We describe the preparation of biodegradable porous silicon nanoparticles (pSiNP) functionalized with cancer cell targeting antibodies and loaded with the hydrophobic anti-cancer drug camptothecin. Orientated immobilization of the antibody on the pSiNP is achieved using novel semicarbazide based bioconjugate chemistry. To demonstrate the generality of this targeting approach, the three antibodies MLR2, mAb528 and Rituximab are used, which target neuroblastoma, glioblastoma and B lymphoma cells, respectively. Successful targeting is demonstrated by means of flow cytometry and immunocytochemistry both with cell lines and primary cells. Cell viability assays after incubation with pSiNPs show selective killing of cells expressing the receptor corresponding to the antibody attached on the pSiNP.

This paper describes a novel transducer principle converting biomolecular binding events into changes in porous silicon structural colour. An enzyme-catalysed reaction, horseradish peroxidase (HRP) mediated oxidation of 3,3′,5,5′-tetramethylbenzidine (TMB), induced an amplified optical detection signal on an antibody-functionalised porous silicon film, which manifested as significant changes in the Fabry–Pérot fringe pattern. Exposing porous silicon to non-oxidised TMB and oxidised TMB products and measuring the resulting effective optical thickness (EOT) changes of the porous silicon layer, an amplified signal from porous silicon was found, caused by an intermediate radical cation produced during the enzymatic-catalysed oxidation of TMB. Finally, it was shown that this system could be used to detect a human immunoglobulin (IgG) at 0.2μg/ml. This work may lead to the development of new biosensors where porous silicon acts as the sensing matrix and transducer element.

We report on surface-engineered microarrays that provide in situ cell sorting, localization, and immobilization of various subsets of human primary lymphocytes, followed by an on-chip bioassay for ionizing-radiation-induced cytogenetic damage. The microarray format eliminates the necessity of separating cell sub-populations by alternative means (such as fluorescence- or magnetic-activated cell sorting) prior to performing informational bioassays. To exemplify the potential of this on-chip cytometry approach, we have integrated the cytokinesis-block micronucleus cytome (CBMNcyt) assay with the microarray platform for analysis of the chromosome damage profile of specific subsets of human peripheral lymphocytes. Microarray results were compared with data obtained from the traditional CBMNcyt assay on heterogeneous lymphocyte populations, and with flow cytometry data. Our results suggest that cytogenetic damage caused by ionizing radiation is not uniformly distributed across all lymphocytes subsets, but rather concentrated in specific subsets. The salient features of our approach are that it requires very small volumes of reagents, allows sorting of lymphocyte subsets in situ, increases parallelism of cell assays and is amenable to high content microscopy analysis. The on-chip cytometry format opens new vistas for advanced cell-based assays, potentially bringing to light important information which remains hidden with conventional assays and hence engendering new discoveries in cell biology.

Recent observations of the deregulated expression of several dipeptidyl peptidase (DP) IV-like enzymes in human cancers have led to presumptions of their pathogenic role in cancer. To further explore this concept we have characterized the expression of all DPIV-like enzymes in chronic lymphocytic leukemia (CLL). We have demonstrated the constitutive expression of DPIV, DP8, DP9, DPII and PEP mRNA and DPIV, DP8 and DP9 protein in CLL. FAP mRNA was not detected in CLL or normal B-lymphocytes. This correlated with an absence of FAP protein on the cell surface. This study also shows that DP8 mRNA expression is significantly upregulated in CLL compared to normal tonsil B-lymphocytes (p < 0.05) which may suggest biological importance in this disease. DP expression could not be correlated with any molecular or clinical prognostic markers for CLL in this cohort including IgVH mutational status, CD38, ZAP-70 or CD49d expression (n = 58). However, the constitutive expression of the DPIV-like enzymes in CLL and their emergence as potent immune regulators makes them candidate therapeutic targets in this disease.

The common neurotrophin receptor P75NTR, its co-receptor sortilin and ligand proNGF, have not previously been investigated in Natural Killer (NK) cell function. We found freshly isolated NK cells express sortilin but not significant amounts of P75NTR unless exposed to interleukin-12 (IL-12), or cultured in serum free conditions, suggesting this receptor is sequestered. A second messenger associated with p75NTR, neurotrophin-receptor-interacting-MAGE-homologue (NRAGE) was identified in NK cells. Cleavage resistant proNGF123 killed NK cells in the presence of IL-12 after 20h and without IL-12 in serum free conditions at 48h. This was reduced by blocking sortilin with neurotensin. We conclude that proNGF induced apoptosis of NK cells may have important implications for limiting the innate immune response.

Background Allergic fungal sinusitis (AFS) is considered a different disease from other polypoid chronic rhinosinusitis diseases (CRS) with eosinophilic mucus (EM) termed eosinophilic mucus chronic rhinosinusitis (EMCRS). To substantiate this, studies on cellular responses to fungi and sinus mucosal inflammatory cell populations in AFS and other EMCRS diseases are. required. This study was designed to examine polyp inflammatory cell populations and peripheral blood fungal-specific T-cell responses in AFS, other EMCRS subgroups (defined later), and polypoid CRS without EM. Methods A prospective study was performed. Clinical characteristics, including CRS symptoms, sinus computed tomography (CT) scans, allergy status, intraoperative endoscopy, presence of EM, and fungal culture results were used to define patient groups. Polyps and peripheral blood were examined for populations of eosinophils, lymphocytes (CD4 + , CD8 + T cells, natural killer cells, and B cells), and neutrophils using immunohistochemistry, cytospin preparations and flow cytometry. Fungal-specific peripheral blood lymphocyte proliferation was examined in AFS patients, other EMCRS patients, CRS patients, and controls. Results There was no significant difference in the percentage of cell populations and fungal-specific lymphocyte proliferation between AFS and other EMCRS diseases. However, AFS and other EMCRS polyps had a higher percentage of eosinophils and CD8 + T cells whereas CRS polyps had higher CD4 4 T cells. Fungal-specific lymphocyte proliferation was significantly greater in AFS and other EMCRS patients regardless of fungal allergy, whereas in CRS and controls, higher proliferation was observed in fungal-allergic individuals. Conclusion These findings question the basis for differentiating AFS from, other EMCRS diseases based on fungal allergy and fungi in EM. Fungal-specific cellular response was present in AFS and other EMCRS diseases, different from, that associated with fungal allergy, suggesting a nonallergic fungal immune response. Increased CD8 + T cells in EMCRS polyps signify a different type of inflammation to CRS that may be driven by CD8 + T cells.

Mitochondria isolated from brain tissue following middle cerebral artery occlusion or during early reperfusion were tested for their ability to generate a membrane potential under standard conditions in vitro. Membrane potential was evaluated based on rhodamine 123 fluorescence in the mitochondria as detected using flow cytometry. Compared with equivalent samples from the contralateral hemisphere, the geometric mean fluorescence was significantly lower in mitochondria prepared from the striatum and perifocal tissue in the cortex at 3 h ischemia. During reperfusion, this property was decreased in mitochondria from tissue in the striatum and cortex that had been part of severely ischemic core tissue during the arterial occlusion. These findings provide additional evidence that mitochondria develop changes during ischemia and reperfusion that are likely to limit their ability to respond to changing energy requirements and contribute to cell dysfunction and cell death. It also demonstrates the ability to gain a sensitive measure of these mitochondrial changes using flow cytometry.

Purpose of review To examine the current evidence for IgE and non-IgE-mediated hypersensitivity mechanisms in acute and chronic rhinosinusitis. Recent findings Epidemiological studies show that classical IgE-mediated allergy is present in a proportion of acute rhinosinusitis patients. There is conflicting evidence whether the prevalence of IgE-mediated allergy is greater in chronic rhinosinusitis than in individuals without chronic rhinosinusitis. Despite presence of classical IgE-mediated allergy, based on elevated allergen-specific serum IgE levels and positive skin prick tests, currently there is no direct evidence for allergy as a major cause of sinonasal inflammation in chronic rhinosinusitis. There is increasing evidence that non-IgE-mediated fungal hypersensitivity and nonallergic IgE-associated inflammation may contribute to the pathogenesis in some forms of chronic rhinosinusitis, including allergic fungal sinusitis. Specific IgE to bacterial superantigens may also be elevated in nasal polyps and modulate eosinophilic inflammation. Recent insights into mucosal immune mechanisms yield intriguing prospects for the roles of mucosal IgE, mast cells and non-IgE-mediated hypersensitivity mechanisms that require further examination in rhinosinusitis. Summary There is a need for further immunological studies of the systemic and mucosal cellular and humoral mechanisms in well defined patient groups and controls to better understand the role of IgE and non-IgE-mediated hypersensitivity mechanisms and nonhypersensitivity functions of IgE in rhinosinusitis.

This review will address the current knowledge of the pathogenic mechanisms in allergic fungal sinusitis (AFS) and the basis for the current classification of a subgroup of chronic rhinosinusitis patients. Special attention is directed to the role of immunoglobulin E (IgE)-mediated fungal allergy in the pathogenesis of AFS. Concepts relating to the mucosal inflammatory response are introduced, as a knowledge of the reactions of the sinus mucosal cells can lead to a better understanding of the mechanisms perpetuating and maintaining the chronic inflammation. Laryngoscope, 2009.

Materials assisting with the efforts of cell isolation are attractive for numerous biomedical applications including tissue engineering and cell therapy. Here, we have developed surface modification methods on microparticles for the purposes of advanced cell separation. Iron oxide nanoparticles were incorporated into 200 ìm polystyrene microparticles for separation of particle-bound cells from non-bound cells in suspension by means of a permanent magnet. The polystyrene microparticles were further encoded with fluorescent quantum dots (QD) as identification tags to distinguish between specific microparticles in a mixture. Cluster of differentiation (CD) antibodies were displayed on the surface of the microparticles through direct adsorption and various methods of covalent attachment. In addition, a protein A coating was used to orientate the antibodies on the microparticle surface and to maximise accessibility of the antigen-binding sites. Microparticles which carried CD antibodies via covalent attachment showed greater cell attachment over those modifications that were only adsorbed to the surface through weak electrostatic interactions. Greatest extent of cell attachment was observed on microparticles modified with protein A - CD antibody conjugates. B and T lymphocytes were successfully isolated from a mixed population using two types of microparticles displaying B and T cell specific CD antibodies, respectively. Our approach will find application in preparative cell separation from tissue isolates and for microcarrier-based cell expansion.

Flow cytometry enables the sequential determination of calcium levels in millions of stimulated lymphocytes over a short period of time. Current algorithms available are not suitable for the statistical analysis of this large amount of data. The authors aimed to develop a robust algorithm that fits a function to median values of measured data and provides an opportunity for statistical comparison between different calcium‐flux measurements. The alteration of calcium signal was monitored in CD4+ cells loaded with calcium binding fluorescent dyes and stimulated with phytohemagglutinin; the alteration of calcium signal was monitored for 10 minutes. The authors also reanalyzed published calcium‐flux data of CD3+ cells and Jurkat cells stimulated with different concentrations of anti‐CD3 and thapsigargin. The authors fitted different functions to the medians of data per time unit and identified hormesis function as the best fitting one. On the basis of the optimally fitting function, the authors calculated the most relevant biological descriptors such as starting value, peak, time to reach the maximum, and time to reach 50% of maximum before and after the peak. Statistically significant differences in cell activation kinetics at different stimulatory concentrations were also demonstrated. This approach enables us to characterize the kinetics and distribution of calcium‐flux data derived by flow cytometry and may be a reliable tool for the characterization of lymphocyte activation (for details see: http://calciumflux.intralab.eu). © 2007 International Society for Analytical Cytology

BACKGROUND:Eosinophilic mucus chronic rhinosinusitis (EMCRS) can be subclassified using the criteria of detection of fungi in eosinophilic mucus and systemic fungal allergy. Allergic fungal sinusitis (AFS), a subgroup of EMCRS characterized by the presence of fungal allergy, is proposed to be an immunoglobulin (Ig)E-driven disease, distinct from other EMCRS subgroups. However, our recent studies cast doubt on the central pathogenic role of allergy in AFS. The purpose of this study was to examine the clinical features of EMCRS patients from the different subcategories to determine the relevance of this classification system.METHOD:The demographic, clinical, and immunologic characteristics of the EMCRS subgroups were examined prospectively and compared with three control groups: healthy volunteers, allergic rhinitis with fungal allergy, and chronic rhinosinusitis without eosinophilic mucus.RESULTS:EMCRS patients with allergy were younger than those without. There was no significant difference in clinicopathologic parameters between EMCRS subgroups. As a single group, EMCRS had a more severe sinus disease compared with chronic rhinosinusitis patients.CONCLUSIONS:AFS was not clinically distinct from other subgroups of EMCRS. However, eosinophilic mucus may mark a more severe and distinct form of sinus disease.

Indo-1 and high-power water-cooled lasers have been the standard for flow cytometric based Ca(2+) flux measurements. With advances in technology and the availability of low-power air-cooled lasers, there is interest in alternative protocols. Here, we have compared Indo-1 with the combination of fluo-3 and Fura Red calcium indicator dyes using low-power air-cooled lasers as the excitation source. The reagents were examined in parallel to detect Ca(2+) flux in peripheral blood T lymphocytes and in a T lymphoblastoid cell line. Ca(2+) flux was detected with a FACSVantage SE equipped with an Omnichrome Series 74 Helium-Cadmium, or a Spectra Physics 177-G1202 Argon ion air-cooled laser. Following determination of optimal loading conditions, Ca(2+) flux was examined in response to membrane receptor stimulation or intracellular Ca(2+) mobilization. Dose dependent Ca(2+) flux to anti-CD3 and thapsigargin was detected with either Indo-1 or with fluo-3 and Fura Red. The profile of the Ca(2+) flux detected by Indo-1 or with fluo-3 and Fura Red appeared similar, with the combination of fluo-3 and Fura Red more sensitive under the particular test conditions. The results clearly demonstrated that Indo-1 could be usefully excited with a low-power air-cooled laser. The alternative use of fluo-3 and Fura Red does not require the availability of a UV capable laser and produced equivalent data.

In this study, p75NTREXONIII knockout mice were used as immune-naive hosts to produce functional antibodies to human p75NTR. Three monoclonal antibodies were produced and named MLR1, MLR2 and MLR3, and isotyped as IgG1, IgG2a and IgG2a, respectively. MLR1 and MLR2 bound to human p75NTR with higher affinity than the well-characterized ME20.4 in ELISA and also recognized p75NTR present on neurons in both rat and mouse. MLR1 and MLR2 bound to nerves known to express p75NTR following injection into Balb/C mice but not p75NTREXONIII knockout mice, indicating the antibodies are directed against the ligand binding extracellular region absent in knockout mice. Both MLR1 and MLR2 partially blocked NGF induced cell death in a mouse cell-line that expresses p75NTR but not TrKA. Importantly, intracerebroventricular injections indicated MLR2 was internalized within the cell bodies of mouse basal forebrain neurons, further demonstrating that this antibody is biologically active.

Objectives/Hypothesis: An immunoglobulin (Ig)E-mediated allergic pathogenesis is presumed in allergic fungal sinusitis (AFS), yet extensive polyps and eosinophilic mucus (EM) in the paranasal sinuses may also occur in the absence of allergy. Although a noninvasive fungal pathogenesis is presumed in all chronic rhinosinusitis with EM (EMCRS), fungal-specific nonallergic immune responses have not been thoroughly investigated. We tested the hypothesis that there is a fungal-specific humoral response in EMCRS and that it is not confined to IgE. Study Design: EMCRS patients were prospectively stratified into subgroups based on the presence or absence of fungi within EM and of fungal-specific systemic IgE. There were 12 AFS, 5 AFS-like, 8 nonallergic fungal eosinophilic sinusitis (NAFES), and 5 nonallergic, nonfungal eosinophilic sinusitis (NANFES) patients. Methods: Alternaria alternata and Aspergillus fumigatus-specific serum IgE, IgG, IgM, and IgA was measured by enzyme-linked immunosorbent assay and compared with strictly defined healthy and disease-control groups. Results: Fungal-specific IgG (Alternaria alternata P = .0002; Aspergillus fumigatus P = .004), and IgA levels (Alternaria alternata P = .0016; Aspergillus fumigatus P = .002) were higher in EMCRS compared with healthy volunteers but not with disease controls. Fungal-specific IgG3 levels were significantly elevated in all the EMCRS subgroups compared with controls for either fungal antigen (P < .0001). Importantly, fungal-specific IgE levels were not significantly different between fungal-allergic EMCRS and disease controls. Conclusions: Fungal-specific immunity characterized by serum IgG3 and not IgE, distinguished the EMCRS subgroups from control groups regardless of the presence of fungus within EM or of systemic fungal allergy. Fungal-specific IgE responses in fungal-allergic EMCRS were no different to those in fungal-allergic controls, thus challenging the presumption of a unique pathogenic role of fungal allergy in "allergic fungal sinusitis.".

The immune system works through leukocytes interacting with each other, with other cells, with tissue matrices, with infectious agents, and with other antigens. These interactions are mediated by cell-surface glycoproteins and glycolipids. Antibodies against these leukocyte molecules have provided powerful tools for analysis of their structure, function, and distribution. Antibodies have been used widely in hematology, immunology, and pathology, and in research, diagnosis, and therapy. The associated CD nomenclature is commonly used when referring to leukocyte surface molecules and antibodies against them. It provides an essential classification for diagnostic and therapeutic purposes. The most recent (8th) Workshop and Conference on Human Leukocyte Differentiation Antigens (HLDA), held in Adelaide, Australia, in December 2004, allocated 95 new CD designations and made radical changes to its aims and future operational strategy in order to maintain its relevance to modern human biology and clinical practice.

There were over 600 antibodies submitted to HLDA8, with many of unknown specificity. Of these, 101 antibodies were selected for a blind panel study that also included 5 negative controls and 27 positive controls of known CD specificity making a total of 133 antibodies in the final panel. Of the 101 unknowns, 31 antibodies were identified during the course of this blind panel study as being specific for known molecules and included some specific for MHC class II antigens, CD45 isoforms and the Dombrock antigen. Several antibody pairs among those in the blind panel were found to have very similar staining patterns and were therefore compared by immunohistochemical and/or Western blot analyses for identity.

The shared rheumatoid epitope (SRE) on the MHC class II antigen-presenting molecule constitutes a probable genetic risk factor for the occurrence of rheumatoid arthritis (RA) and may also determine disease severity. We have used a novel flow cytometric technique to determine the SRE in over 500 predominantly Caucasian patients attending a general rheumatology clinic. This technique has been validated against a polymerase chain reaction (PCR)/SSO molecular method. The SRE was observed in 90% of patients with Felty's syndrome (n=10) and 75% of patients with RA (n=178) as compared with 39% of patients with osteoarthritis or non-inflammatory rheumatic disorders (n=73). Thus, the SRE determined by this method has a sensitivity for RA of 0.75, a specificity of 0.62 and an estimated positive predictive value of 0.02. In our RA cohort, there was no correlation between the functional outcome (health assessment questionnaire score) and SRE status. In conclusion, the determination of the SRE status by a flow cytometric method was found to have only modest sensitivity and specificity for RA; furthermore, the SRE did not correlate with functional outcomes. The clinical utility of the SRE assay is yet to be defined.

Calreticulin (CR) is a highly conserved, calcium-binding protein with a diverse functional repertoire located primarily in the endoplasmic reticulum (ER). A murine monoclonal antibody (mAb) reactive with human CR was produced by immunizing with a maltose-binding protein-CR fusion protein expressed in Escherichia coli. This mAb (FMC75) bound recombinant and native human 60-kDa CR on Western blots but, unlike a polyclonal anti-CR antibody, did not cross-react with mouse CR. FMC75 gave a staining pattern identical to that of the polyclonal antibody on confocal microscopy of cultured cells and was positive on microwave-treated tissue sections embedded in paraffin. Immunohistochemical analysis of a range of normal tissues confirmed the widespread expression of CR, notably in parenchymal epithelial cells, neurons, endothelial cells, and lymphocytes, predominantly of B-cell origin. The pattern of staining was cytoplasmic, not nuclear. Only weak staining was found in stromal cells. This first mAb to be produced against human CR will be a valuable reagent for studying the expression of CR and its putative role in autoimmune disease and malignancy. Recombinant fusion proteins in which the target protein is fused with a foreign moiety may be useful immunogens for breaking tolerance and generating mAbs against extremely conserved proteins such as CR.