Phenotypic and genotypic analyses of enterohemorrhagic Escherichia coli O145 strains from patients in Germany

Enterohemorrhagic Escherichia coli (EHEC) strains of serogroup O145 are emerging as causes of diarrhea and the hemolytic-uremic syndrome. However, there have been few genetic analyses of this EHEC group. We investigated the serotypes, virulence genes, plasmid profiles, pulsed-field gel electrophoresis (PFGE) patterns, and genetic variability of the fliC and eae genes in 120 EHEC O145 strains isolated from cases of hemolytic-uremic syndrome (n = 24) or diarrhea (n = 96) in Germany between 1996 and 2002. Three isolates belonged to serotype O145:H28, one to serotype O145:H25, and 116 were nonmotile (O145:H(-)). One hundred fourteen of the nonmotile strains shared fliC restriction fragment length polymorphism (RFLP) patterns identical to that of the O145:H28 strains. The remaining two nonmotile strains displayed a fliC-RFLP pattern identical to that of the O145:H25 strain. Each of the 117 strains with the fliC-RFLP(H28) pattern harbored eae gamma, whereas the three strains with the fliC-RFLP(H25) pattern possessed eae beta. Five different stx genotypes, six combinations of plasmid-encoded putative virulence genes, 29 plasmid profiles, and 47 PFGE types were identified. Strains within some of the PFGE types could be further subtyped by means of distinct plasmid profiles. These data demonstrate that the EHEC O145 serogroup is comprised of two different serotypes that possess distinct eae types. The heterogeneity of EHEC O145 strains at the chromosomal and plasmid level, in particular the high diversity in PFGE patterns, provides a basis for molecular subtyping of these pathogens.     

Expression and potential role of fibroblast growth factor 2 and its receptors in human embryonic stem cells

Although the detection of several components of the fibroblast growth factor (FGF) signaling pathway in human embryonic stem cells (hESCs) has been reported, the functionality of that pathway and effects on cell fate decisions are yet to be established. In this study we characterized expression of FGF-2, the prototypic member of the FGF family, and its receptors (FGFRs) in undifferentiated and differentiating hESCs; subsequently, we analyzed the effects of FGF-2 on hESCs, acting as both exogenous and endogenous factors. We have determined that undifferentiated hESCs are abundant in several molecular-mass isoforms of FGF-2 and that expression pattern of these isoforms remains unchanged under conditions that induce hESC differentiation. Significantly, FGF-2 is released by hESCs into the medium, suggesting an autocrine activity. Expression of FGFRs in undifferentiated hESCs follows a specific pattern, with FGFR1 being the most abundant species and other receptors showing lower expression in the following order: FGFR1 --> FGFR3 --> FGFR4 --> FGFR2. Initiation of differentiation is accompanied by profound changes in FGFR expression, particularly the upregulation of FGFR1. When hESCs are exposed to exogenous FGF-2, extracellular signal-regulated kinases are phosphorylated and thereby activated. However, the presence or absence of exogenous FGF-2 does not significantly affect the proliferation of hESCs. Instead, increased concentration of exogenous FGF-2 leads to reduced outgrowth of hESC colonies with time in culture. Finally, the inhibitor of FGFRs, SU5402, was used to ascertain whether FGF-2 that is released by hESCs exerts its activities via autocrine pathways. Strikingly, the resultant inhibition of FGFR suppresses activation of downstream protein kinases and causes rapid cell differentiation, suggesting an involvement of autocrine FGF signals in the maintenance of proliferating hESCs in the undifferentiated state. In conclusion from our data, we propose that this endogenous FGF signaling pathway can be implicated in self-renewal or differentiation of hESCs.     

A comparison of the potency of the oxime HLö-7 and currently used oximes (HI-6, pralidoxime, obidoxime) to reactivate nerve agent-inhibited rat brain acetylcholinesterase by in vitro methods

(1) The efficacy of the oxime HL?7 and currently used oximes (pralidoxime, obidoxime, HI-6) to reactivate acetylcholinesterase inhibited by various nerve agents (sarin, tabun, cyclosarin, VX) was tested by in vitro methods. (2) Both H oximes (HL?-7, HI-6) were found to be more efficacious reactivators of sarin and VX-inhibited acetylcholinesterase than pralidoxime and obidoxime. On the other hand, their potency to reactivate tabun-inhibited acetylcholinesterase is very low and does not reach the reactivating efficacy of obidoxime. In the case of cyclosarin, the oxime HI-6 was only found to be able to sufficiently reactivate cyclosarin-inhibited acetylcholinesterase in vitro. (3) Thus, the oxime HL?-7 does not seem to be more efficacious reactivator of nerve agent-inhibited acetylcholinesterase than HI-6 according to in vitro evaluation of their reactivation potency and, therefore, it is not more suitable to be introduced for antidotal treatment of nerve agent-exposed people than HI-6.     

Comparison of couples referred and not referred for genetic counseling in a genetic clinic after the birth of a child with a congenital anomaly: A study in a population in the northeastern Netherlands

After the birth of a child with a congenital anomaly, parents have many questions about cause, prognosis, and recurrence risk. An important means of transmitting such information is referral to a genetic clinic. We were interested in knowing what determines whether or not parents are referred for genetic counseling. Data from the local registration of congenital anomalies in the northeastern Netherlands (birth years 1981–1986; 1,217 children/fetuses) and data of the local genetic clinic were compared. The parents of 204 cases (16.8%) had been referred for genetic counseling. Of the couples referred, 76% were referred within one year after birth, usually by a pediatrician (48%). Parents of children with a single anomaly, recognized syndrome, or multiple anomalies not recognized as a syndrome were referred in 5%, 43%, and 26% of cases, respectively. Parents of liveborn children who died were referred in 38% of cases, parents of liveborn/still-alive and stillborn children in 13% and 22%, respectively. Previous affected sibs and absence of previous livebirths increased the likelihood of referral.     

Long-term elevations of dietary sodium produce parallel increases in the renal excretion of urodilatin and sodium

The effects of dietary sodium intake on the renal excretion of urodilatin and of sodium were examined in six healthy male subjects. The 24-day study period was divided into three phases of 8 days each. Subjects Ingested 2.8 mequiv sodium (kg body weight)^–1 day^–1 during the first phase, 5.6 mequiv (kg body weight)^–1 day^–1 during the second phase, and 8.4 mequiv (kg body weight)^–1 day^–1 during the third phase. The excretion of both sodium (P<0.002) and urodilatin (P<0.006) increased in response to the increasing dietary sodium, while urine flow did not change. Urinary urodilatin excretion correlated closely with renal sodium excretion (P<0.001). Serum aldosterone levels (P<0.01) as well as serum renin levels (P<0.05) significantly decreased with increasing sodium intake. Plasma [Arg]vasopressin levels increased significantly (P<0.05). Plasma atrial natriuretic factor and cGMP levels as well as urinary cGMP excretion rates were unaltered by the changes in sodium intake. We conclude from these results that the renal natriuretic peptide, urodilatin, but not the main cardiac member of the natriuretic peptide family may be involved in the regulation of day-to-day sodium balance.     

Acute changes of coagulation and fibrinolysis parameters after experimental thromboembolic stroke and thrombolytic therapy

Thrombolysis is the only effective pharmaceutical therapy in acute ischemic stroke in humans but has a high risk of intracerebral hemorrhage. We aimed to establish an animal model to study changes of coagulation and fibrinolytic parameters during thromboembolic ischemic stroke and thrombolysis with recombinant tissue plasminogen activator (rt-PA). We used a thromboembolic stroke model in the rat. Animals were treated with rt-PA thrombolysis (n = 10) and compared with untreated (n = 10), sham operated (n = 10) and control animals (n = 20). Coagulation parameters (APTT, PT, TT, fibrinogen, AT III, TAT) and fibrinolytic parameters (t-PA antigen concentration, t-PA activity, PAI-1 concentration, PAI activity, plasminogen, antiplasmin) were measured at two time points (2.5 and 5 h after stroke induction) with a battery of commercially available test kits. We observed an (1) initiation of coagulation and inhibition of fibrinolysis by the operation procedure itself, (2) simultaneous activation of fibrinolysis and its inhibitors after stroke induction and (3) potent initiation of fibrinolysis and consumption of fibrinolysis inhibitors after rt-PA therapy of stroke. We established a model system to monitor coagulation and fibrinolysis during thrombolytic therapy of stroke in the rat. This model may be used to study the influence of these parameters on hemorrhagic stroke transformation and outcome in experimental stroke in future.     

Selection of CMV-specific CD8+ and CD4+ T cells by mini-EBV-transformed B cell lines

Efficient protocols to generate cytomegalovirus (CMV)-specific T cells are required for adoptive immunotherapy. Recombinant Epstein-Barr virus (EBV) vectors called mini-EBV can be used to establish permanent B cell lines in a single step, which present the CMV antigen pp65 in a constitutive manner. These B cell lines, coined pp65 mini-LCL, were successfully used to reactivate and expand CMV-specific cytotoxic T cells. Here we evaluate this pp65 mini-EBV system in closer detail, focusing on (1) the quantification of T cells with specific effector function and (2) the identification of CMV-specific CD4(+) helper T cells. The co-expansion of various functional CMV epitope specificities was demonstrated by IFN-gamma enzyme-linked immunospot assay (ELISPOT) assays and HLA-peptide tetramer staining. Single-cell cloning resulted in both CD4(+) and CD8(+) T cell clones, the majority of which was CMV specific. Thus, mini-LCL present the pp65 antigen on HLA class I and II, mobilizing both arms of the T cell response. Using a peptide library covering the pp65 sequence for further analysis of T cell clones, we identified new pp65 CD8(+) and CD4(+) T cell epitopes.     

Migrating anterior mesoderm cells and intercalating trunk mesoderm cells have distinct responses to Rho and Rac during Xenopus gastrulation.

Rho GTPases have been shown recently to be important for cell polarity and motility of the trunk mesoderm during gastrulation in Xenopus embryos. This work demonstrated that Rho and Rac have both distinct and overlapping roles in regulating cell shape, and the dynamic properties, polarity, and type of protrusive activity of these cells. Overexpression of activated or inhibitory versions of these GTPases also disrupts development of the head in Xenopus embryos. In this study, we have undertaken a detailed analysis of Rho and Rac function in migrating anterior mesendoderm cells. Scanning electron micrographs of these cells in situ revealed that their normal shingle arrangement is disrupted and both the cells and their lamellipodia are disoriented. Anterior mesendoderm explants plated on their natural blastocoel roof matrix, however, still migrated towards the animal pole, although the tendency to move in this direction is reduced compared to controls. Analysis of a number of parameters in time-lapse recordings of dissociated cells indicated that Rho and Rac also have both distinct and overlapping roles in the motility of the prospective head mesoderm; however, their effects differ to those previously seen in the trunk mesoderm. Both GTPases appear to modulate cell polarization, migration, and protrusive activity. Rho alone, however, regulates the retraction of the lagging edge of the cell. We propose that within the gastrulating Xenopus embryo, two types of mesoderm cells that undergo different motilities have distinct responses to Rho GTPases.