MCP-1-dependent signaling in CCR2(-/-) aortic smooth muscle cells

Monocyte chemoattractant protein-1 (MCP-1, CCL2) is a mediator of inflammation that has been implicated in the pathogenesis of a wide variety of human diseases. CCR2, a heterotrimeric G-coupled receptor, is the only known receptor that functions at physiologic concentrations of MCP-1. Despite the importance of CCR2 in mediating MCP-1 responses, several recent studies have suggested that there may be another functional MCP-1 receptor. Using arterial smooth muscle cells (SMC) from CCR2(-/-) mice, we demonstrate that MCP-1 induces tissue-factor activity at physiologic concentrations. The induction of tissue factor by MCP-1 is blocked by pertussis toxin and 1,2-bis(O-aminophenyl-ethane-ethan)-N,N,N',N'-tetraacetic acid-acetoxymethyl ester, suggesting that signal transduction through the alternative receptor is G(alphai)-coupled and dependent on mobilization of intracellular Ca(2+). MCP-1 induces a time- and concentration-dependent phosphorylation of the mitogen-activated protein kinases p42/44. The induction of tissue factor activity by MCP-1 is blocked by PD98059, an inhibitor of p42/44 activation, but not by SB203580, a selective p38 inhibitor. These data establish that SMC possess an alternative MCP-1 receptor that signals at concentrations of MCP-1 that are similar to those that activate CCR2. This alternative receptor may be important in mediating some of the effects of MCP-1 in atherosclerotic arteries and in other inflammatory processes.     

Nonmyeloablative bone marrow transplantation: Infectious complications in 65 recipients of HLA-identical and mismatched transplants.

Infections are a common complication of allogeneic bone marrow transplantation and the leading cause of transplantation-related mortality. It had been hypothesized that transplantation following nonmyeloablative preparative regimens would result in fewer infections by causing less mucosal injury, less graft-versus-host disease, and allowing earlier immune reconstitution. We have retrospectively reviewed the infectious complications of 65 consecutive patients with advanced hematologic malignancies who underwent bone marrow transplantation using a novel preparative regimen consisting of cyclophosphamide, thymic irradiation, and in vivo T-cell depletion. Cytomegalovirus (CMV) infection occurred in 52% of cases in which the donor or recipient had evidence of prior CMV exposure. Using a strategy of preemptive therapy and secondary prophylaxis with ganciclovir, no CMV disease occurred. Infections with gram-positive bacteria predominated over the first 100 days after bone marrow transplantation. Thereafter, the relative proportion of gram-negative infections increased without a significant increase in episodes of neutropenia. The rate of bacterial infections was not influenced by relapse of the underlying malignancy. Seven patients developed infections with Aspergillus species, which was the most common infectious cause of death in these patients. Infections with viruses other than CMV (n=10) and with protozoan organisms (n=2) also occurred. The use of HLA-mismatched donors, the occurrence of grade II-IV acute graft-versus-host disease, and treatment with corticosteroids did not influence the risk of CMV or bacterial or fungal infections in patients who underwent transplantation following this preparative regimen. Overall, the incidence and spectrum of infections in this series was similar to the reported incidence of infections following conventional myeloablative allogeneic stem cell transplantation. We conclude that a quantitative T-cell deficiency in these extensively T-cell depleted patients may be a risk factor for infection, even in the absence of graft-versus-host disease.     

Genetic mediation of infanticide and parental behavior in male and female domestic and wild stock house mice

Infanticide is a reproductive strategy found in many mammals, especially rodents. The proportion of male and female house mice (Mus domesticus) that are either infanticidal or noninfanticidal is strain specific and varies widely from stock to stock. Male house mice also show strain-specific variation in the behavioral mechanisms that inhibit infanticidal individuals from killing their own offspring. The adult offspring generated from reciprocally crossed CF-1 and Wild stock house mice were tested for their behavior toward newborn pups. In male CF-1xWild hybrids, the proportion of infanticidal and noninfanticidal males matched with their maternal phenotype, whereas female CF-1xWild hybrids exhibited a proportion of behaviors typical of the CF-1 phenotype, regardless of their mother's genotype. Our results suggest three conclusions: first, that infanticide is a highly labile and heritable behavior in both sexes; second, that there is a sex difference in the genetic substrate that regulates the inheritance of infanticidal behavior; and third, that selection pressures in male mice may operate independently on the mechanisms that promote spontaneous infanticidal behavior versus the mechanisms that inhibit infanticide.     

Phase Variation of Campylobacter jejuni 81-176 Lipooligosaccharide Affects Ganglioside Mimicry and Invasiveness In Vitro

The outer cores of the lipooligosaccharides (LOS) of many strains of Campylobacter jejuni mimic human gangliosides in structure. A population of cells of C. jejuni strain 81-176 produced a mixture of LOS cores which consisted primarily of structures mimicking GM(2) and GM(3) gangliosides, with minor amounts of structures mimicking GD(1b) and GD(2). Genetic analyses of genes involved in the biosynthesis of the outer core of C. jejuni 81-176 revealed the presence of a homopolymeric tract of G residues within a gene encoding CgtA, an N-acetylgalactosaminyltransferase. Variation in the number of G residues within cgtA affected the length of the open reading frame, and these changes in cgtA corresponded to a change in LOS structure from GM(2) to GM(3) ganglioside mimicry. Site-specific mutation of cgtA in 81-176 resulted in a major LOS core structure that lacked GalNAc and resembled GM(3) ganglioside. Compared to wild-type 81-176, the cgtA mutant showed a significant increase in invasion of INT407 cells. In comparison, a site-specific mutation of the neuC1 gene resulted in the loss of sialic acid in the LOS core and reduced resistance to normal human serum but had no affect on invasion of INT407 cells.     

Detection and Identification of Actinobacillus pleuropneumoniae Serotype 5 by Multiplex PCR

Serotyping of Actinobacillus pleuropneumoniae is based on detection of the serotype-specific capsular antigen. However, not all isolates can be serotyped, and some may cross-react with multiple serotyping reagents. To improve sensitivity and specificity of serotyping and for early detection, a multiplex PCR assay was developed for detection of A. pleuropneumoniae and identification of serotype 5 isolates. DNA sequences specific to the conserved export and serotype-specific biosynthesis regions of the capsular polysaccharide of A. pleuropneumoniae serotype 5 were used as primers to amplify 0.7- and 1.1-kb DNA fragments, respectively. The 0.7-kb fragment was amplified from all strains of A. pleuropneumoniae tested with the exception of serotype 4. The 0.7-kb fragment was not amplified from any heterologous species that are also common pathogens or commensals of swine. In contrast, the 1.1-kb fragment was amplified from all serotype 5 strains only. The assay was capable of amplifying DNA from less than 10² CFU. The A. pleuropneumoniae serotype 5 capsular DNA products were readily amplified from lung tissues obtained from infected swine, although the 1.1-kb product was not amplified from some tissues stored frozen for 6 years. The multiplex PCR assay enabled us to detect A. pleuropneumoniae rapidly and to distinguish serotype 5 strains from other serotypes. The use of primers specific to the biosynthesis regions of other A. pleuropneumoniae serotypes would expand the diagnostic and epidemiologic capabilities of this assay.     

Changes in EEG activity and hypothalamic temperature as indices for non-REM sleep to REM sleep transitions

A shift of physiological regulations from a homeostatic to a non-homeostatic modality characterizes the passage from non-NREM sleep (NREMS) to REM sleep (REMS). In the rat, an EEG index which allows the automatic scoring of transitions from NREMS to REMS has been proposed: the NREMS to REMS transition indicator value, NIV [J.H. Benington et al., Sleep 17 (1994) 28-36]. However, such transitions are not always followed by a REMS episode, but are often followed by an awakening. In the present study, the relationship between changes in EEG activity and hypothalamic temperature (Thy), taken as an index of autonomic activity, was studied within a window consisting of the 60s which precedes a state change from a consolidated NREMS episode. Furthermore, the probability that a transition would lead to REMS or wake was analysed. The results showed that, within this time window, both a modified NIV (NIV(60)) and the difference between Thy at the limits of the window (Thy(D)) were related to the probability of REMS onset. Both the relationship between the indices and the probability of REMS onset was sigmoid, the latter of which saturated at a probability level around 50-60%. The efficacy for the prediction of successful transitions from NREMS to REMS found using Thy(D) as an index supports the view that such a transition is a dynamic process where the physiological risk to enter REMS is weighted at a central level.     

Effect of synthetic lipopeptides formulated in liposomes on the maturation of human dendritic cells

Diacylated (e.g. MALP-2) and triacylated (Pam(3)Cys derivatives) lipopeptides, deriving from the N-terminal moiety of respectively mycoplasmal and E. coli lipoproteins, are powerful adjuvants recognized by Toll-like receptors (TLR) which have been used successfully to trigger cell activation and immune responses. To design liposome-based vaccination constructs in which Th and CTL epitopes are conjugated to synthetic lipopeptide analogues anchored into the bilayers of the vesicles, the peptide moieties of the lipopeptides were functionalized with thiol-reactive groups, such as maleimide (Mal) or bromoacetyl, incorporated into liposomes and reacted with thiol carrying peptide epitopes. Because dendritic cells (DCs) play a key role as antigen-presenting cells in immune responses, in the present study we have evaluated the impact of the functionalization of lipopeptide analogues Pam(2)CAG, Pam(3)CAG and Ol(3)GAG on the phenotypic maturation of human monocyte-derived DCs. The intrinsic cellular activities of the lipopeptide analogues incorporated into liposomes were monitored, in vitro, by measuring the up-regulation of the cell-surface markers CD80, CD83, CD86 and HLA-DR. We found that in some cases their functionalization with thiol-reactive groups led to a loss of activity. The stimulatory potency can be ranked in the following order: Pam(3)CAG>/=Pam(2)CAG-Mal-Th approximately Pam(2)CAG-Mal>Pam(3)CAG-Mal-Th (where Th is a HS-peptide) and no appreciable activity was detected for Pam(3)CAG-Mal, Ol(3)CAG-Mal and Ol(3)CAG-Mal-Th. Our findings indicate that subtle modifications in the peptide moiety of lipopeptides have a great impact on the immunomodulatory properties of these molecules. For the engineering of liposome/lipopeptide-based vaccines, the maleimide derivative of Pam(2)CAG appears to be the best candidate.     

Light and electron microscope studies on the Sarcocystis of Rattus fuscipes,an Australian rat

Summary Light and electron microscope studies on the Sarcocystis of Rattus fuscipes showed that sarcocysts of two types occurred in this rat. These types could be distinguished from each other on the morphology of their cyst walls, on the size and micromorphology of their zoites, as well as by the changes they induced in the host cell. On the basis of these differences, it was concluded that the two sarcocyst types belonged to distinct Sarcocystis species. The possible life histories of the infections occurring in the rats were considered.Abbreviations A Amylopectin - C Conoid - CF Connected Tissue Fibres - CP Capillary - CW Cyst Wall - DC Daughter Cell - DCA Daughter Cell Anlagen - E Erythrocyte - FE Fibrillar Elements - GA Golgi Adjunct - GO Golgi Apparatus - GS Ground Substance - HC Host Cell - HCN Host Cell Nucleus - I Invagination - M Metrocyte - ME Merozoite - MI Mitochondrion - MIH Host Cell Mitochondrion - MN Micronemes - N Nucleus - NE Nucleus of Endothelial Cell - NR Neck Region - NU Nucleolus - OG Osmiophilic Granules - P Papilla - PI Pellicular Invagination - PR Projections - PW Primary Wall - RH Rhoptries - S Septum - SP Sarcoplasm - TS Thread-like Structures - Z Zoites Visiting DAAD Research Fellow from the Department of Parasitology, University of Queensland, AustraliaSupported by the Deutsche Forschungsgemeinschaft     

An inexpensive restraint for small animals

An animal restraint is described that can be built in the laboratory from a piece of Plexiglas and a few inches of Velcro.