Peter J. Macardle

Antibody-Functionalized Porous Silicon Nanoparticles for Vectorization of Hydrophobic Drugs

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.

Fungal-specific humoral response in eosinophilic mucus chronic rhinosinusitis.

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.

Different temporal expression of immunodominant Ro60/60 kDa-SSA and La/SSB apotopes

Opsonization of apoptotic cardiocytes by maternal anti‐Ro/SSA and anti‐La/SSB antibodies contributes to tissue injury in the neonatal lupus syndrome. The objective of the current study was to quantify the surface membrane expression of Ro/La components during different phases of apoptosis and map the Ro/La apotopes (epitopes expressed on apoptotic cells) bound by cognate antibodies. Multi‐parameter flow cytometry was used to define early and late apoptotic populations and their respective binding by monospecific anti‐Ro and anti‐La IgGs. Anti‐Ro60 bound specifically to early apoptotic Jurkat cells and remained accessible on the cell surface throughout early and late apoptosis. In contrast, anti‐La bound exclusively to late apoptotic cells in experiments controlled for non‐specific membrane leakage of IgG. Ro52 was not accessible for antibody binding on either apoptotic population. The immunodominant NH2‐terminal and RNA recognition motif (RRM) epitopes of La were expressed as apotopes on late apoptotic cells, confirming recent in vivo findings. An immunodominant internal epitope of Ro60 that contains the RRM, and is recognized by a majority of sera from mothers of children with congenital heart block (CHB) and patients with primary Sjögrens syndrome, was also accessible as an apotope on early apoptotic cells. The distinct temporal expression of the immunodominant Ro60 and La apotopes indicates that these intracellular autoantigens translocate independently to the cell surface, and supports a model in which maternal antibody populations against both Ro60 and La apotopes act in an additive fashion to increase the risk of tissue damage in CHB.

Eosinophilic mucus chronic rhinosinusitis: Clinical subgroups or a homogeneous pathogenic entity?

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.

ProNGF mediates death of Natural Killer cells through activation of the p75NTR–sortilin complex

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.

The role of allergy in rhinosinusitis.

Purpose of reviewTo examine the current evidence for IgE and non-IgE-mediated hypersensitivity mechanisms in acute and chronic rhinosinusitis. Recent findingsEpidemiological 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. SummaryThere 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.

Functional monoclonal antibodies to p75 neurotrophin receptor raised in knockout mice

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.

Cytometry‐acquired calcium‐flux data analysis in activated lymphocytes

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).

Expression of the costimulator molecules, CD40 and CD154, on lymphocytes from neonates and young children

Differential expression of the costimulator molecules CD40 and CD154 on neonatal lymphocytes may be one explanation for limited T-dependent antibody responses in human neonates. CD40 was expressed at similar levels on resting B cells from adults, young children (2-20 months of age) or cord blood. CD40 expression was higher on cord blood B cells compared to adult B cells after stimulation with PMA and ionomycin, but similar on adult and cord blood B cells activated by CD3-stimulated T cells. In contrast to previous reports, cord blood T cells stimulated with PMA and ionomycin expressed adult levels of CD154 initially, but this expression was more transient on cord blood T cells. When adult and cord blood mononuclear cells were stimulated with CD3 mAb, T cells from some cord blood specimens showed different kinetics of CD154 expression compared with adult T cells. However, some cord blood specimens showed adult patterns of T cell CD154 expression. When mononuclear cells were depleted of B cells and monocytes prior to stimulation with CD3 mAb, the MFI and percentage of T cells expressing CD154 increased, with adult and cord T cells showing similar patterns of expression. These results show some differences in expression of CD40 and CD154 between neonatal and adult lymphocytes, but do not directly account for the relative deficiencies of humoral immunity in neonates.

IgE-mediated fungal allergy in allergic fungal sinusitis

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