Viral vector-mediated transgenic cell therapy in regenerative medicine: safety of the process

Introduction: There are safety concerns regarding viral vectors in regenerative medicine research because of adverse experiences in conventional gene therapy with systemic delivery of recombinant virus. Transgenic cell therapy emerges as an attractive strategy, in which the genes of interest are delivered in vitro into isolated cells first; instead of transgene vectors, these transgenic cells are then implanted back to the host. This ex vivo strategy enables the examination of cell viability and phenotype before subsequent transplantation and prevents to the most extent the potential delivery-related hazards caused by exposure of viral components to the host. The transgenic implants are often localized, thus traceable for safety monitoring except those cases involving systemic distribution of transgenic cells.

Areas covered: The safety of ex vivo process used in viral vector-mediated transgenic cell therapy for regenerative medicine purpose.

Expert opinion: Safety concerns related to viral vector delivery can be dispelled in the majority of regenerative medicine applications by transgenic cell therapy. The ex vivo process executes in vitro transfection before subsequent transplantation of transgenic cells so that it avoids the exposure of viral components (particularly capsids or envelops) to the host, while this exposure is inevitable in conventional in vivo gene therapy. Besides, the practice of localized cell implantation and in vitro manipulation also reinforce the safety of transgenic cell therapy. Given the significantly reduced delivery-related hazard, viral vector-mediated transgenic cell therapy can be generally considered as a safe approach for most regenerative medicine applications.     

Wnt signalling in an in vitro niche model for conjunctival progenitor cells

Mimicking an environment in vitro that is more similar to the stem cell niche in vivo, by co‐culture of mitotically active conjunctival fibroblasts (HCF) with human conjunctival epithelial cells (HCECs), improves the maintenance of epithelial cells with progenitor cell characteristics during in vitro expansion. However, little is known about the pathways controlling the fate of the epithelial progenitor cells during in vitro culture. In this study, differences in gene expression between this in vitro 'niche' model and standard culture conditions, in which growth‐arrested 3 T3 feeder cells and fetal calf serum are used, were explored using a genome level microarray platform, quantitative (q)RT–PCR and western blot. The microarray analysis revealed significant alterations of biological processes involved in cell proliferation, differentiation and cell death. The analysis of stem cell‐related pathways indicated changes in expression of genes involved in the Wnt signalling pathway, and further investigation by qPCR revealed significant downregulation of the Wnt ligands Wnt3, Wnt4, Wnt7B and Wnt10A, Wnt receptor proteins FZD1, LRP5, LRP6, ß‐catenin and TCF7L1 and important Wnt target genes, such as CCND1, also confirmed by western blot and immunocytochemistry. The results indicate that epithelial cell expansion in the HCEC–HCF co‐culture system is accompanied by significant changes in expression of genes involved in the Wnt signalling pathway. This altered pathway activation might be involved in the enhanced maintenance of epithelial progenitor cells in this in vitro 'niche' model. Copyright © 2012 John Wiley & Sons, Ltd.     

Assessment of Chemotherapy-Induced Organ Damage with Ga-68 Labeled Duramycin

Purpose

Evaluation of [⁶⁸Ga]NODAGA-duramycin as a positron emission tomography (PET) tracer of cell death for whole-body detection of chemotherapy-induced organ toxicity.

Procedures

Tracer specificity of Ga-68 labeled NODAGA-duramycin was determined in vitro using competitive binding experiments. Organ uptake was analyzed in untreated and doxorubicin, busulfan, and cisplatin-treated mice 2 h after intravenous injection of [⁶⁸Ga]NODAGA-duramycin. In vivo data were validated by immunohistology and blood parameters.

Results

In vitro experiments confirmed specific binding of [⁶⁸Ga]NODAGA-duramycin. Organ toxicities were detected successfully using [⁶⁸Ga]NODAGA-duramycin PET/X-ray computed tomography (CT) and confirmed by immunohistochemistry and blood parameter analysis. Organ toxicities in livers and kidneys showed similar trends in PET/CT and immunohistology. Busulfan and cisplatin-related organ toxicities in heart, liver, and lungs were detected earlier by PET/CT than by blood parameters and immunohistology.

Conclusion

[⁶⁸Ga]NODAGA-duramycin PET/CT was successfully applied to non-invasively detect chemotherapy-induced organ toxicity with high sensitivity in mice. It, therefore, represents a promising alternative to standard toxicological analyses with a high translational potential.

     

The activin‐βA/BMP‐2 chimera AB204 is a strong stimulator of adipogenesis

Several of the bone morphogenetic proteins (BMPs) have been reported to induce white as well as brown adipogenesis. Here, we characterized the adipogenic potential of AB204, a recombinant chimeric protein of activin‐βA and BMP‐2, in in vitro, ex vivo and in vivo settings. BMP‐2 is generally known to promote adipogenesis. When compared with BMP‐2, which previously showed varying degrees of adipogenesis, AB204 displayed superior in vitro adipogenic differentiation of mouse 3 T3‐L1 pre‐adipocytes and human adipose‐derived stem cells (hASCs). Surprisingly, implantation of hASCs, preconditioned with AB204 for as short a time as 48 h, into the subcutaneous space of athymic nude mice effectively produced fat pads, but not with BMP‐2. When BMP‐2 and AB204 were injected intraperitoneally, AB204 promoted dramatic systemic adipogenesis of C57BL/6 mice on a high‐fat diet very effectively. The results implicate the novel clinical potential of AB204, including induction of fat tissue ex vivo or in vivo for tissue re‐engineering and regenerative medicinal purposes, more than any known natural protein ligand. Copyright © 2015 John Wiley & Sons, Ltd.     

Diagnostic efficacy of the ELISA test for the detection of deamidated anti‐gliadin peptide antibodies in the diagnosis and monitoring of celiac disease

Background and Aim: We evaluated the diagnostic performance of an ELISA test for anti‐gliadin IgA and IgG antibodies, which uses synthetic deamidated gliadin peptides (anti‐gliadin antibodies, AGAs) as coating; the results were compared with a test that uses extracted gliadin (AGAe). Methods: The study was conducted on the sera of 144 patients suffering from celiac disease (CD), including 20 patients with IgA deficiency and 9 who were following a gluten‐free diet (GFD), and 129 controls. Results: In the 115 CD patients (without IgA deficiency), the sensitivity of AGAe IgA and IgG was 32.2 and 60.9%, whereas that of AGAs IgA and IgG was 59.1 and 72.2%. The specificity for AGAe IgA and IgG, and AGAs IgA and IgG was 93.8 and 89.9%, and 96.9% and 99.2%, respectively. Of the 20 patients with CD and IgA deficiency, 7 tested positive for AGAe IgG and 14 for AGAs IgG. The test using deamidated gliadin peptides performed better in terms of sensitivity and specificity than the AGA tests with extracted antigen. Conclusions: The very high specificity of the AGAs IgG test (99.2%) also suggests that patients who test positive with this assay require a thorough followup, even if the anti‐tissue transglutaminase antibodies (anti‐tTG) and anti‐endomysial autoantibodies (EMA) assays are negative. J. Clin. Lab. Anal. 23:165–171, 2009. © 2009 Wiley‐Liss, Inc.     

Skin epithelial cells as possible substitutes for ameloblasts during tooth regeneration

The disappearance of ameloblasts in erupted teeth hampers the implementation of tissue engineering‐based tooth regeneration. We aimed at utilizing skin epithelial cells as the appropriate substitute for ameloblasts. The conversion potential of 1 day postnatal rat skin epithelial cells to ameloblasts was investigated under the induction of dental papillae mesenchymal cells (DPMCs). Induction strategies had been designed both in vitro and in vivo. Markers for ameloblasts had been detected in skin epithelial cells, which showed a columnar appearance with the nuclei located at one side, under indirect co‐culture with DPMCs in vitro. An enamel–dentine‐like and tooth germ‐like structure was formed by recombining skin epithelial pieces or cells with DPMCs after 14 days of implantation in rat renal capsule. Immunohistochemistry and cell labelling analysis further demonstrated that the enamel‐forming cells were skin epithelium‐derived. These results indicated that the skin epithelium‐derived cells from postnatal rats have the potential to convert to functional ameloblasts under effective induction. Copyright © 2012 John Wiley & Sons, Ltd.     

Bread wheat gliadin cytotoxicity: a new three-dimensional cell model

BACKGROUND: In an attempt to clarify the role of gliadin toxicity in the pathogenesis of gluten intolerance (celiac disease), previous in vitro studies have been based on two-dimensional human cell cultures. However, the specific morphological and biochemical properties of in vivo tissue are better maintained in three-dimensional cell cultures (multicellular spheroids, MCS). The aim of this study was to develop a three-dimensional in vitro model to investigate the effects of gliadin on epithelial cells and broaden our understanding of the early tissue damage occurring in celiac disease. METHODS: The three-dimensionally growing Lovo cell line was exposed to increasing concentrations of peptic-tryptic-digested bread wheat gliadin (from 125 to 1000 microg/mL) for 7 days in order to evaluate cell viability (colony-forming assay), and at the standard concentration of 500 microg/mL for 7 days in order to evaluate MCS diameters, volumes and cell morphology using light and electron microscopy. RESULTS: In comparison with the controls, the cell viability of the gliadin-treated MCS was significantly reduced (20-80%), but there was no difference in size. Various degrees of cell damage (autophagic vacuoles and intra-cytoplasmic lipid-like droplets) were detected by both light and electron microscopy. CONCLUSION: This is the first study investigating the effects of gliadin on MCS. Lovo MCS seem to be responsive to gliadin exposure, thus confirming previous results obtained using two-dimensional cell cultures. The data suggest that three-dimensional cell cultures may be useful in broadening our understanding of some of the early effects of gliadin peptides on epithelial cells.     

Gluten-related disorders: certainties,questions and doubts

In the last decade, the ingestion of gluten, a heterogeneous complex of proteins present in wheat, rice, barley and probably in oats, has been associated with clinical disorders, such as celiac disease, wheat allergy and recently to non-celiac gluten sensitivity or wheat intolerance syndrome. Gluten-related disorders, which are becoming epidemiologically relevant with an estimated global prevalence of about 5%, require the exclusion of gluten from the diet. For the past 5 years, an important shift in the availability of gluten-free products, together with increased consumption in the general population, has been recorded and is estimated to be about 12–25%. Many people follow a self-prescribed gluten-free diet, despite the fact that the majority have not first been previously excluded, or confirmed, as having gluten disorders. They rely on claims that a gluten-free diet improves general health. In this review, we provide an overview of the clinical disorders related to gluten or wheat ingestion, pointing out the current certainties, open questions, possible answers and several doubts in the management of these conditions.

• KEY MESSAGE

• Incidence of gluten-related disorders is increased in the last decade and self-diagnosis is frequent with inappropriate starting of a gluten-free diet.

• Gluten and wheat are considered as the most important triggers to coeliac disease, wheat allergy and non-celiac gluten sensitivity.

• Pediatricians, allergologist and gastroenterologist are involved in the management of these conditions and appropriate diagnostic protocols are required.

     

Wnt‐4 potently inhibits capillary outgrowth from rat aorta in 3D culture

Regulation of angiogenesis involves tight cell‐to‐cell and cell‐to‐extracellular‐matrix interactions. Various reports demonstrate that the Wnt signaling pathways participate in this regulation. Using a three‐dimensional aortic ring culture combined with an ex vivo retroviral infection approach, we evaluated the effects of two Wnt growth factors, Wnt‐1 and Wnt‐4, on the formation and growth of new capillaries. Our results show that Wnt‐1 had no effect, whereas Wnt‐4 was a potent inhibitor of capillary outgrowth in vitro.     

Non-contact discrimination of human bone marrow-derived mesenchymal stem cells and fibroblasts using Raman spectroscopy

Background

Human bone marrow-derived mesenchymal stem cells (BM-MSCs) are a promising cell source for regenerative medical applications because they can be easily isolated and expanded ex vivo. However, due to the lack of specific cell surface markers, it is difficult to identify whether ex vivo isolated BM-MSC populations are free of stromal fibroblasts. Bright-field microscopical analyses are insufficient to determine fibroblastic contaminations since these two cell types have similar cell morphologies.

Materials and methods

We employed and compared traditional flow cytometric (FACS) analysis, in vitro differentiation assays, and Raman spectroscopy to distinguish between human BM-MSCs and fibroblasts.

Results

We found that FACS analysis, utilizing previously described fibroblast-identifying antibodies, was inadequate in separating stromal fibroblasts from BM-MSCs as over 75% of the BM-MSCs shared these antigens. In vitro differentiation assays revealed that, in contrast to fibroblasts, BM-MSCs could be successfully differentiated into adipocytes, osteoblasts and chondrocytes. Using this method it was possible to discriminate between the two cell types. However, the need for prolonged in vitro culture periods of up to 4 weeks is a major disadvantage of this test method. Raman spectroscopy, a non-contact technique measuring the wavelength and intensity of inelastic scattered light from molecules by employing high-power near-infrared lasers, distinguished ultra-fast between BM-MSCs and fibroblasts (integration time of 100 s/cell).

Conclusion

Based on the results, we conclude that Raman spectroscopy is a suitable tool for the rapid detection of fibroblastic contaminations in BM-MSC cultures.     

A responsive human triple‐culture model of the air–blood barrier: incorporation of different macrophage phenotypes

Current pulmonary research underlines the relevance of the alveolar macrophage (AM) integrated in multicellular co‐culture‐systems of the respiratory tract to unravel, for example, the mechanisms of tissue regeneration. AMs demonstrate a specific functionality, as they inhabit a unique microenvironment with high oxygen levels and exposure to external hazards. Healthy AMs display an anti‐inflammatory phenotype, prevent hypersensitivity to normally innocuous contaminants and maintain tissue homeostasis in the alveolus. To mirror the actual physiological function of the AM, we developed three different polarized [classically activated (M1) and alternatively activated (M2_wh, wound‐healing; M2_reg, regulatory)] macrophage models using a mixture of differentiation mediators, as described in the current literature. To test their immunological impact, these distinct macrophage phenotypes were seeded on to the epithelial layer of an established in vitro air–blood barrier co‐culture, consisting of alveolar epithelial cells A549 or H441 and microvascular endothelial cells ISO‐HAS‐1 on the opposite side of a Transwell filter‐membrane. IL‐8 and sICAM release were measured as functionality parameters after LPS challenge. The M1 model itself already provoked a severe inflammatory‐like response of the air–blood barrier co‐culture, thus demonstrating its potential as a useful in vitro model for inflammatory lung diseases. The two M2 models represent a ‘non‐inflammatory’ phenotype but still showed the ability to trigger inflammation following LPS challenge. Hence, the latter could be used to establish a quiescent, physiological in vitro air–blood model. Thus, the more complex differentiation protocol developed in the present study provides a responsive in vitro triple‐culture model of the air–blood‐barrier that mimics AM features as they occur in vivo. © 2015 The Authors Journal of Tissue Engineering and Regenerative Medicine Published by John Wiley & Sons, Ltd.     

Preclinical Development of Near-Infrared-Labeled CD38-Targeted Daratumumab for Optical Imaging of CD38 in Multiple Myeloma

Purpose

Cluster of differentiation 38 (CD38) is a promising therapeutic target in multiple myeloma (MM) patients and has resulted in the development of several CD38 immunotherapies. Current methods to evaluate CD38 expression in the preclinical setting include ex vivo flow cytometry and immunohistochemistry, which can be cumbersome and do not give whole-body information. In vivo imaging technologies such as positron emission tomography rely on decay of radioisotopes, limiting the number of molecular interactions observed at any given time point. Here, we demonstrate the use of near-infrared (NIR) fluorescence imaging for spatiotemporal monitoring of CD38 expression in preclinical MM using the anti-CD38 daratumumab (DARA) conjugated to the NIR fluorophore IRDye800CW (DARA-IRDye800).

Procedures

Stability studies with human serum and binding assays with human myeloma cells were performed with DARA-IRDye800. Immunocompromised mice with intra- and extramedullary tumors (n = 5/group) were administered with DARA-IRDye800 for in vivo imaging up to 7 days after injection. Ex vivo biodistribution and flow cytometry studies were performed to validate in vivo imaging results. A separate therapy study was performed in mice with intramedullary tumors that were treated and not treated with DARA at a therapeutic dose (n = 7/group). DARA-IRDye800 was administered for subsequent in vivo and ex vivo imaging in both cohorts of mice.

Results

DARA-IRDye800 maintained stability and had high affinity for CD38 (K_D = 3.5 ± 0.05 nM). DARA-IRDye800 demonstrated a 5- and 18-fold increase in contrast in tumor-bearing regions of mice with extra- and intramedullary MM. Finally, mice treated with therapeutic doses of DARA and imaged with DARA-IRDye800 showed an 11-fold decrease in fluorescence intensities in vivo compared with untreated controls.

Conclusions

Our studies establish DARA-IRDye800 as a promising contrast agent for preclinical evaluation of CD38 expression and for further investigating myeloma engraftment and kinetics in relation to anti-CD38 therapies.

     

Wheat amylase trypsin inhibitors drive intestinal inflammation via activation of toll-like receptor 4

Ingestion of wheat, barley, or rye triggers small intestinal inflammation in patients with celiac disease. Specifically, the storage proteins of these cereals (gluten) elicit an adaptive Th1-mediated immune response in individuals carrying HLA-DQ2 or HLA-DQ8 as major genetic predisposition. This well-defined role of adaptive immunity contrasts with an ill-defined component of innate immunity in celiac disease. We identify the α-amylase/trypsin inhibitors (ATIs) CM3 and 0.19, pest resistance molecules in wheat, as strong activators of innate immune responses in monocytes, macrophages, and dendritic cells. ATIs engage the TLR4–MD2–CD14 complex and lead to up-regulation of maturation markers and elicit release of proinflammatory cytokines in cells from celiac and nonceliac patients and in celiac patients’ biopsies. Mice deficient in TLR4 or TLR4 signaling are protected from intestinal and systemic immune responses upon oral challenge with ATIs. These findings define cereal ATIs as novel contributors to celiac disease. Moreover, ATIs may fuel inflammation and immune reactions in other intestinal and nonintestinal immune disorders.Wheat has only recently been added to the human diet (since the Neolithic age, i.e., roughly 12,000 years ago; Charmet, 2011). Today wheat has become the world’s major staple, and wheat products are widely used ingredients in processed foods. Wheat consumption correlates with certain disorders like wheat allergies and especially celiac disease (Shewry et al., 2003). Celiac disease is a common small intestinal enteropathy caused by dietary gluten in wheat, barley, and rye and affects ∼1% of most populations (Fasano et al., 2003; Green and Cellier, 2007; Di Sabatino and Corazza, 2009; Schuppan et al., 2009) Wheat gluten represents a family of largely water-insoluble storage proteins, subdivided into gliadins and glutenins, whereas other proteins are extractable as water-soluble albumins and salt-soluble globulins (Shewry et al., 2003; Wieser, 2007). Because of their unusual structure, with a high proline and glutamine content, the gluten proteins are partly resistant to intestinal enzymes, which leads to several nondegraded immunogenic peptides that can be sensed by the intestinal immune system. These gluten peptides are bound by the human lymphocyte antigen HLA-DQ2 or HLA-DQ8 (the major and necessary genetic predisposition for celiac disease) on intestinal antigen-presenting cells, and this binding is potentiated by the ubiquitous enzyme tissue transglutaminase (TG2), the celiac disease autoantigen (Dieterich et al., 1997). TG2 deamidates certain glutamine residues in (immunodominant) gluten peptides to glutamic acid, thereby increasing the peptides’ binding affinity to HLA-DQ2 and HLA-DQ8 and the subsequent T cell activation (Dieterich et al., 1997; van de Wal et al., 1998). This results in villus atrophy and crypt hyperplasia of the intestinal mucosa, the histological hallmarks of celiac disease, and frequent nutrient malabsorption and may promote certain celiac disease–associated autoimmune disorders (Green and Cellier, 2007; Di Sabatino and Corazza, 2009; Schuppan et al., 2009).Although several HLA-DQ2– and HLA-DQ8–restricted gluten peptides that trigger the adaptive immune response in celiac disease have been identified (Molberg et al., 1998; Anderson et al., 2000; Shan et al., 2002), only 2–5% of individuals expressing these HLAs develop the disease, indicating additional mechanisms of celiac disease pathogenesis, especially innate immune activation. The innate immune system provides an early response to many microbial and chemical stimuli and is critical for successful priming of adaptive immunity. Responsive innate cells are primarily macrophages, monocytes, DCs, and polymorphonuclear leukocytes that by means of their pattern-recognition receptors, such as TLRs, induce the release of proinflammatory cytokines and chemokines, resulting in recruitment and activation of additional inflammatory cells (Medzhitov, 2007).Thus, peptides p31-43 or p31-49 from α-gliadin that lack adaptive stimulatory capacity were incriminated as triggers of innate immunity as they induced IL-15 and Cox-2 expression in patients’ biopsies (Maiuri et al., 2003) and MHC class I polypeptide–related sequence A (MICA) on intestinal epithelial cells (Hüe et al., 2004). However, these studies were difficult to reproduce in cell culture, and no receptor responsible for the observed effects could be identified.In cell culture, gliadin was reported to induce increased expression of co-stimulatory molecules and the production of proinflammatory cytokines in monocytes and DCs (Nikulina et al., 2004; Cinova et al., 2007). In addition, the chemokine receptor CXCR3 was implicated in increased intestinal epithelial permeability upon gliadin challenge in a MyD88-dependent manner (Thomas et al., 2006; Lammers et al., 2008). However, no defined gliadin peptide was reproducibly identified. Collectively, a clear picture of the role of the innate immune system in celiac disease has not emerged.In this study, we show that members of the nongluten α-amylase/trypsin inhibitor (ATI) family contained in wheat and related cereals are strong inducers of innate immune responses in human and murine macrophages, monocytes, and DCs. ATI family members activate the TLR4–MD2–CD14 complex and elicit strong innate immune effects not only in vitro but also in vivo after oral or systemic challenge. Our findings have broad implications not only for celiac disease but also for other intestinal inflammatory disorders of the gastrointestinal tract.     

Specific interaction of food proteins with apical membranes of the human intestinal cell lines Caco-2 and T84

A comparison between the intestinal epithelial cell lines Caco-2 and T84 was made to assess the influence of enterocytic differentiation on food protein binding capacities of the brush border membrane. Cell morphology and expression of brush border-associated enzymes were studied as differentiation markers. Food protein binding to isolated brush border membranes was measured with a dot blot chemiluminescence assay. Early at confluence, Caco-2 cells exhibited a more differentiated state compared to T84 cells. Brush border membranes of both cell lines bound gliadin peptides, β-lactoglobulin and ovalbumin specifically. Binding capacities increased from gliadin peptides to ovalbumin to β-lactoglobulin. There was correlation of membrane binding capacity with degree of cell differentiation. Due to their similarity to small intestinal epithelial cells, the colon carcinoma cell lines Caco-2 and T84 represent models for studying food protein–enterocytic brush border membrane interactions in relation to varying degrees of cell differentiation.     

Degradation of interleukin 8 by the serine protease MucD of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

We investigated the influence of the type III effector, ExoS, on the host epithelial cell response to Pseudomonas aeruginosa infection, and we found that disruption of the exoS gene caused a significant increase in the amount of interleukin-8 (IL-8) in the culture medium of Caco-2 cells. We show that IL-8 was degraded in the culture medium following infection of the cells with the wild-type (PAO1), but not the exoS knock-out (the ΔexoS) strain. Purified ExoS protein itself did not degrade IL-8. We next show that IL-8 degradation by PAO1 was inhibited by the addition of serine protease inhibitors. These results strongly suggest that a bacterial serine protease that degrades IL-8 is expressed and secreted into the culture medium of Caco-2 cells infected with PAO1, and that the expression of this protein is repressed in cells infected with the ΔexoS strain. The PAO1 genome encodes 28 different protease genes, including two serine proteases: PA3535 and mucD. PA3535 and mucD gene knock-outs were constructed (ΔmucD and ΔPA3535), and ΔmucD but not ΔPA3535 showed reduced IL-8 degradation. To understand the significance of IL-8 degradation, we next evaluated neutrophil infiltration in lungs excised from mice intranasally infected with the P. aeruginosa strains. Increased neutrophil infiltration was observed in PAO1-infected mice, but not in ΔexoS- or ΔmucD-infected mice. Taken together, our results suggest that P. aeruginosa escapes from phagocytic killing due to IL-8 degradation following the secretion of the MucD serine protease, whose expression appears to be influenced by ExoS.     

Positron Emission Tomography Imaging of Small Animals in Anticancer Drug Development

Positron emission tomography (PET) imaging of small animals enables researchers to bridge the gap between in vitro science and in vivo human studies. The imaging paradigm can be established and refined in animals before implementation in humans and image data related to ex vivo assays of biological activity. Small animal PET (saPET) imaging enables assessment of baseline focal pathophysiology, pharmacokinetics, biological target modulation and the efficacy of novel drugs. The potential and challenge of this technology as applied to anticancer drug development is discussed here.     

Bench-to-bedside review: Hypercapnic acidosis in lung injury - from 'permissive' to 'therapeutic'

Modern ventilation strategies for patients with acute lung injury and acute respiratory distress syndrome frequently result in hypercapnic acidosis (HCA), which is regarded as an acceptable side effect ('permissive hypercapnia'). Multiple experimental studies have demonstrated advantageous effects of HCA in several lung injury models. To date, however, human trials studying the effect of carbon dioxide per se on outcome in patients with lung injury have not been performed. While significant concerns regarding HCA remain, in particular the possible unfavorable effects on bacterial killing and the inhibition of pulmonary epithelial wound repair, the potential for HCA in attenuating lung injury is promising. The underlying mechanisms by which HCA exerts its protective effects are complex, but dampening of the inflammatory response seems to play a pivotal role. After briefly summarizing the physiological effects of HCA, a critical analysis of the available evidence on the potential beneficial effects of therapeutic HCA from in vitro, ex vivo and in vivo lung injury models and from human studies will be reviewed. In addition, the potential concerns in the clinical setting will be outlined.     

An in vitro model of gluten-sensitive enteropathy. Effect of gliadin on intestinal epithelial cells of patients with gluten-sensitive enteropathy in organ culture

Jejunal biopsy specimens from patients with gluten-sensitive enteropathy (GSE) (obtained during gluten challenge) as well as from normal individuals and patients with other gastrointestinal abnormalities were cultured in vitro for 48 h in the presence or absence of a peptic-tryptic digest (P-T digest) of gliadin. In the absence of gliadin the alkaline phosphatase activity in the biopsy specimens obtained from normal control individuals increased from an initial value of 384 +/- 83 U to a 48 h value of 561 +/- 151 U (mean +/- SD) (difference significant at P < 0.01). The initial alkaline phosphatase activity of specimens obtained from patients with GSE was strikingly lower than that of normals, 117 +/- 79 U, and increased to a 48 h value of 399 +/- 203 U (difference significant at P < 0.01). The biochemical change in cultured biopsy specimens of GSE patients correlated with increases in the length and regularity of brush borders of epithelial cells as seen with the electron microscope. In the presence of a P-T digest of gliadin, the alkaline phosphatase activity of biopsy specimens of control individuals increased from an initial value of 384 +/- 83 U to a 48 h value of 578 +/- 156 U. In contrast, the alkaline phosphatase activity of biopsy specimens of patients with GSE in exacerbation showed a markedly diminished increase in activity during 48 h of culture; in this case the initial activity was 117 +/- 79 U and the final activity was 203 +/- 93 U. This inhibitory effect on increase of alkaline phosphatase activity during organ culture was specific in that a P-T digest of casein (a protein not toxic in vivo to patients with GSE) had no effect on alkaline phosphatase increases in culture. Finally, these results obtained with biopsy specimens taken from patients with GSE in exacerbation were compared with results obtained from patients with GSE in remission. Alkaline phosphatase activity of specimens obtained from the latter group of patients also increased during culture but in this instance P-T digest of gliadin in the culture medium had no significant inhibitory effect. In conclusion, the inhibitory effect of gliadin on intestinal epithelial cells in organ culture represents an in vitro model of gluten-sensitive enteropathy. Inasmuch as this effect of gliadin is not seen in cultures of specimens taken from patients in remission, it appears that gliadin is not directly toxic to GSE jejunal mucosa per se, but rather toxicity requires the participation of an endogenous effector mechanism which must first be stimulated in vivo.     

Celiac disease‐associated antibodies in patients with psoriasis and correlation with HLA Cw6

Etiopathology of psoriasis is not completely understood. Patients with psoriasis show elevated sensitivity to gluten. The aim of this study was to see the expression of celiac disease (CD)‐associated antibodies gliadin IgA, gliadin IgG, and tissue transglutaminase IgA, and their correlation with HLA Cw6 in patients with psoriasis. The study comprised 56 patients with psoriasis and 60 healthy controls (HC). The levels of antibodies were detected by using ELISA technique and HLA Cw6 typing was carried out by microcytotoxicity method. HLA Cw6 was significantly expressed in psoriasis cases when compared with HC (P<0.05). CD‐associated antibodies gliadin IgA/IgG and tissue transglutaminase IgA were significantly higher in the serum of patient with psoriasis when compared with HC (P<0.05, <0.05, and 0.01, respectively). Serum anti tissue transglutaminase IgA (anti tTG IgA) was significantly higher in females when compared with males and expressed more in elderly patients. There was a significant positive correlation among the antibodies (anti gliadin IgA with anti gliadin IgG: r=0.67, P<0.05; anti gliadin IgA with anti tTG IgA: r=0.45, P<0.05, anti gliadin IgG with anti tTG IgA: r=0.26, P<0.05, respectively), whereas insignificant with HLA Cw6. Our study concludes that latent CD or CD‐associated antibodies were present in patients with psoriasis and also concludes that HLA Cw6 has no association with expression of these antibodies in patients with psoriasis. J. Clin. Lab. Anal. 24:269–272, 2010. © 2010 Wiley‐Liss, Inc.