Necrotic tumor cell death in vivo impairs tumor-specific immune responses

The manner in which cells die is believed to have a major impact on the nature of immune responses to their released Ags. In this study, we present the first direct analysis of tumor-specific immune responses to in vivo occurring tumor cell death through apoptosis or necrosis. Mice bearing thymidine kinase-transfected tumors were treated either with ganciclovir to induce tumor cell apoptosis in vivo or a vascular targeting agent, ZD6126, to induce tumor cell necrosis in vivo. In contrast to tumor apoptosis, induction of necrosis reduced the frequency and impaired the function of tumor-specific CD8(+) T cells. Adoptive transfer of lymphocytes from mice with apoptotic tumors into tumor-challenged mice resulted in a significant tumor protection, which was absent when splenocytes were transferred from mice with necrotic tumors. Anti-CD40 treatment reversed impaired Ag-specific CD8(+) T cell responses in these mice. These observations have not only fundamental importance for the development of immunotherapy protocols but also help to understand the underlying mechanism of in vivo immune responses to tumor cell death.     

Dual selection pressure by drugs and HLA class I-restricted immune responses on human immunodeficiency virus type 1 protease

To determine the influence of human immunodeficiency virus type 1 (HIV-1)-specific CD8+ T cells on the development of drug resistance mutations in the HIV-1 protease, we analyzed protease sequences from viruses from a human leukocyte antigen class I (HLA class I)-typed cohort of 94 HIV-1-positive individuals. In univariate statistical analyses (Fisher's exact test), minor and major drug resistance mutations as well as drug-associated polymorphisms showed associations with HLA class I alleles. All correlations with P values of 0.05 or less were considered to be relevant without corrections for multiple tests. A subset of these observed correlations was experimentally validated by enzyme-linked immunospot assays, allowing the definition of 10 new epitopes recognized by CD8+ T cells from patients with the appropriate HLA class I type. Several drug resistance-associated mutations in the protease acted as escape mutations; however, cells from many patients were still able to generate CD8+ T cells targeting the escape mutants. This result presumably indicates the usage of different T-cell receptors by CD8+ T cells targeting these epitopes in these patients. Our results support a fundamental role for HLA class I-restricted immune responses in shaping the sequence of the HIV-1 protease in vivo. This role may have important clinical implications both for the understanding of drug resistance pathways and for the design of therapeutic vaccines targeting drug-resistant HIV-1.     

Missing the target? A critical view on butterfly conservation efforts on calcareous grasslands in south-western Germany

Butterflies are strongly declining on grassland habitats of Central Europe. Therefore, the success of conservation measures on high quality grassland habitats is controversially discussed. We compared the changes in butterfly diversity and community structure on six managed calcareous grasslands with eight unmanaged vineyard fallows. We obtained strong losses of species diversity and remarkable shifts of community compositions on both habitat types. However, the changes on vineyard fallows were only slightly more severe but more stochastic than on the calcareous grasslands. The shifts in community composition with respect to functional species traits were rather similar between the two different grassland types so that complex butterfly communities evolved into generalist-dominated ones. Connectivity was higher among vineyard fallows than among calcareous grasslands. Consequently, conservation measures on calcareous grasslands only partly achieved their goal to maintain the high species diversity and functional complexity still observed in the 1970s. The negative impacts of eutrophication and monotonisation of the landscape as well as climate change are affecting all habitats, independently from management concepts. Therefore, management on conservation sites can buffer against these effects, but is not sufficient for a full compensation.     

Value of stool examination in patients with diarrhoea.

Findings of stool examinations in 1593 patients with diarrhoea due to a single enteric pathogen--enterotoxigenic Escherichia coli rotavirus, Shigella, Campylobacter jejuni, Vibrio cholerae 0:1, Entamoeba histolytica, or Giardia lamblia--were reviewed to determine how well they predicted the agent associated with the diarrhoea. Specimens were examined visually for blood and mucus, tested for pH, and examined under a microscope for the presence of red and white blood cells, parasites, and stool fat. Although visible blood was more common in specimens from patients infected with Shigella (51%) and Ent histolytica (39%) than in those from patients infected with other agents (6%; p less than 0.01), patients infected with Shigella were most likely to have numerous faecal leucocytes (greater than 50/high power field: 39% v 8% of all patients and 7% of patients infected with Ent histolytica, p less than 0.01 in both cases). Patients infected with enterotoxigenic E coli, rotavirus, V cholerae 0:1, or C jejuni had loose stools with fewer red or white cells. Patients infected with rotavirus and C jejuni were more likely to have acid stools with 3 to 4+ fat, but these findings were related to young age and breast feeding. Stool examination is most useful in establishing a diagnosis of dysentery and in helping to distinguish between patients infected with Shigella and Ent histolytica; it is of limited usefulness in discriminating between pathogens causing watery diarrhoea.     

Zum Einflu? einer Autobahn im Bau und w?hrend des Betriebs auf die Brutbiologie von Kohlmeisen (Parus major) und Blaumeisen (P. caeruleus)

Zusammenfassung Im Februar 1992 wurde an der in Bau befindlichen Autobahn BAB 66 zwischen Steinau und Schlüchtern (5021'N, 0931'E) ein Nistkastenkontrollgebiet mit 310 Nistkästen eingerichtet, um die Auswirkungen der Autobahn vor und nach Inbetriebnahme auf die Brutbiologie von Meisen zu untersuchen. Als Vergleichsgebiete dienen ein direkt benachbartes sowie ein ca. 5 km entferntes Untersuchungsgebiet. Zum Vergleich der Gewichtsentwicklung der Nestlinge wurde ein drittes autobahnfernes Gebiet herangezogen.In einzelnen Jahren finden sich zwischen den Gebieten Unterschiede in der Zusammensetzung der Brutpopulation, der Besetzungsrate der Nistkästen, dem Legebeginn, der Gelegegröße, der Schlüpfrate oder im Bruterfolg. Diese Unterschiede treten in verschiedenen Jahren und in unterschiedlichen Gebieten auf, so daß sich keine generelle Abweichung des Gebietes an der Autobahn von den straßenfernen Gebieten feststellen läßt. Die Eröffnung der Autobahn hatte keinen Effekt auf die untersuchten Parameter.Die Gewichte der Nestlinge im Gebiet an der Autobahn waren im Jahr 1996 nur am 12. und 13. Nestlingstag signifikant geringer als die Gewichte der Nestlinge in einem autobahnfernen gleichartig strukturierten Biotop. Die Ausflugsgewichte der Jungvögel an der A66 liegen jedoch ca. 2 g höher als in städtischen Biotopen.Störungen durch den Straßenverkehr, wie sie von anderen Autoren berichtet werden, ließen sich in unserem Untersuchungsgebiet nicht feststellen. In diesem Zusammenhang werden die geringe Störungsempfindlichkeit der Kohl- und Blaumeisen sowie der bisher geringe Verkehr auf der Autobahn diskutiert.

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The impact of a motorway in construction and after opening to traffic on the breeding biology of Great Tit (Parus major) and Blue Tit (P. caeruleus)

Summary In February 1992, a study area (A 66) with 310 nestboxes was installed along the construction line of a four laned motorway between Steinau and Schlüchtern (5021'N, 0931'E). One area in the immediate neighbourhood (VG1) and one 5 km distant (VG2) from the study area at the motorway (see Fig. 1) served as controls for comparison. In all three areas, the nestboxes were checked weekly during the breeding season from the beginning of May until July. Species composition of the birds using nestboxes, rate of occupation of nestboxes, and, for Great Tits (Parus major) and Blue Tits (P. caeruleus), date of the first egg, clutch size, hatching rate and number of birds fledged were recorded. The study included 5 breeding seasons from 1992 to 1996, 3 years during the period of construction and 2 years after the opening of the motorway to traffic in December 1994. In 1996, the nestling weight of 20 broods of Great Tits at the A66 was recorded daily and compared with that of all nestlings in another study area (VG3, see Fig. 1).The data were analyzed separately for each year. Distributions of frequencies were analyzed with the x² test, the other data with the Kruskal-Wallis-Test (H-Test). In case of significant differences between the study areas, the Mann Whitney U-test was used to determine which study area differed from the others. Nestlings bodymass was compared using the t-test.The species composition was similar in all three areas (Fig. 2), and it did not change after the opening of the motorway to traffic. The rate of occupation of the nestboxes, dates of laying of the first egg, clutch size, hatching rate and breeding success vary between years and between study areas (Table 1 to 5); data that differ significantly from those of two other areas are indicated by bold print. Such differences were observed in various years and involved all three areas. In study area A66, clutch size of Great Tits was higher in 1993; hatching success of Great Tits was lower in 1994 and that of Blue Tits was lower in 1993. However, there was no general trend that area A66 at the motorway was different from the other areas, either before or after being opened to the traffic. The weight of nestlings in study area A66 at the motorway was similar to that in comparable area nearby; only on day 12 and 13 after hatching was it slightly lower (Tab. 6). The last measurement on day 15 indicates a nestling weight of 16.2 g for the area at the motorway, which is in normal for deciduous forest habitats and approximately 2 g higher than that recorded in urban habitats.In brief, the motorway did not affect the species composition, rate of occupied nestboxes or the various breeding parameters; in particular, the moving traffic after opening did not seem to have any effect. The breeding parameters from the study area at the motorway were typical for the Schlüchtern region. Density reductions in breeding populations, as discussed by several authors, or disturbances caused by moving traffic, as previously reported, were not observed in our study. However, our key species, Great Tits and Blue Tits, are not generally very sensitive to interferences. Also, the level of traffic on the motorway was still rather low. This must be considered when our findings are generalized.

     

Transforming Growth Factor-β (TGF-β) Expression Is Increased in the Subsynovial Connective Tissue in a Rabbit Model of Carpal Tunnel Syndrome

Carpal tunnel syndrome (CTS) is an idiopathic disease that results from increased fibrosis of the subsynovial connective tissue (SSCT). A recent study found overexpression of both transforming growth factor-β (TGF-β) and connective tissue growth factor (CTGF) in the SSCT of CTS patients. This study investigated TGF-β and CTGF expression in a rabbit model of CTS, in which SSCT fibrosis is induced by a surgical injury. Levels of TGF-β1 and CTGF at 6, 12, 24 weeks after injury were determined by immunohistochemistry A significant increase in TGF-β1 and a concomitant significant increase in CTGF were found at 6 weeks, in addition to higher cell density compared to normal (all p<0.05), Interestingly, CTGF expression was reduced at 12 and 24 weeks, suggesting that an initial insult results in a time limited response. We conclude that this rabbit model mimics the fibrosis found in human CTS, and may be useful to study pathogenetic mechanisms of CTS in vivo.     

Colmation and Depth Filtration within Streambeds: Retention of Particles in Hyporheic Interstices

Colmation refers to the retention processes that can lead to the clogging of the top layer of channel sediments and decolmation refers to the resuspension of deposited fine particles. Internal colmation, clogging of the interstices directly below the armor layer, may form a thin seal that disconnects surface water from hyporheic water by inhibiting exchange processes. The settling of particles under low flow conditions can cause external colmation. Colmated channel sediments are characterized by reduced porosity and hydraulic conductivity as well as by a consolidated texture. The term ‘depth filtration’ refers to the transport and storage of fine matrix sediments in interstitial layers. Depth filtration is of significance for the transport of colloidal and fine particulate inorganic as well as organic matter within the hyporheic interstices and into the alluvial aquifer. The role of depth filtration is assessed for the content (given in mg per liter) of matrix fine particles retained in the coarse framework sediment of a gravel-bed river in Switzerland. Sediment samples were taken by freeze-coring with liquid nitrogen down to 70 cm depth and by piezometers down to 150 cm depth. Seventy-two percent of the mobile matrix fine particles were smaller than 0.1 mm and 50% were smaller than 0.03 mm. The content of fines tended to increase with depth, although higher accumulations were found at intermediate depths in sediments influenced by exfiltrating ground water. Interstitial detrital particles >90 μm showed vertical distribution patterns opposing those of total particles. These relationships revealed a differential significance of import, storage, and transport within three types of hydrological exchange zones (infiltration, horizontal advection, exfiltration) in the cross-section of the stream.     

Synthetic substrate analogues for UDP-GlcNAc: Manα1-6R β(1-2)-N-acetylglucosaminyltransferase II. Substrate specificity and inhibitors for the enzyme

UDP-GlcNAc: Man1-6R (1-2)-N-acetylglucosaminyltransferase II (GlcNAc-T II; EC 2.4.1.143) is a key enzyme in the synthesis of complexN-glycans. We have tested a series of synthetic analogues of the substrate Man1-6(GlcNAc1-2Man1-3)Man-O-octyl as substrates and inhibitors for rat liver GlcNAc-T II. The enzyme attachesN-acetylglucosamine in 1-2 linkage to the 2-OH of the Man1-6 residue. The 2-deoxy analogue is a competitive inhibitor (K _i=0.13mm). The 2-O-methyl compound does not bind to the enzyme presumably due to steric hindrance. The 3-, 4- and 6-OH groups are not essential for binding or catalysis since the 3-, 4- and 6-deoxy and -O-methyl derivatives are all good substrates. Increasing the size of the substituent at the 3-position to pentyl and substituted pentyl groups causes competitive inhibition (K _i=1.0–2.5mm). We have taken advantage of this effect to synthesize two potentially irreversible GlcNAc-T II inhibitors containing a photolabile 3-O-(4,4-azo)pentyl group and a 3-O-(5-iodoacetamido)pentyl group respectively. The data indicate that none of the hydroxyls of the Man1-6 residue are essential for binding although the 2- and 3-OH face the catalytic site of the enzyme. The 4-OH group of the Man-O-octyl residue is not essential for binding or catalysis since the 4-deoxy derivative is a good substrate; the 4-O-methyl derivative does not bind. This contrasts with GlcNAc-T I which cannot bind to the 4-deoxy-Man- substrate analogue. The data are compatible with our previous observations that a bisectingN-acetylglucosamine at the 4-OH position prevents both GlcNAc-T I and GlcNAc-T II catalysis. However, in the case of GlcNAc-T II, the bisectingN-acetylglucosamine prevents binding due to steric hindrance rather than to removal of an essential OH group. The 3-OH of the Man1-3 is an essential group for GlcNAc-T II since the 3-deoxy derivative does not bind to the enzyme. The trisaccharide GlcNAc1-2Man1-3Man-O-octyl is a good inhibitor (K _i=0.9mm). The above data together with previous studies indicate that binding of the GlcNAc1-2Man1-3Man- arm of the branched substrate to the enzyme is essential for catalysis. Abbreviations: GlcNAc-T I, UDP-GlcNAc:Man1-3R (1-2)-N-acetylglucosaminyltransferase I (EC 2.4.1.101); GlcNAc-T II, UDP-GlcNAc:Man1-6R (1-2)-N-acetylglucosaminyltransferase II (EC 2.4.1.143); MES, 2-(N-morpholino)ethane sulfonic acid monohydrate.     

Absence of MMACHC in peripheral retinal cells does not lead to an ocular phenotype in mice

Combined methylmalonic aciduria with homocystinuria (cblC type) is a rare disease caused by mutations in the MMACHC gene. MMACHC encodes an enzyme crucial for intracellular vitamin B₁₂ metabolism, leading to the accumulation of toxic metabolites e.g. methylmalonic acid (MMA) and homocysteine (Hcy), and secondary disturbances in folate and one-carbon metabolism when not fully functional. Patients with cblC deficiency often present in the neonatal or early childhood period with a severe multisystem pathology, which comprises a broad spectrum of treatment-resistant ophthalmological phenotypes, including retinal degeneration, impaired vision, and vascular changes. To examine the potential function of MMACHC in the retina and how its loss may impact disease, we performed gene expression studies in human and mouse, which showed that local expression of MMACHC in the retina and retinal pigment epithelium is relatively stable over time. To study whether functional MMACHC is required for retinal function and tissue integrity, we generated a transgenic mouse lacking Mmachc expression in cells of the peripheral retina. Characterization of this mouse revealed accumulation of cblC disease related metabolites, including MMA and the folate-dependent purine synthesis intermediates AICA-riboside and SAICA-riboside in the retina. Nevertheless, fundus appearance, morphology, vasculature, and cellular composition of the retina, as well as ocular function, remained normal in mice up to 6 or 12 months of age. Our data indicates that peripheral retinal neurons do not require intrinsic expression of Mmachc for survival and function and questions whether a local MMACHC deficiency is responsible for the retinal phenotypes in patients.