Targeting self-antigens through allogeneic TCR gene transfer

Adoptive transfer of T-cell receptor (TCR) genes has been proposed as an attractive approach for immunotherapy in cases where the endogenous T-cell repertoire is insufficient. While there are promising data demonstrating the capacity of TCR-modified T cells to react to foreign antigen encounter, the feasibility of targeting tumor-associated self-antigens has not been addressed. Here we demonstrate that T-cell receptor gene transfer allows the induction of defined self-antigen-specific T-cell responses, even when the endogenous T-cell repertoire is nonreactive. Furthermore, we show that adoptive transfer of T-cell receptor genes can be used to induce strong antigen-specific T-cell responsiveness in partially MHC-mismatched hosts without detectable graft versus host disease. These results demonstrate the feasibility of using a collection of "off the shelf" T-cell receptor genes to target defined tumor-associated self-antigens and thereby form a clear incentive to test this immunotherapeutic approach in a clinical setting.     

Aphasia blog talk: How does stroke and aphasia affect the carer and their relationship with the person with aphasia?

Background: Carers of stroke survivors with aphasia are at risk of experiencing negative bio-psychosocial consequences and reduced quality of life. So far, in aphasia studies, this has mainly been explored through qualitative interviews and questionnaires. Unsolicited first-person narratives in the form of blogs offer a novel and rich source of data to examine how stroke and aphasia affect the carer and their relationship with the person with aphasia.

Aims: This study explored how carers of people with aphasia perceive their roles and responsibilities; it also examined the consequences of carrying out these carer roles and duties, in terms of both the carer’s own well-being and their relationship with the person with aphasia; furthermore, it investigated facilitative factors in their adaptation to the carer role.

Methods & Procedures: Publically available blogs written by carers of aphasic stroke survivors, which included information on how stroke and aphasia affect the carer and their relationship with the person with aphasia, were analysed using the Framework Method.

Outcomes & Results: The search resulted in nine carer blogs. The number of posts per blog ranged from 13 to 241. For blogs containing over 90 posts, the first and last 30 relevant posts were collected and analysed. New roles and extra responsibilities identified by participants included having to act as therapists, nurses, counsellors, and administrators and carrying out tasks usually assigned to the other person in the relationship. The extra tasks and duties impacted on the carers’ quality of life and their relationship with the person with aphasia in negative ways, such as leading to physical and mental exhaustion, health issues, feeling lonely, and resentful of their circumstances; however, participants also identified positive changes such as new closeness, new appreciation of life, and pride in achievements. A variety of strategies emerged from the data that helped carers adjust to their new roles. Strategies included positive reframing, allocating time to oneself, and seeking support from family and friends. The activity of blogging was also mentioned as having beneficial effects on the carers’ well-being.

Conclusions: The study provides further evidence for the specific challenges faced by carers of people with stroke and aphasia; it thus confirms the importance of addressing carer related needs in intervention and considering psychosocial well-being for both the carer and the person with aphasia.     

Kinetics of inflammatory response of astrocytes induced by TLR 3 and TLR4 ligation

Toll-like receptors (TLRs) are sentinels of the innate immune system that recognize an array of exogenous and endogenous pathogenic molecules. The ligation of the receptors triggers inflammatory response necessary for pathogen elimination and for the healing process. In the present study we examined inflammatory response of astrocytes elicited by the ligation of TLR3 and TLR4. Astrocytic cultures established from newborn rat brains were exposed to double stranded RNA (dsRNA) and lipopolysaccharide (LPS), the ligands for TLR3 and TLR4, respectively. The expression of cytokine genes was determined by RNase protection assay, and the generation of nitric oxide (NO) was measured by Griess technique. Both ligands upregulated the expression of several cytokines (i.e., IL-1alpha, IL-1beta, IL-6, TNFalpha, GM-CSF, LTbeta, and TGFbeta3) and downregulated the expression of MIF, but have no effect on the expression of IL-2, IL-3, IL-4, IL-5, IL-10, TGFbeta1, TGFbeta2, TNFbeta, and IFNgamma. Although dsRNA upregulated the expression of IFNbeta, LPS did not indicating that the TRIF-dependent branch of TLR4 signaling is inactive in astrocytes. Proinflammatory response as seen from upregulated cytokine expression and NO generation reached a peak within the first day of exposure, and was subsequently abrogated. The cells also became refractory to subsequent stimulation by the ligands indicating the existence of negative feedback mechanisms that control proinflammatory response in astrocytes.     

Presence of six different lesion types suggests diverse mechanisms of tissue injury in neuromyelitis optica

Neuromyelitis optica (NMO) is an autoimmune disease targeting aquaporin 4 (AQP4), localized mainly at the astrocytic foot processes. Loss of AQP4 and glial fibrillary acidic protein (GFAP) was reported, but the pathological significance of astrocytopathy is still controversial. Here we show that active lesions in NMO display a wide spectrum of pathology even within a single tissue block of an individual patient. We have distinguished six different lesion types. The first reflects complement deposition at the surface of astrocytes, associated with granulocyte infiltration and astrocyte necrosis and followed by demyelination, global tissue destruction and the formation of cystic, necrotic lesions (lesion type 2). Such destructive lesions lead to Wallerian degeneration in lesion-related tracts (lesion type 3). Around active NMO lesions AQP4 may selectively be lost in the absence of aquaporin 1 (AQP1) loss or other structural damage (lesion type 4). Another pattern is characterized by clasmatodendrosis of astrocytes, defined by cytoplasmic swelling and vacuolation, beading and dissolution of their processes and nuclear alterations resembling apoptosis, which was associated with internalization of AQP4 and AQP1 and astrocyte apoptosis in the absence of complement activation. Such lesions give rise to extensive astrocyte loss, which may occur in part in the absence of any other tissue injury, such as demyelination or axonal degeneration (lesion type 5). Finally, lesions with a variable degree of astrocyte clasmatodendrosis are found, which show plaque-like primary demyelination that is associated with oligodendrocyte apoptosis, but with preservation of axons (lesion type 6). In active multiple sclerosis (MS) lesions astrocytes reveal changes of reactive protoplasmatic or fibrillary gliosis. Only in a subset of lesions, in patients with aggressive disease, loss of AQP4 is observed in the initial stage of their formation, which is associated with retraction of astrocyte processes in the absence of complement deposition, granulocyte infiltration or loss of AQP1 or astrocytes. Our data underline the primary assault of astrocytes in NMO lesions, but also indicate that different mechanisms of tissue injury operate in parallel in the same patient and even within the same lesion.     

Comparative evaluation of DNA damage by genotoxicants in primary rat cells applying the comet assay

Various compounds known to cause DNA damage (hydrogen peroxide, visible light-excited methylene blue, N-nitrosomorpholine and benzo[a]pyrene) were tested with different primary rat cells (lymphocytes, testicular cells, type II pneumocytes and hepatocytes) to determine the range of induced DNA damage applying the comet assay. A dose-dependent increase of DNA breaks was observed after treatment with hydrogen peroxide in all cell types studied. The most prominent effect was observed in lymphocytes, whereas only a slight increase of DNA breaks was observed in hepatocytes. Visible light-excited methylene blue caused significant oxidative DNA damage, which did not significantly differ between the cell types used with the exception of hepatocytes, for which a lower level of DNA damage was observed. N-Nitrosomorpholine and benzo[a]pyrene induced a moderate but significant increase of DNA strand breaks in pneumocytes and hepatocytes while in lymphocytes no effect was observed. Our results clearly demonstrate that due to their differential function which is also expressed by the level of drug metabolizing and/or antioxidant enzymes, freshly isolated rat cells (lymphocytes, testicular cells, type II pneumocytes and hepatocytes) respond differently to the exposure to genotoxic agents as detected by comet assay.     

Cellular uptake and cytotoxic potential of respirable bentonite particles with different quartz contents and chemical modifications in human lung fibroblasts

Considering the biological reactivity of pure quartz in lung cells, there is a strong interest to clarify the cellular effects of respirable siliceous dusts, like bentonites. In the present study, we investigated the cellular uptake and the cytotoxic potential of bentonite particles (Ø< 10 μm) with an α-quartz content of up to 6% and different chemical modifications (activation: alkaline, acidic, organic) in human lung fibroblasts (IMR90). Additionally, the ability of the particles to induce apoptosis in IMR90-cells and the hemolytic activity was tested. All bentonite samples were tested for endotoxins with the in vitro-Pyrogen test and were found to be negative. Cellular uptake of particles by IMR90-cells was studied by transmission electron microscopy (TEM). Cytotoxicity was analyzed in IMR90-cells by determination of viable cells using flow cytometry and by measuring of the cell respiratory activity. Induced apoptotic cells were detected by AnnexinV/Propidiumiodide-staining and gel electrophoresis. Our results demonstrate that activated bentonite particles are better taken up by IMR90-cells than untreated (native) bentonite particles. Also, activated bentonite particles with a quartz content of 5–6% were more cytotoxic than untreated bentonites or bentonites with a quartz content lower than 4%. The bentonite samples induced necrotic as well as apoptotic cell death. In general, bentonites showed a high membrane-damaging potential shown as hemolytic activity in human erythrocytes. We conclude that cellular effects of bentonite particles in human lung cells are enhanced after chemical treatment of the particles. The cytotoxic potential of the different bentonites is primarily characterized by a strong lysis of the cell membrane.