIAP-2 block apoptotic events in bladder cancer cells.

Background: The Inhibitors of Apoptosis (IAPs) are negative regulators of apoptosis and their overexpression renders cells resistant to a variety of apoptotic stimuli. We investigated the mRNA expression levels of IAPs in a panel of bladder tumour cells, selected as being sensitive or resistant to death receptor-mediated apoptosis.

Materials And Methods: The mRNA expression of IAPs was quantified in a RNase protection assay (RPA).