Escherichia coli Nissle 1917 distinctively modulates T-cell cycling and expansion via toll-like receptor 2 signaling

Although the probiotic Escherichia coli strain Nissle 1917 has been proven to be efficacious for the treatment of inflammatory bowel diseases, the underlying mechanisms of action still remain elusive. The aim of the present study was to analyze the effects of E. coli Nissle 1917 on cell cycling and apoptosis of peripheral blood and lamina propria T cells (PBT and LPT, respectively). Anti-CD3-stimulated PBT and LPT were treated with E. coli Nissle 1917-conditioned medium (E. coli Nissle 1917-CM) or heat-inactivated E. coli Nissle 1917. Cyclin B1, DNA content, and caspase 3 expression were measured by flow cytometry to assess cell cycle kinetics and apoptosis. Protein levels of several cell cycle and apoptosis modulators were determined by immunoblotting, and cytokine profiles were determined by cytometric bead array. E. coli Nissle 1917-CM inhibits cell cycling and expansion of peripheral blood but not mucosal T cells. Bacterial lipoproteins mimicked the effect of E. coli Nissle 1917-CM; in contrast, heat-inactivated E. coli Nissle 1917, lipopolysaccharide, or CpG DNA did not alter PBT cell cycling. E. coli Nissle 1917-CM decreased cyclin D2, B1, and retinoblastoma protein expression, contributing to the reduction of T-cell proliferation. E. coli Nissle 1917 significantly inhibited the expression of interleukin-2 (IL-2), tumor necrosis factor alpha, and gamma interferon but increased IL-10 production in PBT. Using Toll-like receptor 2 (TLR-2) knockout mice, we further demonstrate that the inhibition of PBT proliferation by E. coli Nissle 1917-CM is TLR-2 dependent. The differential reaction of circulating and tissue-bound T cells towards E. coli Nissle 1917 may explain the beneficial effect of E. coli Nissle 1917 in intestinal inflammation. E. coli Nissle 1917 may downregulate the expansion of newly recruited T cells into the mucosa and limit intestinal inflammation, while already activated tissue-bound T cells may eliminate deleterious antigens in order to maintain immunological homeostasis.     

Inverse regulation of the ADAM-family members, decysin and MADDAM/ADAM19 during monocyte differentiation

Two members of the ADAM (a disintegrin and metalloprotease)-family, MADDAM and decysin, were described as dendritic cell (DC) maturation markers. We are interested in monocyte differentiation and investigated in particular the expression pattern of both genes during the differentiation of human monocytes into DC and macrophages (MAC). Both genes are weakly expressed in freshly isolated monocytes. In immature DC decysin mRNA was absent, even after induction of the terminal differentiation of DC by CD40L or tumour necrosis factor-alpha (TNF-alpha). Only in DC maturated by lipopolysaccharide (LPS) strong signals of decysin mRNA were detected. However, MADDAM mRNA was expressed in immature DC and the expression was markedly increased after induction of the terminal DC differentiation by various stimuli. In contrast, MAC showed a high constitutive decysin mRNA expression, but expressed no MADDAM mRNA. On protein level similar results of MADDAM expression were obtained. Stimulation of MAC by LPS did not induce MADDAM mRNA expression, while decysin mRNA expression was strongly increased. Further investigations revealed that the well-known inducer of MAC differentiation, 1alpha,25-dihydroxyvitamin D3 up-regulated decysin mRNA expression during the differentiation of primary monocytes and myelomonocytic THP-1 cells into MAC. In vivo decysin mRNA expression was only detected in human colon, but not in other tissues we examined. Accordingly, isolated intestinal MAC expressed decysin mRNA. In conclusion, decysin and MADDAM mRNA expression were regulated in an opposite way during monocyte differentiation: MADDAM mRNA and protein was mainly detected in DC, whereas decysin mRNA expression was mainly found in MAC. Therefore only MADDAM, but not decysin is a suitable marker for human monocyte-derived DC.     

The clinical-grade 42-kilodalton fragment of merozoite surface protein 1 of Plasmodium falciparum strain FVO expressed in Escherichia coli protects Aotus nancymai against challenge with homologous erythrocytic-stage parasites

A 42-kDa fragment from the C terminus of major merozoite surface protein 1 (MSP1) is among the leading malaria vaccine candidates that target infection by asexual erythrocytic-stage malaria parasites. The MSP1(42) gene fragment from the Vietnam-Oak Knoll (FVO) strain of Plasmodium falciparum was expressed as a soluble protein in Escherichia coli and purified according to good manufacturing practices. This clinical-grade recombinant protein retained some important elements of correct structure, as it was reactive with several functional, conformation-dependent monoclonal antibodies raised against P. falciparum malaria parasites, it induced antibodies (Abs) that were reactive to parasites in immunofluorescent Ab tests, and it induced strong growth and invasion inhibitory antisera in New Zealand White rabbits. The antigen quality was further evaluated by vaccinating Aotus nancymai monkeys and challenging them with homologous P. falciparum FVO erythrocytic-stage malaria parasites. The trial included two control groups, one vaccinated with the sexual-stage-specific antigen of Plasmodium vivax, Pvs25, as a negative control, and the other vaccinated with baculovirus-expressed MSP1(42) (FVO) as a positive control. Enzyme-linked immunosorbent assay (ELISA) Ab titers induced by E. coli MSP1(42) were significantly higher than those induced by the baculovirus-expressed antigen. None of the six monkeys that were vaccinated with the E. coli MSP1(42) antigen required treatment for uncontrolled parasitemia, but two required treatment for anemia. Protective immunity in these monkeys correlated with the ELISA Ab titer against the p19 fragment and the epidermal growth factor (EGF)-like domain 2 fragment of MSP1(42), but not the MSP1(42) protein itself or the EGF-like domain 1 fragment. Soluble MSP1(42) (FVO) expressed in E. coli offers excellent promise as a component of a vaccine against erythrocytic-stage falciparum malaria.     

Repeat sequences in block 2 of Plasmodium falciparum merozoite surface protein 1 are targets of antibodies associated with protection from malaria

Human antibodies to the block 2 region of Plasmodium falciparum merozoite surface protein 1 (MSP1) are associated with a reduced prospective risk of clinical malaria. Block 2 is highly polymorphic, but all known alleles can be grouped into three major types. Two of these types (the K1-like and MAD20-like types) contain type-specific sequences (found in all alleles of a particular type) that flank polymorphic tripeptide repeats. These repeats contain both type-specific and subtype-specific sequences. To evaluate the antibody recognition of these parts of block 2, a new panel of six recombinant proteins was used (fused type-specific flanking sequences and two representative repeat sequences for each of the K1-like and MAD20-like types separately). Extensive testing of these antigens and full-length block 2 antigens showed that human serum immunoglobulin G antibodies induced by infection can recognize (i) type-specific epitopes in the repeats, (ii) subtype-specific epitopes in the repeats, or (iii) type-specific epitopes in flanking sequences. A large prospective study in The Gambia showed that antibodies to the repeats are strongly associated with protection from clinical malaria. The results are important for design of a vaccine to induce protective antibodies, and they address hypotheses about repeat sequences in malaria antigens.     

Prognostic significance of del(20q) in patients with hematological malignancies

Deletions of the long arm of chromosome 20 represent a common chromosomal abnormality associated with myeloid malignancies, in particular with myeloproliferative disorders (MPD), myelodysplastic syndromes (MDS), and acute myeloid leukemia (AML). Using G-banding cytogenetic techniques, we found clones with del(20q) in 36 patients with hematological malignancies examined in our laboratory during the years 2001–2003: in 23 patients as a sole cytogenetic aberration and in 13 patients together with other chromosomal changes. Fluorescence in situ hybridization (FISH) with a probe specific for the 20q12 region was used in all cases to confirm the presence of the clone with deletion. For patients with additional or complex chromosomal rearrangements, multicolor FISH (M-FISH) analysis was performed. Statistical evaluation of the prognostic impact of sex, age, diagnosis, and karyotype was performed. The survival time correlated with the type of chromosomal aberration; no significant differences in survival were found for sex, age, and diagnosis.     

Evaluation of a dipstick ELISA and a rapid immunochromatographic test for diagnosis of Dengue virus infection

Here we report standardization of a dipstick enzyme-linked immunosorbent assay (ELISA, Dipstick ELISA) and its comparative evaluation with a commercial Rapid PanBio Immunochromatographic test (IC test) for detection of Dengue (DEN) virus-specific IgM and IgG antibodies in patient sera. Among crude and purified viral antigens prepared from mouse brains or cell cultures, a DEN virus type 2 antigen purified from cell cultures by sucrose density gradient centrifugation was found superior in terms of the signal/ noise (S/N) ratio in the assay system. The sensitivity of detection of the virus by specific IgM antibody was improved by removal of IgG from patient sera prior to testing. The evaluation of the Dipstick ELISA by use of 156 serum samples revealed an overall accordance of 96% and 93% with the IC test in detection of IgM antibodies to DEN viruses (IgM antibodies) and IgG antibodies to DEN viruses (IgG antibodies), respectively. The sensitivity of the Dipstick ELISA and the IC test with reference to the mu-capture ELISA was 83% and 87%, respectively, with a specificity of 98% in both cases. The sensitivity of the Dipstick ELISA with reference to the IC test in detecting IgM and IgG antibodies was 84% and 94%, respectively, and the specificity of the Dipstick ELISA was 98% and 92%, respectively.     

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.     

The direct early diagnosis of cystic fibrosis by the detection of the deltaF508 CFTR gene mutation in a prematurely delivered boy

The suspicion of prenatal meconium ileus syndrome was raised in a pregnancy in a family with no history of cystic fibrosis because of significantly higher maternal serum alpha-fetoprotein in the 16th and 19th week of gestation, dispersed areas with increased echogenity in the fetal abdomen, slight fetal ascites in the 24th-25th weeks of gestation, decreased amniotic fluid gamma-glutamyltranspeptidase (GGT) activity and alpha-fetoprotein level in the 25th-26th weeks, and normal 46,XY karotype of the fetus. The detection of a homozygous deltaF508 cystic fibrosis transmembrane regulator (CFTR) gene mutation, by means of PCR from a small amount of white blood cells and urine sediment cells, substantiated the diagnosis of cystic fibrosis in a prematurely delivered boy in the 28th week of gestation. The repeated sweat test was unsuccessful. The autopsy examination confirmed the diagnosis of cystic fibrosis. Fetal meconium ileus syndrome was complicated by peritonitis and by formation of a meconium pseudocyst. Direct PCR typing improves postnatal diagnostic possibilities in the early neonatal period in prematurely delivered babies when the sweat test is difficult to perform.     

Posttraumatic stress disorder (PTSD) and posttraumatic stress symptoms (PTSS) in families of adolescent childhood cancer survivors

OBJECTIVE: To describe rates and concordance of posttraumatic stress disorder (PTSD) and posttraumatic stress symptoms (PTSS) in adolescent childhood cancer survivors and their mothers and fathers. METHOD: Participants were 150 adolescent survivors of childhood cancer, 146 mothers, and 103 fathers who completed the Impact of Events Scale-Revised, the Posttraumatic Stress Disorder Reaction Index, and the PTSD module of the Structured Clinical Interview for the Diagnostic and Statistical Manual of Mental Disorders, fourth edition. RESULTS: PTSS are common in families of childhood cancer survivors. Parents reported more symptomatology than former patients. Mothers and fathers had relatively equal rates of current PTSD and levels of PTSS. Nearly 30% of mothers met diagnostic criteria since their child's diagnosis, with 13.7% currently experiencing PTSD. Nearly 20% of families had at least one parent with current PTSD. Ninety-nine percent of the sample had at least one family member reexperiencing symptoms. CONCLUSIONS: Both PTSD and PTSS help in understanding the experience of adolescent cancer survivors and their families. Within families of childhood cancer survivors, it is likely that some member may be experiencing treatable bothersome memories, arousal, or avoidance specific to the cancer experience.     

The time-course of ribavirin-provoked changes of basal and AMPH-induced motor activities in rats

The time-course of changes of basal and amphetamine (AMPH)-induced locomotor and stereotypic activities in adult male Wistar rats after a single ribavirin injection was studied. In the first set of experiments, 10, 20 or 30 mg ribavirin/kg body weight (b.w.) were injected i.p. to rats and their basal motor activities were recorded every 10 min for 2 h and compared with those of saline-treated controls. In the second set of experiments, the animals were pretreated with ribavirin and 20 min later i.p. injected with AMPH (1.5 mg/kg b.w.). The controls received AMPH 20 min after the saline injection. Motor activity was recorded after the first injection and until 120 min after AMPH administration. Ribavirin did not significantly affect the time-course of either basal locomotor or stereotypic activities. Pretreatment with any of the applied ribavirin doses decreased the AMPH-induced hyperlocomotor response. However, the most pronounced effect was observed with ribavirin doses of 20 mg/kg and 30 mg/kg when administered during the first 10 min and 30 min after the AMPH injection respectively. In contrast, the stereotypic activities of these animals were only slightly changed. These results indicate a different susceptibility of regions in the basal ganglia to ribavirin.