Epicutaneous immunotherapy protects cashew-sensitized mice from anaphylaxis

Background

The prevalence of tree nut allergy has increased worldwide, and cashew has become one of the most common food allergens. More critically, cashew allergy is frequently associated with severe anaphylaxis. Despite the high medical need, no approved treatment is available and strict avoidance and preparedness for prompt treatment of allergic reactions are considered dual standard of care. In the meantime, Phase III study results suggest investigational epicutaneous immunotherapy (EPIT) may be a relevant and safe treatment for peanut allergy and may improve the quality of life for many peanut allergic children.

Objective

We aimed to evaluate the capacity of EPIT to provide protection against cashew-induced anaphylaxis in a relevant mouse model.

Methods

The efficacy of EPIT was evaluated by applying patches containing cashew allergens to cashew-sensitized mice. As negative control, sham mice received patches containing excipient. Following treatment, mice were challenged orally to cashew and anaphylactic symptoms, as well as plasmatic levels of mast-cell proteases (mMCP)-1/7, were quantified.

Results

Of 16 weeks of EPIT significantly protects against anaphylaxis by promoting a faster recovery of challenged mice. This protection was characterized by a significant reduction of temperature drop and clinical symptoms, 60 minutes after challenge. This was associated with a decrease in mast-cell reactivity as attested by the reduction of mMCP-1/7 in plasma, suggesting that EPIT specifically decrease IgE-mediated anaphylaxis.

Conclusion

We demonstrate that EPIT markedly reduced IgE-mediated allergic reactions in a mouse model of cashew allergy, which suggests that EPIT may be a relevant approach to treating cashew allergy.

     

The effect of automated landmark identification on morphometric analyses

Morphometric analysis of anatomical landmarks allows researchers to identify specific morphological differences between natural populations or experimental groups, but manually identifying landmarks is time‐consuming. We compare manually and automatically generated adult mouse skull landmarks and subsequent morphometric analyses to elucidate how switching from manual to automated landmarking will impact morphometric analysis results for large mouse (Mus musculus) samples (n = 1205) that represent a wide range of ‘normal’ phenotypic variation (62 genotypes). Other studies have suggested that the use of automated landmarking methods is feasible, but this study is the first to compare the utility of current automated approaches to manual landmarking for a large dataset that allows the quantification of intra‐ and inter‐strain variation. With this unique sample, we investigated how switching to a non‐linear image registration‐based automated landmarking method impacts estimated differences in genotype mean shape and shape variance‐covariance structure. In addition, we tested whether an initial registration of specimen images to genotype‐specific averages improves automatic landmark identification accuracy. Our results indicated that automated landmark placement was significantly different than manual landmark placement but that estimated skull shape covariation was correlated across methods. The addition of a preliminary genotype‐specific registration step as part of a two‐level procedure did not substantially improve on the accuracy of one‐level automatic landmark placement. The landmarks with the lowest automatic landmark accuracy are found in locations with poor image registration alignment. The most serious outliers within morphometric analysis of automated landmarks displayed instances of stochastic image registration error that are likely representative of errors common when applying image registration methods to micro‐computed tomography datasets that were initially collected with manual landmarking in mind. Additional efforts during specimen preparation and image acquisition can help reduce the number of registration errors and improve registration results. A reduction in skull shape variance estimates were noted for automated landmarking methods compared with manual landmarking. This partially reflects an underestimation of more extreme genotype shapes and loss of biological signal, but largely represents the fact that automated methods do not suffer from intra‐observer landmarking error. For appropriate samples and research questions, our image registration‐based automated landmarking method can eliminate the time required for manual landmarking and have a similar power to identify shape differences between inbred mouse genotypes.     

Suppressed Cell-Mediated Immunity and Monocyte and Natural Killer Cell Activity Following Allogeneic Immunization of Women with Spontaneous Recurrent Abortion

Spontaneous recurrent abortion (SRA) has been treated by means of immunization with paternal or third-party white blood cells, yet the immunological basis for SRA and for the role of immunization protocols in pregnancy outcome remains controversial. To elucidate this question, nine women with SRA were immunized with paternal mononuclear cells and studied before and 2 weeks after immunization. Seven women who became pregnant gave birth to live newborns. Secretion of the T helper 1 cytokines IL-2 and interferon- by patients' mononuclear cells decreased, while production of IL-10 increased. The levels of natural killer and lymphokine-activated killer cell-mediated cytotoxicity were markedly decreased. Monocyte functions such as secretion of IL-l, tumor necrosis factor a, IL-6, and cytotoxic activity decreased concurrently with elevations in IL-10 and transforming growth factor secretion. Production of IL-12, a pivotal regulatory cytokine, decreased. Furthermore, B7/1 expression on patients' mononuclear cells was downregulated. This resulted in a decrease in monocyte costimulatory activity of purified T cells with soluble anti-CD3, paralleled by a decline in allogeneic proliferative responses. These results suggest that the improved pregnancy success rate in women with SRA following immunization may be partly related to suppression of cell-mediated immunity and monocyte and natural killer cell activity.     

Surface chemistry modulates focal adhesion composition and signaling through changes in integrin binding

Biomaterial surface properties influence protein adsorption and elicit diverse cellular responses in biomedical and biotechnological applications. However, the molecular mechanisms directing cellular activities remain poorly understood. Using a model system with well-defined chemistries (CH₃, OH, COOH, NH₂) and a fixed density of the single adhesive ligand fibronectin, we investigated the effects of surface chemistry on focal adhesion assembly and signaling. Surface chemistry strongly modulated integrin binding and specificity—₅β₁ integrin binding affinity followed the pattern OH>NH₂=COOH>CH₃, while integrin _Vβ₃ displayed the relationship COOH>NH₂OH=CH₃. Immunostaining and biochemical analyses revealed that surface chemistry modulates the structure and molecular composition of cell-matrix adhesions as well as focal adhesion kinase (FAK) signaling. The neutral hydrophilic OH functionality supported the highest levels of recruitment of talin, -actinin, paxillin, and tyrosine-phosphorylated proteins to adhesive structures. The positively charged NH₂ and negatively charged COOH surfaces exhibited intermediate levels of recruitment of focal adhesion components, while the hydrophobic CH₃ substrate displayed the lowest levels. These patterns in focal adhesion assembly correlated well with integrin ₅β₁ binding. Phosphorylation of specific tyrosine residues in FAK also showed differential sensitivity to surface chemistry. Finally, surface chemistry-dependent differences in adhesive interactions modulated osteoblastic differentiation. These differences in focal adhesion assembly and signaling provide a potential mechanism for the diverse cellular responses elicited by different material properties.     

Interferon-α activates cytotoxic function but inhibits interleukin-2-mediated proliferation and tumor necrosis factor-α secretion by immature human natural killer cells

Natural killer (NK) cells play an important role in host defense mechanisms against infection and neoplasia. Interferon- (IFN-) has been shown to activate NK cells and to augment their cytotoxic activity, albeit its role in the maturation pathway of NK cells has not been elucidated. The present study examined whether IFN- activates the immature NK subset (Free cells) to become cytotoxic and also ascertained whether IFN- uses the same pathway of activation as that mediated by interleukin-2 (IL-2). Incubation of sorted Free cells overnight with IFN- resulted in augmentation of their cytotoxic function against NK sensitive target cells. The enhanced cytotoxic activity was not accompanied by a new recruitment of NK-target binder cells but by an increase in the frequency of killer cells in the conjugate fraction. Activation of the Free subset by IFN- resulted in upregulation of CD69, CD11b, and CD2 surface expression and stimulated secretion of IFN-. Unlike IL-2, IFN- did not stimulate the Free cells to proliferate or secrete TNF- and activation of cytotoxicity and modulation of surface antigens by IFN- were independent of TNF-. The failure of IFN- to stimulate secretion and proliferation by Free cells appeared to be mediated by negative signals. This was corroborated in experiments demonstrating that when Free cells were cultured with both IFN- and IL-2, a significant inhibition was observed for both the IL-2 dependent secretion of TNF- and proliferation. These results demonstrate that IFN- serves as both an activator and a regulator of NK function. Further, activation of the immature Free NK cells by IL-2 and IFN- proceeds by TNF--dependent and independent pathways, respectively. The findings also support our contention that the mechanism of activation of the cytotoxic machinery of NK cells is not linked to the mechanism of activation of cytokine secretion and/or proliferation.Abbreviations used IFN interferon - IL interleukin - PBL peripheral blood leukocytes - PE phycoerythrin - PE-GAM PE-conjugated Fab2 goat anti-mouse IgG - NK natural killer - NRS normal rabbit serum - TNF tumor necrosis factor - FCS fetal calf serum - FITC fluorescein isothiocyanate - PBS phosphate-buffered saline - MACS magnetic cell sorting - ELISA enzyme-linked immunosorbent assay - BSA bovine serum albumin - PKC protein kinase C - mAb monoclonal antibody - PBMC peripheral blood mononuclear cells - BCLL B-chronic lymphocytic leukemia - E effector - T target     

Humoral indices of stress in rats

Rats, bearing chronic venous cannulas, were subjected to 30 sec of constant current grid shock at 1 of 6 intensities (0, 0.25, 0.5, 1, 2, 4 mA), after being allowed to acclimate to the test chamber overnight. Blood, sampled before and after shock, was assayed for epinephrine, norepinephrine and corticosterone. Peak levels of both catecholamines increased in a stepwise fashion (i.e., monotonically) with increasing magnitude of stress, as reflected by current intensity of foot shock. Plasma corticosterone did not increase monotonically but instead showed similar increases in the 5 groups of rats that actually received shock. These data support earlier work which indicate that plasma corticosterone is not a sensitive index of stress; this is probably the case because of the relatively narrow range of responsiveness of the adrenal cortex to ACTH. In contrast, both plasma catecholamines appear to satisfy some of the requisites for a sensitive visceral index of stress.     

The functional anatomy of the human anterior talofibular ligament in relation to ankle sprains

The anterior talofibular ligament is the most commonly injured ligament in the ankle. Despite considerable interest in the clinical outcome of treatment protocols, we do not know whether the distinctive pattern of localization of the injuries relates to regional differences in the structure and molecular composition of the ligament. To address this issue, ligaments were examined by histology and immunohistochemistry. Differences in the structure of its two attachments (i.e. entheses) were evaluated with quantitative, morphometric techniques, and regional differences in the distribution of collagens, glycosaminoglycans and proteoglycans were determined qualitatively by immunolabelling. Morphometric analyses showed that bone density was less at the fibular attachment, but that enthesis fibrocartilage was more prominent. Immunohistochemistry revealed the presence of a fibrocartilage (containing type II collagen and aggrecan) at the site where the ligament wraps around the lateral talar articular cartilage in a plantarflexed and inverted foot: the fibrocartilage is regarded as an adaptation to resisting compression. We propose that avulsion fractures are less common at the talar end of the ligament because (1) bone density is greater here than at the fibular enthesis, and (2) stress is dissipated away from the talar enthesis by the 'wrap-around' fibrocartilaginous character of the ligament near the talar articular facet.