Visualization and microscopic quantification of HCMV-peptide-specific cytotoxic T lymphocytes using tetramer binding

To monitor the frequencies of virus-specific cytotoxic T lymphocytes (CTLs), FACS analyses were performed detecting lymphocyte-specific surface molecules and tetramer binding, as marker for peptide-specificity. Aim of this investigation was to establish an alternative protocol for the quantification of virus-specific CTLs using tetramer binding and microscopic analyzing. The frequencies of HCMV-pp65-peptide-specific CTLs in the blood of eight different HLA-A*0201-positive, HCMV-IgG antibody-positive donors were analyzed with both methods. Using FACS analyses, a median of 0.8% and, using the microscopic analyses, a median of 3.0% was detected in the CD3+CD8+ cells. After enrichment of HCMV-pp65-peptide-specific CTLs using the interferon-gamma secretion assay followed by expansion in cell culture, a median of 90.6% using FACS analyses and a median of 87.1% using the microscopic analyses was detected. Thus, the staining protocol presented in this investigation is an alternative approach to detect and to quantify virus-specific CTLs in low as well as in high frequencies.     

Gene profiling reveals increased expression of uteroglobin and other anti-inflammatory genes in glucocorticoid-treated nasal polyps

BACKGROUND: Treatment with local glucocorticoids (GCs) decreases symptoms and the size of nasal polyps. This might depend on the downregulation of proinflammatory genes, as well as the upregulation of anti-inflammatory genes. OBJECTIVE: We sought to identify GC-regulated anti-inflammatory genes in nasal polyps. METHODS: Affymetrix DNA microarrays were used to analyze the expression of 22,283 genes in 4 nasal polyps before and after local treatment with fluticasone (400 microg/d). Expression of uteroglobin and mammaglobin B was analyzed with real-time PCR in 6 nasal polyps and in nasal biopsy specimens from 6 healthy control subjects. RESULTS: Two hundred three genes had changed in expression in treated polyps, and 139 had known functions: 54 genes were downregulated, and 85 were upregulated. Genes associated with inflammation constituted the largest single functional group. These genes affected key steps in inflammation (eg, immunoglobulin production; antigen processing and presentation; and the chemoattraction and activation of granulocytes, T cells, and B cells). Several proinflammatory genes were downregulated. In contrast, some anti-inflammatory genes were upregulated. The gene that increased most in terms of expression was uteroglobin. This was confirmed with real-time PCR. By contrast, expression of uteroglobin was lower in untreated polyps than in healthy nasal mucosa. Immunohistochemical investigation showed staining of uteroglobin in the epithelium and in seromucous glands in control subjects and in nasal polyps. CONCLUSION: Upregulation of anti-inflammatory genes, such as uteroglobin, might contribute to the effects of local treatment with GCs in nasal polyps.     

Horizontal optokinetic ocular nystagmus in wildtype (B6CBA+/+) and weaver mutant mice

Summary Horizontal optokinetic nystagmus (OKN) evoked by a random dot pattern moving at a constant speed around the animal was investigated in wild-type mice and Weaver mutants (cerebellar impairment) by means of chronically implanted EOG-electrodes. The shape of OKN in the homozygotic Weaver mouse was clearly different from that in normal mice. The OKN in the mutant showed inconstant velocity during the slow phase. Nystagmus frequency of the mutant was significantly below that of normal controls for velocities of 1.4 to 25 degrees · s^-1. In one group of normals the mean slow-phase gain was relatively constant for stimulus angular velocities between 1.4 and 15 degrees · s^-1 and declined thereafter. In a second group the mean slow-phase gam decreased gradually between stimulus angular velocities from 1.4 to 15 degrees · s^-1 and thereafter with a steeper slope. In mutants gain decreases with increasing stimulus velocity over the entire range tested (1.4 to 42 degrees · s^-1). Normals and mutants with one eye occluded exhibited strong OKN when the pattern was moved in a temporonasal direction; little response was obtained by stimuli moving in a naso-temporal direction.     

Amygdala-prefrontal coupling depends on a genetic variation of the serotonin transporter

Major depression is conditionally linked to a polymorphism of the human serotonin transporter gene (SLC6A4). During the presentation of aversive, but not pleasant, pictures, healthy carriers of the SLC6A4 short (s) allele showed stronger activation of the amygdala on functional magnetic resonance imaging. s carriers also showed greater coupling between the amygdala and the ventromedial prefrontal cortex, which may contribute to the abnormally high activity in the amygdala and medial prefrontal cortex seen in major depression.     

Effect of a brief cognitive training programme in patients with long-lasting back pain evaluated as unfit for surgery

The aim of the study was to evaluate the effect of cognitive intervention (information and physical exercise), on patients with long-lasting back pain referred for surgical evaluation at an orthopaedic hospital, but evaluated as unfit for surgery. One hundred and fifty-two patients were randomized to a five days intervention or control. The intervention had no significant effects on pain. At three-month follow-up, the patients in the intervention group used significantly more active strategies to cope with the back pain compared to the control group. This effect seemed to increase over time, being more pronounced at one-year follow-up evaluation.     

Apoptotic cell death in mouse models of GM2 gangliosidosis and observations on human Tay-Sachs and Sandhoff diseases

Tay-Sachs and Sandhoff diseases are autosomal recessive neurodegenerative diseases resulting from the inability to catabolize GM2 ganglioside by beta-hexosaminidase A (Hex A) due to mutations of the alpha subunit (Tay-Sachs disease) or beta subunit (Sandhoff disease) of Hex A. Hex B (beta beta homodimer) is also defective in Sandhoff disease. We previously developed mouse models of both diseases and showed that Hexa-/- (Tay-Sachs) mice remain asymptomatic to at least 1 year of age while Hexb-/- (Sandhoff) mice succumb to a profound neurodegenerative disease by 4-6 months of age. Here we find that neuron death in Hexb-/- mice is associated with apoptosis occurring throughout the CNS, while Hexa-/- mice were minimally involved at the same age. Studies of autopsy samples of brain and spinal cord from human Tay-Sachs and Sandhoff diseases revealed apoptosis in both instances, in keeping with the severe expression of both diseases. We suggest that neuron death is caused by unscheduled apoptosis, implicating accumulated GM2 ganglioside or a derivative in triggering of the apoptotic cascade.      

Erythropoietin and renin substrate in cerebellar haemangioblastoma

We examined eight cerebellar haemangioblastoma tumours from eight patients, aged 16-63 years, 5 females and 3 males. Preoperative haemoglobin values exceeded 180 g/l in four patients, and 150 g/l in four. All high Hb values were normalized upon surgical removal of the tumours. All tumours contained scattered cells which stained positively with antisera against pure human urinary erythropoietin and plasma renin substrate. We conclude that cerebellar haemangioblastomas produce immunoreactive erythropoietin, which shares common antigenic determinants with renin substrate.     

In vitro phosphorylation of SV40 large T antigen

Phosphorylation of simian virus 40 large T antigen (large T) was investigated in vitro. "Autophosphorylation" of large T resulted in the modification of Ser106, Ser112, Ser123, Thr124, either Ser676, Ser677, or Ser679, and Thr701. All of these residues were also found to be phosphorylated in vivo. Reaction of large T with purified casein kinase I resulted in phosphorylation of Ser123, possibly Thr124, and either Ser676, Ser677, or Ser679, while purified casein kinase II phosphorylated Ser106 and possibly Ser112. Submolar amounts of phosphate were transferred to large T indicating that only a fraction of large T served as substrate for the casein kinases. Removal of serine-bound phosphate did not affect the subsequent autophosphorylation or phosphorylation by casein kinase I and II. No phosphorylation at in vivo sites was observed with the cAMP-, cGMP-, or Ca2+/phospholipid-dependent protein kinases, or with the protease-activated kinase I and II.