[Chimeric antigen receptors (CARs): universal tools in adoptive cell therapy]. / Chimäre Antigenrezeptoren (CAR) ­ universelle Werkzeuge in der zellulären Immuntherapie.

BACKGROUND: The observation that tumor-infiltrating lymphocytes (TIL) after ex vivo amplification can control tumors in the long term led to the concept of redirecting patients' cytolytic T­cells by a receptor with defined specificity against the tumor. OBJECTIVES: Development of a recombinant receptor-signal molecule (chimeric antigen receptor, CAR) to increase selectivity and enhance anti-tumor immunity. METHODS: Description of a prototype CAR, overview of the modular composition and further development of CAR technology for use in adoptive immune cell therapy. RESULTS: Intensive research over the last two decades has shown how CAR-mediated T­cell activation is influenced by factors such as binding affinity, the epitope of the target antigen, its expression density and accessibility on the tumor cells, as well as by the signaling domains and their combination to induce T­cell activation. The quality and duration of the T­cell response can be specifically modulated by modifying the modular composition of the CAR; CAR T­cells can act as "biopharmaceutical factories" (T-cells redirected for unrestricted cytokine-mediated killing, TRUCK) in the tissue by CAR-mediated release of transgenic therapeutic proteins. CONCLUSION: Adoptive CAR T­cell therapy has shown clinical efficacy in the treatment of hematological malignancies; the treatment of solid tumors, however, is more challenging. Allogeneic CAR T­cell technology is aimed at generating "off-the-shelf" CAR T­cells that are accessible for a large number of patients. A further promising approach is the use of CAR T­cells for other therapeutic applications such as the treatment of autoimmune diseases.

A new microscopic method to analyse desiccation-induced volume changes in aeroterrestrial green algae.

Aeroterrestrial green algae are exposed to desiccation in their natural habitat, but their actual volume changes have not been investigated. Here, we measure the relative volume reduction (RVRED ) in Klebsormidium crenulatum and Zygnema sp. under different preset relative air humidities (RH). A new chamber allows monitoring RH during light microscopic observation of the desiccation process. The RHs were set in the range of ∼4 % to ∼95% in 10 steps. RVRED caused by the desiccation process was determined after full acclimation to the respective RHs. In K. crenulatum, RVRED (mean ± SE) was 46.4 ± 1.9%, in Zygnema sp. RVRED was only 34.3 ± 2.4% at the highest RH (∼95%) tested. This indicates a more pronounced water loss at higher RHs in K. crenulatum versus Zygnema sp. By contrast, at the lowest RH (∼4%) tested, RVRED ranged from 75.9 ± 2.7% in K. crenulatum to 83.9 ± 2.2% in Zygnema sp. The final volume reduction is therefore more drastic in Zygnema sp. These data contribute to our understanding of the desiccation process in streptophytic green algae, which are considered the closest ancestors of land plants.

Evolution of public attitudes about mental illness: a systematic review and meta-analysis.

OBJECTIVE: To explore whether the increase in knowledge about the biological correlates of mental disorders over the last decades has translated into improved public understanding of mental illness, increased readiness to seek mental health care and more tolerant attitudes towards mentally ill persons. METHOD: A systematic review of all studies on mental illness-related beliefs and attitudes in the general population published before 31 March 2011, examining the time trends of attitudes with a follow-up interval of at least 2 years and using national representative population samples. A subsample of methodologically homogeneous studies was further included in a meta-regression analysis of time trends. RESULTS: Thirty-three reports on 16 studies on national time trends met our inclusion criteria, six of which were eligible for a meta-regression analysis. Two major trends emerged: there was a coherent trend to greater mental health literacy, in particular towards a biological model of mental illness, and greater acceptance of professional help for mental health problems. In contrast, however, no changes or even changes to the worse were observed regarding the attitudes towards people with mental illness. CONCLUSION: Increasing public understanding of the biological correlates of mental illness seems not to result in better social acceptance of persons with mental illness.

The loss of sadness: the public's view.

OBJECTIVE: With the exception of bereavement, the diagnosis of major depressive disorder in the DSM-IV does not take into account the context in which the symptoms occur. Recent criticism has maintained that common sense suggests making a distinction between depression as mental disorder and sorrow as 'normal' reaction to social stress. This study sets out to investigate whether the public does in fact make this distinction. METHOD: In spring 2009, a population-based survey was conducted by phone in the city of Vienna (n = 1205). A fully structured interview was carried out which began with the presentation of a vignette describing a diagnostically unlabeled case of depression, with or without provision of information about preceding stressful life events. RESULTS: Respondents presented with vignettes containing information on loss events were less likely to define depressive symptoms as indication of mental illness. They were also not as willing to recommend professional help. And if they were, they tended to less frequently recommend seeking help from someone with a medical background. CONCLUSION: In contrast to the conceptualization in the DSM-IV, the public tends to perceive depressive symptoms differently depending on the context in which they occur.

Congenital Central Hypoventilation Syndrome due to PHOX2b gene defects: inheritance from asymptomatic parents.

BACKGROUND: Congenital Central Hypoventilation Syndrome (CCHS, Ondine's curse) is a rare syndrome of dysfunction of the autonomic nervous system characterized by a decreased response to hypercarbia requiring mechanical ventilation in most cases. CCHS is an autosomal-dominant disease associated with tumors of neural crest origin, segmental aganglionosis of the colon, and diffuse autonomic dysregulation symptoms. Most cases of CCHS are caused by de novo heterozygous in-frame expansions within in the PHOX2b gene. PATIENTS AND MAIN RESULTS: Here we report two families in which a PHOX2b defect was inherited from an asymptomatic parent. In family 1 an asymptomatic mother carried a mild mutation (15 bp expansion within the polyalanine repeat) also found in her daughter who was symptomatic immediately after birth but did not require mechanical ventilation. In family 2, two newborn infants with respiratory failure due to insufficient respiratory drive requiring mechanical ventilation were born to asymptomatic parents. A 39 pb expansion within the PHOX2b polyalanine repeat was found in one patient in whom DNA was available, but not in blood leukocytes from any parent. Microsatellite analyses confirmed the identity of the parents, such that a germline mosaicism has to be deduced. CONCLUSIONS: Carriers of mild PHOX2b mutations causing disease in their offspring may be asymptomatic; Modifier genes determining the clinical course may exist. Germline mosaicism may lead to CCHS in children from unaffected parents. Genetic counseling should include these variations.

Mental health literacy and attitude towards people with mental illness: a trend analysis based on population surveys in the eastern part of Germany.

BACKGROUND: There is growing evidence that mental health literacy has improved in western countries in recent years. The question arises as to whether this trend is paralleled by an improvement of attitudes towards people with mental illness. AIM: To examine the development of mental health literacy and the desire for social distance towards people with schizophrenia and major depressive disorder in Eastern Germany over a time period of eight years. METHOD: A trend analysis was carried out using data from two population surveys conducted in the eastern part of Germany in 1993 and 2001. By means of a fully structured interview psychiatric labelling, causal beliefs, help-seeking and treatment recommendations as well as the desire for social distance was assessed. RESULTS: While there was an increase in the mental health literacy of the public, the desire for social distance from people with major depression and schizophrenia remained unchanged or even increased. CONCLUSIONS: The assumption underlying a number of anti-stigma campaigns, namely that educating people about mental disorders may automatically lead to the improvement of their attitudes towards the mentally ill, appears questionable.

Investigating cytoskeletal function in chloroplast protrusion formation in the arctic-alpine plant Oxyria digyna.

Arctic and alpine plants like Oxyria digyna have to face enhanced environmental stress. This study compared leaves from Oxyria digyna collected in the Arctic at Svalbard (78 degrees N) and in the Austrian Alps (47 degrees N) at cellular, subcellular, and ultrastructural levels. Oxyria digyna plants collected in Svalbard had significantly thicker leaves than the samples collected in the Austrian Alps. This difference was generated by increased thickness of the palisade and spongy mesophyll layers in the arctic plants, while epidermal cells had no significant size differences between the two habitats. A characteristic feature of arctic, alpine, and cultivated samples was the occurrence of broad stroma-filled chloroplast protrusions, 2 - 5 microm broad and up to 5 microm long. Chloroplast protrusions were in close spatial contact with other organelles including mitochondria and microbodies. Mitochondria were also present in invaginations of the chloroplasts. A dense network of cortical microtubules found in the mesophyll cells suggested a potential role for microtubules in the formation and function of chloroplast protrusions. No direct interactions between microtubules and chloroplasts, however, were observed and disruption of the microtubule arrays with the anti-microtubule agent oryzalin at 5 - 10 microM did not alter the appearance or dynamics of chloroplast protrusions. These observations suggest that, in contrast to studies on stromule formation in Nicotiana, microtubules are not involved in the formation and morphology of chloroplast protrusions in Oxyria digyna. The actin microfilament-disrupting drug latrunculin B (5 - 10 microM for 2 h) arrested cytoplasmic streaming and altered the cytoplasmic integrity of mesophyll cells. However, at the ultrastructural level, stroma-containing, thylakoid-free areas were still visible, mostly at the concave sides of the chloroplasts. As chloroplast protrusions were frequently found to be mitochondria-associated in Oxyria digyna, a role in metabolite exchange is possible, which may contribute to an adaptation to alpine and arctic conditions.

Photosynthetic efficiency, desiccation tolerance and ultrastructure in two phylogenetically distinct strains of alpine Zygnema sp. (Zygnematophyceae, Streptophyta): role of pre-akinete formation.

Two newly isolated strains of green algae from alpine regions were compared physiologically at different culture ages (1, 6, 9 and 15 months). The strains of Zygnema sp. were from different altitudes ('Saalach' (S), 440 m above sea level (a.s.l.), SAG 2419 and 'Elmau-Alm' (E-A), 1,500 m a.s.l., SAG 2418). Phylogenetic analysis of rbcL sequences grouped the strains into different major subclades of the genus. The mean diameters of the cells were 23.2 µm (Zygnema S) and 18.7 µm (Zygnema E-A) but were reduced significantly with culture age. The photophysiological response between the strains differed significantly; Zygnema S had a maximal relative electron transport rate (rETR max) of 103.4 µmol electrons m(-2) s(-1), Zygnema E-A only 61.7 µmol electrons m(-2) s(-1), and decreased significantly with culture age. Both strains showed a low-light adaption and the absence of strong photoinhibition up to 2,000 µmol photons m(-2) s(-1). Photosynthetic oxygen production showed similar results (P max Zygnema S, 527.2 µmol O2 h(-1) mg(-1) chlorophyll (chl.) a, Zygnema E-A, 390.7 µmol O2 h(-1) mg(-1) chl. a); the temperature optimum was at 35 °C for Zygnema S and 30 °C for Zygnema E-A. Increasing culture age moreover leads to the formation of pre-akinetes, which accumulate storage products as revealed by light and transmission electron microscopy. Desiccation at 84 % relative air humidity (RH) lead to a reduction of the effective quantum yield of photosystem II (PSII) (ΔFv/Fm') to zero between 90 to 120 min (Zygnema S) and between 30 to 60 min (Zygnema E-A), depending on the culture age. A partial recovery of ΔFv/Fm' was only observed in older cultures. We conclude that pre-akinetes are crucial for the aeroterrestrial lifestyle of Zygnema.

Free calcium in Micrasterias: local gradients are not detected in growing lobes.

Intracellular free Ca2+ ([Ca2+]) has been measured in growing unicells of two species of the green alga, Micrasterias, which have been injected with the indicator dye fura-2-dextran. Ratiometric imaging of Micrasterias denticulata yields levels of 170 to 200 nM [Ca2+] but fails to reveal a significant [Ca2+] gradient associated with the tips of growing lobes, or in any other region of the cell. In Micrasterias muricata slight elevations from a basal value of 350 to 500 nM have been observed, but these might be due to a general inward leakage of Ca2+ at the plasma membrane which is enhanced at the narrow lobes of this cell because of their greater relative surface to volume ratio. Experimental perturbation of the intracellular [Ca2+] with injection of the ion or the addition of the non-fluorescent ionophore, Br-A23187, reveal that [Ca2+] elevations can be generated and indicate that if they naturally occurred, the image system would have detected them. Further evidence that [Ca2+] gradients are lacking derives from studies with BAPTA-type buffers. Injection of 5,5'-dibromo BAPTA and 4,4'-difluoro BAPTA, which in several other systems are the most effective at dissipating intracellular [Ca2+] gradients, have no effect on development of Micrasterias. Taken together, these studies indicate that lobe outgrowth in Micrasterias does not occur in association with marked localized [Ca2+] gradients.

Characterization and functional analysis of the nucleotide binding fold in human peroxisomal ATP binding cassette transporters.

The 70-kDa peroxisomal membrane protein (PMP70) and the adrenoleukodystrophy protein (ALDP) are half ATP binding cassette (ABC) transporters in the peroxisome membrane. Mutations in the ALD gene encoding ALDP result in the X-linked neurodegenerative disorder adrenoleukodystrophy. Plausible models exist to show a role for ATP hydrolysis in peroxisomal ABC transporter functions. Here, we describe the first measurements of the rate of ATP binding and hydrolysis by purified nucleotide binding fold (NBF) fusion proteins of PMP70 and ALDP. Both proteins act as an ATP specific binding subunit releasing ADP after ATP hydrolysis; they did not exhibit GTPase activity. Mutations in conserved residues of the nucleotidases (PMP70: G478R, S572I; ALDP: G512S, S606L) altered ATPase activity. Furthermore, our results indicate that these mutations do not influence homodimerization or heterodimerization of ALDP or PMP70. The study provides evidence that peroxisomal ABC transporters utilize ATP to become a functional transporter.

Genomic organization and chromosomal localization of the human peroxisomal membrane protein-1-like protein (PXMP1-L) gene encoding a peroxisomal ABC transporter.

The cDNA of the peroxisomal membrane protein-1-like protein (PXMP1-L, synonyms: PMP69, P70R), a novel peroxisomal ATP binding cassette transporter of yet unknown function, has recently been cloned. The best known peroxisomal member of this protein family is the adrenoleukodystrophy protein, defects of which are the underlying cause of X-linked adrenoleukodystrophy (X-ALD). Here we describe the complete exon-intron structure (19 exons and 18 introns covering 16.0 kb) of the human PXMP1-L gene, transcript variants, the localization on chromosome 14q24 by cytogenetic analysis and sequencing of the putative promoter region. PXMP1-L has been proposed to play a role as a modifier in determining the phenotypic variations observed in X-ALD. The data presented will enable sequence analysis of the PXMP1-L gene in X-ALD patients and facilitate the analysis of PXMP1-L function.

Cloning and characterization of the gene encoding the human peroxisomal assembly protein Pex3p.

Proteins essential for the assembly of functional peroxisomes are designated peroxins and are encoded by PEX genes. In yeast, Pex3p was previously identified as a peroxisomal integral membrane protein indispensable for peroxisome biogenesis and integrity. Here we report the cloning of the orthologous human PEX3 gene. It encodes a polypeptide of 373 amino acids (42 kDa) and is expressed in all tissues examined. As shown by transfection of epitope tagged constructs and immunofluorescence analysis, human Pex3p is localized at the peroxisome. The N-terminal 40 amino acids were revealed to be sufficient to target a GFP reporter protein to the peroxisome. A positively charged five amino acid sequence within this N-terminal region is highly conserved from yeast to human Pex3p. Overexpression of human Pex3p leads to proliferation of ER membranes in COS7 cells. Since disruption of human peroxins has been shown to result in peroxisomal biogenesis disorders, PEX3 is another candidate gene being involved in this disease group.

The mouse gene encoding the peroxisomal membrane protein 1-like protein (PXMP1-L): cDNA cloning, genomic organization and comparative expression studies.

PXMPI-L (synonyms: PMP69, P70R) is a peroxisomal protein that belongs to the ABC-transporter superfamily. Its closest homolog is the peroxisomal membrane protein 1 (PMP70). We have cloned the mouse PXMP1-L gene. It encodes a 606 amino acid protein. In contrast to the human and the rat, mouse PXMP1-L is predominantly expressed in the liver. The mouse PXMP1-L gene consists of 19 exons and spans 21 kb of genomic sequence. No obvious peroxisome proliferator response element has been found in 1.1 kb of the putative promoter region. No coordination of constitutive or fenofibrate-induced expression of PXMP1-L with other peroxisomal ABC transporters was observed so that an obligate exclusive heterodimer formation is not likely to occur. The data presented will be particularly useful for the generation of a mouse model defective in PXMP1-L in order to elucidate the yet unknown function of this protein.

Association and linkage studies of CRH and PENK genes in bipolar disorder: a collaborative IGSLI study.

Corticotropin-releasing hormone (CRH) and proenkephalin (PENK) are hypothalamic peptides involved in the stress response and hypothalamic-pituitary axis regulation. Previous research has implicated these peptides in the pathogenesis of affective disorders. In this study we investigated two polymorphisms located in the genes that code for CRH and PENK by means of association and linkage analyses. A total of 138 bipolar patients and 108 controls were included in the association study. In addition, 24 families were available for linkage analysis, including six families of probands with documented periodic positivity of dexamethasone suppression tests (DST) during remission. We found no association of bipolar disorder with either gene. Similarly, we did not find any evidence of linkage (P = 0.56 for CRH and 0.52 for PENK) in the entire sample or in the subsample of families of DST positive probands. In conclusion, our study does not support the hypothesis that genes coding for CRH or PENK contribute to the genetic susceptibility to bipolar disorder. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 96:178-181, 2000.

MAOA: association and linkage studies with lithium responsive bipolar disorder.

A number of association studies have investigated the role of the monoamine oxidase A (MAOA) gene in the susceptibility to bipolar disorder. Although some studies have reported positive findings, there remains some controversy, because results from different studies have not been consistent. A common explanation for inconsistencies between studies is genetic heterogeneity. We have focused on lithium responsive bipolar disorder as a way to reduce heterogeneity. In this study, we investigated the role of MAOA in lithium responsive bipolar patients using association and linkage study designs. The investigation used 138 patients and 108 normal controls. In addition, 25 families were also studied. Our results were not supportive of a major role of MAOA in the predisposition to bipolar disorder.

Polyglutamine coding genes in bipolar disorder: lack of association with selected candidate loci.

BACKGROUND: Several studies have suggested that expanded trinucleotide repeats, particularly CAG, may have a role in the etiology of BD. Results obtained with the repeat expansion detection technique (RED) have indicated that bipolar patients have an excess of expanded CAG repeats. However, it is not clear which loci account for this difference. METHODS: Using lithium-responsive bipolar patients in order to reduce heterogeneity, we investigated five loci that are expressed in the brain and contain translated CAG repeats. A sample of 138 cases and 108 controls was studied. Genotypes were coded quantitatively or qualitatively and repeat distributions were compared. RESULTS: No difference was found in allele distribution between cases and controls for any of the loci studied. In one locus - L10378 - patients had a tendency to present shorter alleles (28.1 versus 27.9 repeats; t=2.55, df=205, P=0.011), however, this difference disappeared after correction for multiple testing. LIMITATIONS: The study has limitations common to most candidate gene association studies, that is, limited number of loci investigated and limited power to detect loci that account for a small proportion of the total genetic variability. CONCLUSIONS: Our results suggest that the loci investigated have no major role in the genetic predisposition to bipolar disorder.

The effect of ultraviolet radiation on ultrastructure and photosynthesis in the red macroalgae Palmaria palmata and Odonthalia dentata from Arctic waters.

In radiation exposure experiments, the effects of mild artificial UV conditions (4.7 W m(-2) UV-A and 0.20 W m(-2) UV-B) plus PAR (25 - 30 micromol photons m(-2) s(-1)) on photosynthesis and ultrastructure of two red algal species from the Arctic have been investigated. While Palmaria palmata was collected from the upper sublittoral of the Kongsfjord (Spitsbergen, Norway), Odonthalia dentata represents a typical deepwater species at this high latitude. After 6 h and 24 h exposure to UV, chlorophyll fluorescence of photosystem II (PS II efficiency, F(v)/F(m)) was determined as an indicator for photosynthetic performance, and the relative electron transport rates in response to increasing photon fluence rates were recorded. In parallel, tissue samples were prepared for the transmission electron microscope (TEM). The presented data clearly demonstrate a significant influence of experimental UV on photosynthetic performance. Photochemical efficiency of PS II of both red algal species decreased to about one third of the initial value under UV. While the PI (photosynthesis-irradiance) curve parameter alpha (positive slope at limiting photon fluence rates) strongly decreased in both plants, the I(k) values (initial value of light-saturated photosynthetic rate) increased 3 - 5-fold. Palmaria palmata does not appear to become photoinhibited under these conditions, but O. dentata showed strong photoinhibition. The TEM results demonstrated that the photosynthetic apparatus was severely influenced by UV in both species, because thylakoid membranes appeared wrinkled, lumen dilatations occurred, and the outer membranes were altered. Moreover, mitochondria were damaged, and numerous plasma vesicles were observed. In conclusion, both red algal species are negatively affected by UV on the physiological and ultrastructural level. However, the differences in photoinhibitory responses correlate well with the vertical depth zonation of P. palmata and O. dentata in the Arctic Kongsfjord.

Monoclonal antibody to thyroid transcription factor-1: production, characterization, and usefulness in tumor diagnosis.

Thyroid transcription factor-1 (TTF-1), a member of the NKx2 family of homeodomain transcription factors, is expressed in epithelial cells of the thyroid gland and the lung. To produce monoclonal antibodies specific for TTF-1, the polypeptide was expressed in E. coli and purified utilizing affinity chromatography of a polyhistidine-tagged TTF-1 fusion protein. Splenocytes from BALB/c mice immunized with recombinant TTF-1 were fused with P3x/63Ag8.653 myeloma cells to produce hybridomas. Tissue culture supernatant was screened for anti TTF-1 activity by ELISA employing recombinant TTF-1 as antigen. Hybridomas producing high-affinity antibodies were subcloned by limiting dilution. Antibodies from tissue culture fluid from an IgG1 clone (8G7G3/1) that stained the nuclei of paraffin-embedded human thyroid tissues were precipitation-purified and further characterized. The antibody stained a single 40-kDa polypeptide in immunoblots of nuclear extracts or lysates of cell lines known to express TTF-1 mRNA. MAb 8G7G3/1 also stained nuclei of tissue in a highly specific manner consistent with the pattern of expression obtained with an established polyclonal TTF-1 antibody and by in situ hybridization. MAb 8G7G3/1 was used for TTF-1 immunohistochemistry of human adenocarcinomas of the lung, colon, and breast as well as small cell carcinomas of the lung. TTF-1 was detected in primary lung adenocarcinomas and small cell carcinomas and was absent in colon and breast carcinomas. These findings demonstrate that anti-TTF-1 MAb 8G7G3/1 specifically binds TTF-1 in cell extracts and tissues and can be used to distinguish between lung and nonlung origin of a tumor.

Gender and attitudes towards people with schizophrenia. Results of a representative survey in the Federal Republic of Germany.

Based on the observation that the course of schizophrenia appears to be more unfavourable in men than in women, we examined whether male suffers are exposed to more negative and less positive emotional reactions and are met with a greater amount of rejection by their environment than their female counterparts. Data from a representative survey conducted in the 'old' Federal Republic of Germany during 1990 did not yield the expected gender difference with regard to emotional reactions. There were, however, some gender differences on the side of the respondents: Women expressed more feelings of anxiety and tended to show more prosocial reactions. Social distance tended to be slightly more pronounced towards men than towards women. This applied to both schizophrenia and alcoholism. The gender of the respondents, on the other hand, was of no importance for social distance.

Depression and quality of life: results of a follow-up study.

BACKGROUND: Although there is sufficient evidence that the quality of life of people suffering from depression is reduced hardly anything is known about their quality of life after the remission of a depressive episode. AIMS: We therefore set out to study the quality of life of patients with depression (ICD-10 F32, F33) one, four and seven months after discharge from hospital. For comparison, a random sample of the general population was studied in addition. METHOD: Quality of life was assessed by means of the WHOQOL-100, a self-administered questionnaire developed by WHO. RESULTS: Although, shortly after discharge, quality of life of patients whose depression remitted was better than that of patients with persisting depression it was still slightly worse than that of the general population. During the subsequent six months, there was no further improvement of quality of life, i.e. even at the end of the follow-up period there was a slight lack of quality of life, especially as concerns the level of independence, spirituality/religion/personal beliefs and physical health. CONCLUSIONS: Thus, what already had been reported based on the objective assessment of quality of life, namely that depression implies a persisting impairment of social functioning and living conditions, can be replicated to some extent from the point of view of the patients themselves.