Sarcoma of follicular dendritic cells in the dorsal mediastinum

Follicular dendritic cell sarcomas (FDCSs) are very rare and usually originate in lymph nodes. We report an exceedingly rare case with localization in the dorsal mediastinum and, for the first time, provide positron emission tomography (PET) data for this tumor. This report describes the case of a 76-year-old man with a clinically aggressive tumor in the dorsal mediastinum. Computed tomography scan revealed displacement of soft tissue and lymph nodes. PET showed that the tumor had a high proliferation rate. Investigation of the successfully removed tumor mass revealed reactivity of the tumor cells for follicular dendritic cell markers and desmosomes linking adjacent tumor cells at the ultrastructural level. Marked atypia, a high mitotic rate, and areas of coagulative necrosis were found. The tumor in our case revealed the typical features and thus was classified as FDCS. In contrast to previous reports in the literature, preoperative imaging, histology, and immunohistochemistry studies indicated at least an intermediate degree of malignancy. Nevertheless, the patient made a good postoperative recovery and remained apparently disease-free 2 years later.     

T Cells Recognize Multiple GAD65 and Proinsulin Epitopes in Human Type 1 Diabetes, Suggesting Determinant Spreading

Human type 1 diabetes is thought to be mediated by autoreactive T cells specific for antigens expressed by pancreatic beta cells. However, it is unclear which autoantigens and determinants thereof are the targets of the autoimmune attack. Using comprehensive peptide libraries that cover the entire sequence of two major candidate autoantigens, GAD65 and proinsulin, we measured the in vivo frequencies of peptide-specific, IFN-gamma-producing memory T cells in 27 diabetic patients, 14 high risk individuals, and 15 partially HLA-matched healthy controls. Compared to the controls, both a higher number of determinants on the islet cell antigens were recognized and the frequencies of peptide specific cells were increased in patients and high risk individuals. Inclusion of signal enhancing anti-CD28 antibody further accentuated this difference. Considerable heterogeneity in peptide recognition was seen even in DRB1*04, DQB1*0302 matched individuals. Unlike its peptides, the GAD protein antigen did not recall a T cell memory response. The highly heterogeneous recognition of a multitude of peptide determinants on both autoantigens, occurring in the absence of protein recognition, and the low functional avidity of the memory cells involved jointly suggest that the autoimmune T cell repertoire in human type 1 diabetes primarily targets cryptic determinants engaged by determinant spreading.     

CD4+ and CD8+ cells in cryopreserved human PBMC maintain full functionality in cytokine ELISPOT assays

The frequency and the cytokine signature of antigen-specific T cells in the blood reflect the magnitude and the quality of T cell immunity in vivo. Recently, cytokine enzyme-linked immunospot (ELISPOT) assays performed on freshly isolated peripheral blood mononuclear cells (PBMC) emerged as a promising tool for monitoring these key parameters, providing direct feedback information on the efficacy of vaccinations and immune therapies. However, performing ELISPOT assays with freshly isolated cells is not readily feasible in the context of clinical trials. The ability to obtain valid ELISPOT data on cryopreserved samples would greatly enhance ex vivo immune monitoring capabilities. We have therefore systematically studied antigen-specific T cell responses in freshly isolated PBMC and after cryopreservation. Four healthy donors were selected that displayed T cell responses to six recall antigens. The antigen reactive T cells were defined as CD4 or CD8 cells, and their cytokine effector class was established measuring interferon (IFN)-gamma, interleukin (IL)-2, IL-4 and IL-5. The donors were bled at three different time points, and their PBMC were tested fresh and after freeze-thawing. The results showed that the frequencies and type 1/type 2 cytokine signatures of recall antigen-specific CD4 and CD8 cells are unaffected after cryopreservation. In contrast to these data obtained on human PBMC, cryopreservation of murine spleen cells causes a decrease in cytokine secretion.     

Sequential gene deletions in Hypocrea jecorina using a single blaster cassette

In Hypocrea jecorina (anamorph: Trichoderma reesei) multiple gene deletions are limited by the number of readily available selection markers. We have therefore constructed a blaster cassette which enables successive gene knock-outs in H. jecorina. This 3.5 kb pyr4 blaster cassette contains the H. jecorina pyr4 marker gene encoding orotidine-5′-monophosphate (OMP) decarboxylase flanked by two direct repeats of the Streptoalloteichus hindustanus bleomycin gene (Sh ble), which facilitate the excision of the blaster cassette by homologous recombination after each round of deletion. Functionality of this pyr4 blaster cassette was demonstrated by deletion of the glk1 encoding glucokinase and hxk1 encoding hexokinase. 1.4–1.8 kb of the non-coding flanking regions of both target genes were cloned into the respective blaster cassettes and transformation of a pyr4 negative H. jecorina strain with the two cassettes resulted in 10–13% of the transformants in the deletion of one of the two kinase genes. For excision of the pyr4 blaster cassettes, Δglk1 strains were selected for growth in the presence of 5-fluoroorotic acid. Recombination between the two Sh ble elements resulted in uridine auxotrophic strains which retained their respective glucokinase negative phenotype. Subsequent transformation of one of these auxotrophic Δglk1 strains with the hexokinase blaster cassette resulted in pyr4 prototrophic strains deleted in both glk1 and hxk1. Δglk1 strains showed reduced growth on d-glucose and d-fructose whereas Δhxkl strains showed reduced compact growth on d-glucose but were unable to grow on d-fructose as carbon source. The double Δglk1Δhxk1 deletion strain was completely unable to grow on either d-glucose or d-fructose. Nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession numbers DQ068384 (H. jecorina glk1) and DQ068385 (H. jecorina hxk1).     

Three novel human VMD2-like genes are members of the evolutionary highly conserved RFP-TM family

The RFP-TM protein family was first described in Caenorhabditis elegans as hypothetical transmembrane proteins containing a conserved 350-400 amino acid domain including the invariant peptide motif RFP. The VMD2 gene underlying Best disease was shown to represent the first human member of the RFP-TM protein family. More than 97% of the disease-causing mutations are located in the N-terminal RFP-TM domain implying important functional properties. Here, we have identified three novel VMD2-related human genes (VMD2L1, VMD2L2 and VMD2L3) demonstrating a high degree of conservation in their respective RFP-TM domains. Each of the VMD2-like proteins has a unique C-terminus that lack similarity to other proteins or motifs. By FISH analysis, VMD2L1 was localised to chromosome 19p13.2-p13.12, VMD2L2 to 1p32.3-p33 and VMD2L3 to 12q14.2-q15. RT-PCR analyses revealed tissue-restricted expression of the three genes with both VMD2L1 and VMD2L2 abundantly transcribed in colon. VMD2L1 is present in the retinal pigment epithelium while VMD2L3 shows predominant expression in skeletal muscle.     

Generalized AA-amyloidosis in a 58-year-old Caucasian woman with an 18-month history of gastrointestinal tuberculosis

 We report on a 58-year-old Caucasian woman who went to a general practitioner about recurrent abdominal pain, night sweats and weight loss of a few weeks’ duration. Once gynaecological disease had been ruled out, the patient was admitted to hospital with severe abdominal pain and intestinal obstruction and a right-sided hemicolectomy was performed. Following the investigation of osteolytic lumbar vertebrae, 18 months after visiting the general practitioner the patient was finally found to be suffering from generalized AA-amyloidosis secondary to gastrointestinal tuberculosis. This had been misinterpreted as Crohn’s disease. Re-examination of the specimens from the right-sided hemicolectomy demonstrated that scanty deposits of AA-amyloid were present 9 months after the first presentation. AA-amyloid can thus be present in serious inflammatory disease even during the first 9 months after the initial clinical presentation. Received: 23 June 1998 / Accepted: 19 August 1998     

Detection of a trigger zone of bradykinin-induced fast calcium waves in PC12 neurites

 Bradykinin and caffeine were used as two different agonists to study inositol 1,4,5-trisphosphate (IP₃)-sensitive and caffeine/ryanodine-sensitive intracellular Ca²⁺ release in the outgrowing neurites of nerve-growth-factor (NGF)-treated rat phaeochromocytoma cells (PC12). Changes in neuritic intracellular free Ca²⁺ ([Ca²⁺]_i) in single cells were measured after loading with a 1:1 mixture of the acetoxymethyl (AM) ester of the Ca²⁺-sensitive dyes Fura-red and Fluo-3, in combination with confocal microscopy. Bradykinin-induced Ca²⁺ release was blocked by U73211, a specific phospholipase C inhibitor. Caffeine-induced Ca²⁺ release was very low in neurites at rest. It increased after the cells were preloaded with Ca²⁺. The Ca²⁺ signal evoked at high concentrations of bradykinin (>500 nM) arose from a trigger zone in the proximal part of the neurite, as a bi-directional wave towards the growth cone and cell body. The speed of neuritic Ca²⁺ waves was reduced in cells loaded with the Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-tetraacetic acid/AM. Preloading of Ca²⁺ stores led to increased bradykinin-induced Ca²⁺ release, as seen for caffeine, and faster Ca²⁺ wave speeds. Caffeine evoked a simultaneous [Ca²⁺]_i rise along the neurites of Ca²⁺ preloaded cells. Higher Ca²⁺ signal amplitudes and faster Ca²⁺ wave speeds, but no longer-lasting IP₃-induced [Ca²⁺]_i signals, correlated with increased caffeine-induced Ca²⁺ release in the neurites. At low concentrations of bradykinin (<1.0 nM), the Ca²⁺ signals ceased to propagate as complete Ca²⁺ waves. Instead, repetitive stochastic Ca²⁺ release events (neuritic Ca²⁺ puffs) were observed. Neuritic Ca²⁺ puffs spread across only a few microns, at a slower speed than neuritic Ca²⁺ waves. These Ca²⁺ puffs represent elementary Ca²⁺ release units, whereby the released Ca²⁺ ions form these elementary events into the shape of a Ca²⁺ wave. Received: 16 April 1996 / Received after revision and accepted: 13 May 1996     

Divergent regulation of vascular endothelial growth factor and of erythropoietin gene expression in vivo

There is accumulating evidence from in vitro experiments that the gene expression of the vascular endothelial growth factor (VEGF) is, like that of the erythropoietin (EPO) gene, regulated by the oxygen tension and by divalent cations such as cobalt. Since the information about the regulation of VEGF gene expression in vivo is rather scarce, this study aimed to examine the influence of hypoxia and of cobalt on VEGF gene expression in different rat organs and to compare it with that on EPO gene expression. To this end male Sprague-Dawley rats were exposed to carbon monoxide (0.1% CO), hypoxia (8% O₂ ) or to cobalt chloride (12 and 60 mg/kg s.c.) for 6 h. mRNA levels for VEGF- 188, -164, and -120 amino acid isoforms in lungs, hearts, kidneys and livers were semiquantitated by RNase protection. For these organs we found a rank order of VEGF mRNA abundance of lung >> heart > kidney = liver. EPO mRNA levels were semiquantitated in kidneys and livers. Hypoxia, CO and cobalt increased EPO mRNA levels 60-fold, 140-fold and 5-fold, respectively, in the kidneys, and 11-fold, 11-fold and 3-fold, respectively, in the livers. None of these manoeuvres caused significant changes of VEGF mRNA in lung, heart or kidneys. Only in the livers did hypoxia lead to a significant (50%) increase of VEGF mRNA. These findings suggest that, in contrast to the in vitro situation, the expression of the VEGF gene in normal rat tissues is rather insensitive to hypoxia. In consequence, the in vivo regulation of the VEGF and the EPO genes appear to differ substantially, suggesting that the regulation of the VEGF and EPO genes may not follow the same essential mechanisms in vivo. Received: 31 July 1995/Received after revision: 20 November 1995/Accepted: 27 November 1995     

The effect of crosslinking in elastomers investigated by NMR analysis of 13C edited transverse 1H NMR relaxation

The transverse nuclear ¹H magnetization decay in poly(styrene-co-butadiene) (SBR) is investigated by editing ¹³C NMR spectra. This technique allows for the assignment of localized ¹H dynamical information by discriminating the chemical sites based on their chemical shift in the ¹³C dimension. Here, the homo- and heteronuclear dipolar couplings contribute to the ¹H NMR relaxation giving additional information to a homonuclear experiment. In this heteronuclear 2D experiment two prominent peaks are observed in the ¹³C dimension, which correspond to CH and CH₂ groups, respectively. The decay rate in the ¹H dimension is found for both groups to scale with the crosslink density. An additional ultra-fast magnetization decay is reported. The effect of the carbon black filler is investigated for this component. The analysis of the ¹³C NMR edited transverse ¹H magnetization relaxation is a useful tool in combining high resolution NMR spectra with information on molecular dynamics, providing insight into crosslink density and filler effects.