A combination of local inflammation and central memory T cells potentiates immunotherapy in the skin

Adoptive T cell therapy uses the specificity of the adaptive immune system to target cancer and virally infected cells. Yet the mechanism and means by which to enhance T cell function are incompletely described, especially in the skin. In this study, we use a murine model of immunotherapy to optimize cell-mediated immunity in the skin. We show that in vitro - derived central but not effector memory-like T cells bring about rapid regression of skin-expressing cognate Ag as a transgene in keratinocytes. Local inflammation induced by the TLR7 receptor agonist imiquimod subtly yet reproducibly decreases time to skin graft rejection elicited by central but not effector memory T cells in an immunodeficient mouse model. Local CCL4, a chemokine liberated by TLR7 agonism, similarly enhances central memory T cell function. In this model, IL-2 facilitates the development in vivo of effector function from central memory but not effector memory T cells. In a model of T cell tolerogenesis, we further show that adoptively transferred central but not effector memory T cells can give rise to successful cutaneous immunity, which is dependent on a local inflammatory cue in the target tissue at the time of adoptive T cell transfer. Thus, adoptive T cell therapy efficacy can be enhanced if CD8+ T cells with a central memory T cell phenotype are transferred, and IL-2 is present with contemporaneous local inflammation. Copyright © 2012 by The American Association of Immunologists, Inc.

Targeting antigen to diverse APCs inactivates memory CD8+ T cells without eliciting tissue-destructive effector function

Memory T cells develop early during the preclinical stages of autoimmune diseases and have traditionally been considered resistant to tolerance induction. As such, they may represent a potent barrier to the successful immunotherapy of established autoimmune diseases. It was recently shown that memory CD8+ T cell responses are terminated when Ag is genetically targeted to steady-state dendritic cells. However, under these conditions, inactivation of memory CD8+ T cells is slow, allowing transiently expanded memory CD8+ T cells to exert tissue-destructive effector function. In this study, we compared different Ag-targeting strategies and show, using an MHC class II promoter to drive Ag expression in a diverse range of APCs, that CD8+ memory T cells can be rapidly inactivated by MHC class II+ hematopoietic APCs through a mechanism that involves a rapid and sustained downregulation of TCR, in which the effector response of CD8+ memory cells is rapidly truncated and Ag-expressing target tissue destruction is prevented. Our data provide the first demonstration that genetically targeting Ag to a broad range of MHC class II+ APC types is a highly efficient way to terminate memory CD8+ T cell responses to prevent tissue-destructive effector function and potentially established autoimmune diseases. Copyright © 2010 by The American Association of Immunologists, Inc.

Human parvovirus B19: tissue persistence and prevalence of prototypic and new variants

Human parvovirus B19 is a minute ssDNA virus causing a wide variety of diseases, including erythema infectiosum, arthropathy, anemias, and fetal death. After primary infection, genomic DNA of B19 has been shown to persist in solid tissues of not only symptomatic but also of constitutionally healthy, immunocompetent individuals. In this thesis, the viral DNA was shown to persist as an apparently intact molecule of full length, and without persistence-specific mutations. Thus, although the mere presence of B19 DNA in tissue can not be used as a diagnostic criterion, a possible role in the pathogenesis of diseases e.g. through mRNA or protein production can not be excluded. The molecular mechanism, the host-cell type and the possible clinical significance of B19 DNA tissue persistence are yet to be elucidated. In the beginning of this work, the B19 genomic sequence was considered highly conserved. However, new variants were found: V9 was detected in 1998 in France, in serum of a child with aplastic crisis. This variant differed from the prototypic B19 sequences by ~10 %. In 2002 we found, persisting in skin of constitutionally healthy humans, DNA of another novel B19 variant, LaLi. Genetically this variant differed from both the prototypic sequences and the variant V9 also by ~10%. Simultaneously, B19 isolates with DNA sequences similar to LaLi were introduced by two other groups, in the USA and France. Based on phylogeny, a classification scheme based on three genotypes (B19 types 1-3) was proposed. Although the B19 virus is mainly transmitted via the respiratory route, blood and plasma-derived products contaminated with high levels of B19 DNA have also been shown to be infectious. The European Pharmacopoeia stipulates that, in Europe, from the beginning of 2004, plasma pools for manufacture must contain less than 104 IU/ml of B19 DNA. Quantitative PCR screening is therefore a prerequisite for restriction of the B19 DNA load and obtaining of safe plasma products. Due to the DNA sequence variation among the three B19 genotypes, however, B19 PCR methods might fail to detect the new variants. We therefore examined the suitability of the two commercially available quantitative B19 PCR tests, LightCycler-Parvovirus B19 quantification kit (Roche Diagnostics) and RealArt Parvo B19 LC PCR (Artus), for detection, quantification and differentiation of the three B19 types known, including B19 types 2 and 3. The former method was highly sensitive for detection of the B19 prototype but was not suitable for detection of types 2 and 3. The latter method detected and differentiated all three B19 virus types. However, one of the two type-3 strains was detected at a lower sensitivity. Then, we assessed the prevalence of the three B19 virus types among Finnish blood donors, by screening pooled plasma samples derived from >140 000 blood-donor units: none of the pools contained detectable levels of B19 virus types 2 or 3. According to the results of other groups, B19 type 2 was absent also among Danish blood-donors, and extremely rare among symptomatic European patients. B19 type 3 has been encountered endemically in Ghana and (apparently) in Brazil, and sporadical cases have been detected in France and the UK. We next examined the biological characteristics of these virus types. The p6 promoter regions of virus types 1-3 were cloned in front of a reporter gene, the constructs were transfected into different cell lines, and the promoter activities were measured. As a result, we found that the activities of the three p6 promoters, although differing in sequence by >20%, were of equal strength, and most active in B19-permissive cells. Furthermore, the infectivity of the three B19 types was examined in two B19-permissive cell lines. RT-PCR revealed synthesis of spliced B19 mRNAs, and immunofluorescence verified the production of NS1 and VP proteins in the infected cells. These experiments suggested similar host-cell tropism and showed that the three virus types are strains of the same species, i.e. human parvovirus B19. Last but not least, the sera from subjects infected in the past either with B19 type 1 or type 2 (as evidenced by tissue persistence of the respective DNAs), revealed in VP1/2- and VP2-EIAs a 100 % cross-reactivity between virus types 1 and 2. These results, together with similar studies by others, indicate that the three B19 genotypes constitute a single serotype.

Growth factor-loaded microparticles for tissue engineering: The discrepancies of in vitro characterization assays

Efficient and effective growth factor (GF) delivery is an ongoing challenge for tissue regeneration therapies. The accurate quantification of complex molecules such as GFs, encapsulated in polymeric delivery devices, is equally critical and just as complex as achieving efficient delivery of active GFs. In this study, GFs relevant to bone tissue formation, vascular endothelial growth factor (VEGF) and bone morphogenetic protein 7 (BMP-7), were encapsulated, using the technique of electrospraying, into poly(lactic-co-glycolic acid) microparticles that contained poly(ethylene glycol) and trehalose to assist GF bioactivity. Typical quantification procedures, such as extraction and release assays using saline buffer, generated a significant degree of GF interactions, which impaired accurate assessment by enzyme-linked immunosorbent assay (ELISA). When both dry BMP-7 and VEGF were processed with chloroform, as is the case during the electrospraying process, reduced concentrations of the GFs were detected by ELISA; however, the biological effect on myoblast cells (C2C12) or endothelial cells (HUVECs) was unaffected. When electrosprayed particles containing BMP-7 were cultured with preosteoblasts (MC3T3-E1), significant cell differentiation into osteoblasts was observed up to 3 weeks in culture, as assessed by measuring alkaline phosphatase. In conclusion, this study showed how electrosprayed microparticles ensured efficient delivery of fully active GFs relevant to bone tissue engineering. Critically, it also highlights major discrepancies in quantifying GFs in polymeric microparticle systems when comparing ELISA with cell-based assays.

Microneurovascular free muscle transfer with cross-over nerve grafts in facial reanimation

Microneurovascular free muscle transfer with cross-over nerve grafts in facial reanimation Loss of facial symmetry and mimetic function as seen in facial paralysis has an enormous impact on the psychosocial conditions of the patients. Patients with severe long-term facial paralysis are often reanimated with a two-stage procedure combining cross-facial nerve grafting, and 6 to 8 months later with microneurovascular (MNV) muscle transfer. In this thesis, we recorded the long-term results of MNV surgery in facial paralysis and observed the possible contributing factors to final functional and aesthetic outcome after this procedure. Twenty-seven out of forty patients operated on were interviewed, and the functional outcome was graded. Magnetic resonance imaging (MRI) of MNV muscle flaps was done, and nerve graft samples (n=37) were obtained in second stage of the operation and muscle biopsies (n=18) were taken during secondary operations.. The structure of MNV muscles and nerve grafts was evaluated using histological and immunohistochemical methods ( Ki-67, anti-myosin fast, S-100, NF-200, CD-31, p75NGFR, VEGF, Flt-1, Flk-1). Statistical analysis was performed. In our studies, we found that almost two-thirds of the patients achieved good result in facial reanimation. The longer the follow-up time after muscle transfer the weaker was the muscle function. A majority of the patients (78%) defined their quality of life improved after surgery. In MRI study, the free MNV flaps were significantly smaller than originally. A correlation was found between good functional outcome and normal muscle structure in MRI. In muscle biopsies, the mean muscle fiber diameter was diminished to 40% compared to control values. Proliferative activity of satellite cells was seen in 60% of the samples and it tended to decline with an increase of follow-up time. All samples showed intramuscular innervation. Severe muscle atrophy correlated with prolonged intraoperative ischaemia. The good long-term functional outcome correlated with dominance of fast fibers in muscle grafts. In nerve grafts, the mean number of viable axons amounted to 38% of that in control samples. The grafted nerves characterized by fibrosis and regenerated axons were thinner than in control samples although they were well vascularized. A longer time between cross facial nerve grafting and biopsy sampling correlated with a higher number of viable axons. P75Nerve Growth Factor Receptor (p75NGFR) was expressed in every nerve graft sample. The expression of p75NGFR was lower in older than in younger patients. A high expression of p75NGFR was often seen with better function of the transplanted muscle. In grafted nerve Vascular Endothelial Growth Factor (VEGF) and its receptors were expressed in nervous tissue. In conclusion, most of the patients achieved good result in facial reanimation and were satisfied with the functional outcome. The mimic function was poorer in patients with longer follow-up time. MRI can be used to evaluate the structure of the microneurovascular muscle flaps. Regeneration of the muscle flaps was still going on many years after the transplantation and reinnervation was seen in all muscle samples. Grafted nerves were characterized by fibrosis and fewer, thinner axons compared to control nerves although they were well vascularized. P75NGFR and VEGF were expressed in human nerve grafts with higher intensity than in control nerves which is described for the first time.

GATA Transcription Factors in Tissue Homeostasis and Pathology of the Gastrointestinal Tract and Liver

Mammalian gastrointestinal tract and liver are self-renewing organs that are able to sustain themselves due to stem cells present in their tissues. In constant, inflammation-related epithelial damage, vigorous activation of stem cells may lead to their uncontrolled proliferation, and further, to cancer. GATA-4, GATA-5, and GATA-6 regulate cell proliferation and differentiation in many mammalian organs. Lack of GATA-4 or GATA-6 leads to defective endodermal development and cell differentiation. GATA-4 and GATA-5 are considered the ones with tumor suppressive functions, whereas GATA-6 is more related to tumor promotion. In the digestive system their roles in inflammation and tumor-related molecular pathways remain unclear. In this study, we examined the GATA-related molecular pathways involved in normal tissue organization and renewal and in inflammation-related epithelial repair in the gastrointestinal tract and liver. The overall purpose of this study was to elucidate the relation of GATA factors to gastrointestinal and hepatic disease pathology and to evaluate their possible clinical significance in tumor biology. The results indicated distinct expression patterns for GATA-4, GATA-5, and GATA-6 in the human and murine gastrointestinal tract and liver, and their involvement in the regulation of intestine-specific genes. GATA-5 was confined to the intestines of suckling mice, suggesting an association with postnatal enzymatic changes. GATA-4 was upregulated in bowel inflammation concomitantly with TGF-β signaling. In gastrointestinal tumors, GATA-4 was restricted to benign neoplasias of the stomach, while GATA-6 was detected especially at the invasive edges of malignant tumors throughout the gut. In the liver, GATA-4 was upregulated in pediatric tumors along with erythropoietin (Epo), which was detected also in the sera of tumor patients. Furthermore, GATA-4 was enhanced in areas of vigorous hepatic regeneration in patients with tyrosinemia type I. These results suggest a central role for GATA-4 in pediatric tumor biology of the liver. To conclude, GATA-4, GATA-5, and GATA-6 are associated with normal gastrointestinal and hepatic development and regeneration. The appearance of GATA-4 along with TGF-β-signaling in the inflammatory bowel suggests a protective role in the response to inflammation-related epithelial destruction. However, in extremely malignant pediatric liver tumors, GATA-4 function is unlikely to be tumor-suppressing, probably due to the nature of the very primitive multipotent tumor cells. GATA-4, along with its possible downstream factor Epo, could be utilized as novel hepatic tumor markers to supplement the present diagnostics. They could also serve a function in future biological therapies for aggressive pediatric tumors.

Spiral-Wave Turbulence and Its Control in the Presence of Inhomogeneities in Four Mathematical Models of Cardiac Tissue

Regular electrical activation waves in cardiac tissue lead to the rhythmic contraction and expansion of the heart that ensures blood supply to the whole body. Irregularities in the propagation of these activation waves can result in cardiac arrhythmias, like ventricular tachycardia (VT) and ventricular fibrillation (VF), which are major causes of death in the industrialised world. Indeed there is growing consensus that spiral or scroll waves of electrical activation in cardiac tissue are associated with VT, whereas, when these waves break to yield spiral- or scroll-wave turbulence, VT develops into life-threatening VF: in the absence of medical intervention, this makes the heart incapable of pumping blood and a patient dies in roughly two-and-a-half minutes after the initiation of VF. Thus studies of spiral- and scroll-wave dynamics in cardiac tissue pose important challenges for in vivo and in vitro experimental studies and for in silico numerical studies of mathematical models for cardiac tissue. A major goal here is to develop low-amplitude defibrillation schemes for the elimination of VT and VF, especially in the presence of inhomogeneities that occur commonly in cardiac tissue. We present a detailed and systematic study of spiral- and scroll-wave turbulence and spatiotemporal chaos in four mathematical models for cardiac tissue, namely, the Panfilov, Luo-Rudy phase 1 (LRI), reduced Priebe-Beuckelmann (RPB) models, and the model of ten Tusscher, Noble, Noble, and Panfilov (TNNP). In particular, we use extensive numerical simulations to elucidate the interaction of spiral and scroll waves in these models with conduction and ionic inhomogeneities; we also examine the suppression of spiral- and scroll-wave turbulence by low-amplitude control pulses. Our central qualitative result is that, in all these models, the dynamics of such spiral waves depends very sensitively on such inhomogeneities. We also study two types of control chemes that have been suggested for the control of spiral turbulence, via low amplitude current pulses, in such mathematical models for cardiac tissue; our investigations here are designed to examine the efficacy of such control schemes in the presence of inhomogeneities. We find that a local pulsing scheme does not suppress spiral turbulence in the presence of inhomogeneities; but a scheme that uses control pulses on a spatially extended mesh is more successful in the elimination of spiral turbulence. We discuss the theoretical and experimental implications of our study that have a direct bearing on defibrillation, the control of life-threatening cardiac arrhythmias such as ventricular fibrillation.

Spectroscopy of tissue triglyceride composition : In vitro and in vivo studies

Lipid analysis is commonly performed by gas chromatography (GC) in laboratory conditions. Spectroscopic techniques, however, are non-destructive and can be implemented noninvasively in vivo. Excess fat (triglycerides) in visceral adipose tissue and liver is known predispose to metabolic abnormalities, collectively known as the metabolic syndrome. Insulin resistance is the likely cause with diets high in saturated fat known to impair insulin sensitivity. Tissue triglyceride composition has been used as marker of dietary intake but it can also be influenced by tissue specific handling of fatty acids. Recent studies have shown that adipocyte insulin sensitivity correlates positively with their saturated fat content, contradicting the common view of dietary effects. A better understanding of factors affecting tissue triglyceride composition is needed to provide further insights into tissue function in lipid metabolism. In this thesis two spectroscopic techniques were developed for in vitro and in vivo analysis of tissue triglyceride composition. In vitro studies (Study I) used infrared spectroscopy (FTIR), a fast and cost effective analytical technique well suited for multivariate analysis. Infrared spectra are characterized by peak overlap leading to poorly resolved absorbances and limited analytical performance. In vivo studies (Studies II, III and IV) used proton magnetic resonance spectroscopy (1H-MRS), an established non-invasive clinical method for measuring metabolites in vivo. 1H-MRS has been limited in its ability to analyze triglyceride composition due to poorly resolved resonances. Using an attenuated total reflection accessory, we were able to obtain pure triglyceride infrared spectra from adipose tissue biopsies. Using multivariate curve resolution (MCR), we were able to resolve the overlapping double bond absorbances of monounsaturated fat and polyunsaturated fat. MCR also resolved the isolated trans double bond and conjugated linoleic acids from an overlapping background absorbance. Using oil phantoms to study the effects of different fatty acid compositions on the echo time behaviour of triglycerides, it was concluded that the use of long echo times improved peak separation with T2 weighting having a negligible impact. It was also discovered that the echo time behaviour of the methyl resonance of omega-3 fats differed from other fats due to characteristic J-coupling. This novel insight could be used to detect omega-3 fats in human adipose tissue in vivo at very long echo times (TE = 470 and 540 ms). A comparison of 1H-MRS of adipose tissue in vivo and GC of adipose tissue biopsies in humans showed that long TE spectra resulted in improved peak fitting and better correlations with GC data. The study also showed that calculation of fatty acid fractions from 1H-MRS data is unreliable and should not be used. Omega-3 fatty acid content derived from long TE in vivo spectra (TE = 540 ms) correlated with total omega-3 fatty acid concentration measured by GC. The long TE protocol used for adipose tissue studies was subsequently extended to the analysis of liver fat composition. Respiratory triggering and long TE resulted in spectra with the olefinic and tissue water resonances resolved. Conversion of the derived unsaturation to double bond content per fatty acid showed that the results were in accordance with previously published gas chromatography data on liver fat composition. In patients with metabolic syndrome, liver fat was found to be more saturated than subcutaneous or visceral adipose tissue. The higher saturation observed in liver fat may be a result of a higher rate of de-novo-lipogenesis in liver than in adipose tissue. This thesis has introduced the first non-invasive method for determining adipose tissue omega-3 fatty acid content in humans in vivo. The methods introduced here have also shown that liver fat is more saturated than adipose tissue fat.

Matrix Metalloproteinases and their Tissue Inhibitors as Biomarkers in Ulcerative Colitis and Crohn s Disease

Matrix metalloproteinases (MMPs) represent a family of 23 metalloendopeptidases, collectively capable of degrading all components of the extracellular matrix. MMPs have been implicated in several inflammatory processes such as arthritis, atherosclerosis, and even carcinomas. They are also involved in several beneficial activities such as epithelial repair. MMPs are inhibited by endogenous tissue inhibitors of matrix metalloproteinases (TIMP). In this study, MMPs were investigated in intestinal mucosa of inflammatory bowel diseases (IBD), chronic intestinal disorders. The main focus was to characterize mucosal inflammation in the intestine, but also cutaneous pyoderma gangrenosum (PG), to assess similarites with IBD inflammation. MMPs and TIMPs were mainly examined in colonic mucosa, in adult Crohn s disease (CD), and paediatric CD, ulcerative colitis (UC), and indeterminate colitis (IC). Ileal pouch mucosa of proctocolectomized paediatric onset IBD patients was also investigated to characterize pouch mucosa. The focus was on finding specific MMPs that could act as markers to differentiate between different IBD disorders, and MMPs that could be implied as markers for tissue injury, potentially serving as targets for MMP-inhibitors. All examinations were performed using immunohistochemistry. The results show that immunosuppressive agents decrease stromal expression of MMP-9 and -26 that could serve as specific targets for MMP-inhibitors in treating CD. In paediatric colonic inflammation, MMP-10 and TIMP-3 present as molecular markers for IBD inflammation, and MMP-7 for CD. MMP expression in the the pouch mucosa could not be classified as strictly IBD- or non-IBD-like. For the first time, this study describes the expression of MMP-3, -7, -9, -12, and TIMP-2 and -3 in pouch mucosa. The MMP profile in PG bears resemblance to both intestinal IBD inflammation and cutaneous inflammation. Based on the results, MMPs and their inhibitors emerge as promising tools in the differential diagnosis of IBD and characterization of the disease subtype, although further research is necessary. Furthermore, the expression of several MMPs in pouch has been described for the first time. While further research is warranted, the findings contribute to a better understanding of events occurring in IBD mucosa, as well as pyoderma gangrenosum.

An introduction to ophthalmic biomaterials and their role in tissue engineering and regenerative medicine

This chapter presents a brief history of the development of ophthalmic biomaterials. Particularities in the development of ophthalmic biomaterials are discussed and some of their historic priorities within the general field of biomaterials are revealed or emphasized. The chapter then discusses the role and integration of ophthalmic biomaterials in tissue engineering and regenerative medicine applications.