In vivo imaging of human burn injuries with polarization-sensitive optical coherence tomography

The accurate determination of burn depth is critical in the clinical management of burn wounds. Polarization-sensitive optical coherence tomography (PS-OCT) has been proposed as a potentially non-invasive method for determining burn depth by measuring thermally induced changes in the structure and birefringence of skin, and has been investigated in pre-clinical burn studies with animal models and ex vivo human skin. In this study, we applied PS-OCT to the in-vivo imaging of two pediatric burn patients. Deep and superficial burned skins along with contralateral controls were imaged in 3D. The imaging size was 8 mm × 6 mm × 2 mm in width, length, and depth in the air respectively, and the imaging time was approximately 6 s per volume. Superficially burned skins exhibited the same layered structure as the contralateral controls, but more visible vasculature and reduced birefringence compared to the contralateral controls. In contrast, a deeply burned skin showed loss of the layered structure, almost absent vasculature, and smaller birefringence compared to superficial burns. This study suggested the vasculature and birefringence as parameters for characterizing burn wounds.     

Neoplastic transformation of human bronchial cells by lead chromate particles

Particulate hexavalent chromium (Cr(VI)) is a well-established human lung carcinogen with widespread exposure among people in occupational settings and the general public. However, no studies have examined the chromate-induced malignant transformation of human lung epithelial cells, its predominant target. Human papillomavirus-immortalized human bronchial epithelial (BEP2D) cells were used to better understand the mechanisms involved in human bronchial carcinogenesis induced by particulate chromate. We found that aneuploid cells increased in a concentration-dependent manner after chronic exposure to lead chromate. Moreover, chronic exposure to lead chromate induced BEP2D cell transformation. Transformed BEP2D cells developed through a series of sequential steps, including altered cell morphology, loss of cell contact inhibition and anchorage-independent growth. Specifically, a 5-day exposure to lead chromate induced foci formation with 0, 1, 5, and 10 microg/cm2 lead chromate inducing 0, 7, 3, and 15 foci in 10 dishes. Anchorage independence was observed in cell lines derived from these foci. These foci-derived cells also showed centrosome amplification and increases in aneuploid metaphases. Our study demonstrates that particulate Cr(VI) is able to transform human bronchial epithelial cells, and that chromosome instability may play an important role in particulate Cr(VI)-induced neoplastic transformation.     

<Emphasis Type="Italic">Ex Vivo</Emphasis> Methods for Informing Computational Models of the Mitral Valve

Computational modeling of the mitral valve (MV) has potential applications for determining optimal MV repair techniques and risk of recurrent mitral regurgitation. Two key concerns for informing these models are (1) sensitivity of model performance to the accuracy of the input geometry, and, (2) acquisition of comprehensive data sets against which the simulation can be validated across clinically relevant geometries. Addressing the first concern, ex vivo micro-computed tomography (microCT) was used to image MVs at high resolution (~40 micron voxel size). Because MVs distorted substantially during static imaging, glutaraldehyde fixation was used prior to microCT. After fixation, MV leaflet distortions were significantly smaller (p < 0.005), and detail of the chordal tree was appreciably greater. Addressing the second concern, a left heart simulator was designed to reproduce MV geometric perturbations seen in vivo in functional mitral regurgitation and after subsequent repair, and maintain compatibility with microCT. By permuting individual excised ovine MVs (n = 5) through each state (healthy, diseased and repaired), and imaging with microCT in each state, a comprehensive data set was produced. Using this data set, work is ongoing to construct and validate high-fidelity MV biomechanical models. These models will seek to link MV function across clinically relevant states.     

A role for MHC class II antigen processing in B cell development

For mature B cells, the encounter with foreign antigen results in the selective expansion of the cells and their differentiation into antibody secreting cells or memory B cells. The response of mature B cells to antigen requires not only antigen binding to and signaling through the B cell antigen receptor (BCR) but also the processing and presentation of the BCR bound antigen to helper T cells. Thus, in mature B cells, the ability to process and present antigen to helper T cells plays a critical role in determining the outcome of antigen encounter. In immature B cells, the binding of antigen results in negative selection of the B cell, inducing apoptosis, anergy or receptor editing. Negative selection of immature B cells requires antigen induced signaling through the BCR, analogous to the signaling function of the BCR in mature B cells. However, the role of class II antigen processing and presentation in immature B cells is less well understood. Current evidence indicates that the ability to process and present antigen bound to the BCR is a late acquisition of developing B cells, suggesting that during negative selection B cells may not present BCR bound antigen and interact with helper T cells. However, the expression of class II molecules is an early acquisition of B cells and recent evidence indicates that the expression of class II molecules early in development is required for the generation of long lived mature B cells. Here we review our current understanding of the processing and presentation of antigen by mature B cells and the role for antigen processing and class II expression during B cell development.     

Tetanus toxin fragment C expressed in live Salmonella vaccines enhances antibody responses to its fusion partner Schistosoma haematobium glutathione S-transferase

Tetanus toxoid has been used widely as an adjuvant. The atoxic fragment C from tetanus toxin (TetC) is potently immunogenic when expressed in Salmonella vaccine strains and has been used as a fusion partner for antigens (Ag). However, there has been no formal comparison of the immunomodulatory impact of TetC on its fusion partners. In this study, we have addressed this important issue. The protective 28-kDa glutathione S-transferase (GST) from Schistosoma haematobium (Sh28GST) was expressed either as a fusion to TetC or as the full-length Sh28GST alone in a nonvirulent aroA-attenuated strain of Salmonella enterica serovar Typhimurium. The Sh28GST proteins were soluble and stably expressed in Salmonella, as evaluated by Western blotting with TetC and/or Sh28GST antisera. Mice were immunized orally with a single dose of the live recombinant Salmonella. The constructs were stable in mice but, dramatically, only the strain expressing the TetC-Sh28GST fusion elicited significant antibody (Ab) responses directed against Sh28GST as determined by enzyme-linked immunosorbent assay. An analysis of the isotype profiles showed that these mice also produced anti-Sh28GST immunoglobulin A and GST-neutralizing assays revealed high levels of neutralizing Abs in sera. These are important correlates of protection in schistosomiasis. In addition, stimulation of spleen cells from immunized mice with Sh28GST Ag showed that both strains, expressing Sh28GST alone or the TetC-Sh28GST fusion, were able to stimulate the secretion of Th1-related cytokines (gamma interferon and interleukin 2) to comparable levels. Thus, TetC has modulated the immune responses generated against its fusion partner, Sh28GST, by markedly enhancing the Ab responses elicited. These results have important implications in the rational development of live vaccines.     

Anti-Apical-Membrane-Antigen-1 Antibody Is More Effective than Anti-42-Kilodalton-Merozoite-Surface-Protein-1 Antibody in Inhibiting Plasmodium falciparum Growth,as Determined by the In Vitro Growth Inhibition Assay

Apical membrane antigen 1 (AMA1) and the 42-kDa merozoite surface protein 1 (MSP1₄₂) are leading malaria vaccine candidates. Several preclinical and clinical trials have been conducted, and an in vitro parasite growth inhibition assay has been used to evaluate the biological activities of the resulting antibodies. In a U.S. phase 1 trial with AMA1-C1/Alhydrogel plus CPG 7909, the vaccination elicited anti-AMA1 immunoglobulin G (IgG) which showed up to 96% inhibition. However, antibodies induced by MSP1₄₂-C1/Alhydrogel plus CPG 7909 vaccine showed less than 32% inhibition in vitro. To determine whether anti-MSP1₄₂ IgG had less growth-inhibitory activity than anti-AMA1 IgG in vitro, the amounts of IgG that produced 50% inhibition of parasite growth (Ab₅₀) were compared for rabbit and human antibodies. The Ab₅₀s of rabbit and human anti-MSP1₄₂ IgGs were significantly higher (0.21 and 0.62 mg/ml, respectively) than those of anti-AMA1 IgGs (0.07 and 0.10 mg/ml, respectively) against 3D7 parasites. Ab₅₀ data against FVO parasites also demonstrated significant differences. We further investigated the Ab₅₀s of mouse and monkey anti-AMA1 IgGs and showed that there were significant differences between the species (mouse, 0.28 mg/ml, and monkey, 0.14 mg/ml, against 3D7 parasites). Although it is unknown whether growth-inhibitory activity in vitro reflects protective immunity in vivo, this study showed that the Ab₅₀ varies with both antigen and species. Our data provide a benchmark for antibody levels for future AMA1- or MSP1₄₂-based vaccine development efforts in preclinical and clinical trials.The scourge of malaria remains a global health problem, and 2.4 billion people live in areas at risk of infection with Plasmodium falciparum, the most virulent species of malaria in humans (10). An effective vaccine directed to blood-stage malaria parasites, which are responsible for the pathology associated with this disease, is of importance to control or eliminate the disease (8, 32). Apical membrane antigen 1 (AMA1) and the 42-kDa merozoite surface protein 1 fragment (MSP1₄₂) are leading vaccine candidates, and various evidence from epidemiologic and animal studies supports these proteins as potential vaccine targets (8, 33). Both proteins are expressed on the surface of merozoites (the invasive form of the blood-stage parasite) and are thought to have an important role in the invasion process; MSP1 is responsible for initial attachment and AMA1 for reorientation (9, 18, 25).In addition to the range of preclinical studies in animals, multiple phase 1 clinical trials have been conducted with either AMA1 or MSP1₄₂ (6, 7, 15, 16, 22, 23, 24, 26, 28, 30, 31, 33). However, the lack of a validated animal model or an in vitro correlate of protection makes it difficult to assess the efficacy of a vaccine candidate in terms of clinical protection. As of today, performing a large phase 2 trial is the only way to measure efficacy of a blood-stage vaccine; a trial with over 1,000 children is required to obtain precise estimates of efficacy against mild or uncomplicated clinical malaria disease (21). An in vitro parasite growth inhibition assay (GIA; also referred to as the invasion inhibition assay) is one of the few assays that can measure the biological activity of antibodies against parasites (or infected erythrocytes). Therefore, the GIA has been used in preclinical and phase 1 studies as one of the immunological measurements. A recent study showed that in Kenyan children and adults, time to infection after drug treatment was significantly associated with the growth-inhibitory activity measured by an in vitro GIA (5). Another study showed that individuals with high-level MSP1₁₉-specific invasion-inhibitory antibody had a 66% reduction in the risk of blood-stage infection during a 12-week follow-up after drug treatment (13).In preclinical studies and also several clinical trials in malaria-naive populations, we have shown that when the anti-AMA1 or -MSP1₄₂ antibody units (as measured by enzyme-linked immunosorbent assay [ELISA]) of total immunoglobulin G (IgG) were plotted against the percent inhibition (as measured by GIA), there was a strong correlation between antibody units and percent inhibition, with the relationship following a symmetrical sigmoid curve (16, 20, 22, 29). In a U.S. phase 1 trial, AMA1-C1 vaccine (a mixture of FVO and 3D7 allelic forms of AMA1 formulated with Alhydrogel plus CPG 7909) elicited anti-AMA1 IgGs. The IgGs showed up to 96% inhibition of growth against P. falciparum 3D7 parasites (22). However, antibodies induced by MSP1₄₂-C1 vaccine (a mixture of FVO and 3D7 allelic forms of MSP1₄₂ formulated with Alhydrogel plus CPG 7909) in a U.S. trial showed less than 32% inhibition in vitro (L. Martin et al., 56th Annu. Meet. Am. Soc. Trop. Med. Hyg., abstr. 213, p. 62, 2007). Therefore, in this study, we attempted to test the hypothesis that anti-MSP1₄₂ antibody has less biological activity than anti-AMA1 antibody in the GIA.To our knowledge, there is no study which has directly compared the amount of IgG that gives 50% inhibition of parasite growth (Ab₅₀) between anti-AMA1 and anti-MSP1₄₂ antibodies in the GIA. Although there is an argument as to whether in vitro growth-inhibitory activity can be a surrogate marker for in vivo clinical protection for AMA1- and/or MSP1-based vaccines, the GIA is currently one of a few biological assays widely used to estimate the potential of blood-stage vaccines. In this study, the Ab₅₀s for these two vaccine candidates were compared using both rabbit and human antibodies. In addition, for preclinical studies with AMA1- and/or MSP1-based vaccines in the future, we investigated the Ab₅₀s of anti-AMA1 antibodies in two more species (mouse and monkey) to determine whether there are differences in biological activities of antibodies between species. This study showed that the Ab₅₀s of anti-AMA1 IgGs were significantly lower than those of anti-MSP1₄₂ IgGs and that there were significant differences in Ab₅₀s between species.     

Oxidative modification and down-regulation of Pin1 in Alzheimer's disease hippocampus: A redox proteomics analysis

Alzheimer disease (AD) is characterized neuropathologically by intracellular neurofibrillary tangles (NFT) and of extracellular senile plaques (SP), the central core of which is amyloid beta-peptide (Abeta) derived from amyloid precursor protein (APP), a transmembrane protein. AD brain has been reported to be under oxidative stress that may play an important role in the pathogenesis and progression of AD. The present proteomics study is focused on identification of a specific target of protein oxidation in AD hippocampus that has relevance to the role of oxidative stress in AD. Here, we report that the protein, Pin1, is significantly down-regulated and oxidized in AD hippocampus. The identity of Pin1 was confirmed immunochemically. Analysis of Pin1 activity in AD brain and separately as oxidized pure Pin1 demonstrated that oxidation of Pin1 led to loss of activity. Pin1 has been implicated in multiple aspects of cell cycle regulation and dephosphorylation of tau protein as well as in AD. The in vivo oxidative modification of Pin1 as found by proteomics in AD hippocampus in the present study suggests that oxidative modification may be related to the known loss of Pin1 isomerase activity that could be crucial in AD neurofibrillary pathology. Taken together, these results provide evidence supporting a direct link between oxidative damage to neuronal Pin1 and the pathobiology of AD.     

The cytotoxicity and genotoxicity of soluble and particulate cobalt in human lung epithelial cells

Cobalt is a toxic metal used in various industrial applications leading to adverse lung effects by inhalation. Cobalt is considered a possible human carcinogen with the lung being a primary target. However, few studies have considered cobalt‐induced toxicity in human lung cells, especially normal lung epithelial cells. Therefore, in this study, we sought to determine the cytotoxicity and genotoxicity of particulate and soluble cobalt in normal primary human lung epithelial cells. Cobalt oxide and cobalt chloride were used as representative particulate and soluble cobalt compounds, respectively. Exposure to both particulate and soluble cobalt induced a concentration‐dependent increase in cytotoxicity, genotoxicity, and intracellular cobalt ion levels. Based on intracellular cobalt ion levels, we found that soluble and particulate cobalt induced similar cytotoxicity while soluble cobalt was more genotoxic than particulate cobalt. These data indicate that cobalt compounds are cytotoxic and genotoxic to human lung epithelial cells. Environ. Mol. Mutagen. 57:282–287, 2016. © 2016 Wiley Periodicals, Inc.