The tumour marker CA 195 in colorectal and pancreatic cancer.

The aim of this study was to measure the serum level of the tumour markers CA 195 and CEA in patients with either colorectal or pancreatic cancer both before and at serial intervals after operation. CA 195 and CEA were measured in 199 patients with colorectal cancer and 52 patients with pancreatic cancer. The median concentrations of CA 195 were 3.0 u/ml (interquartile range 3.0-4.5 u/ml) in patients with a Dukes' stage A lesion, 5.8 u/ml (3.0-18.2 u/ml) in patients with a Dukes' stage B lesion, 6.1 u/ml (3.0-24.7 u/ml) in patients with a Dukes' stage C and 23.8 u/ml (11.1-409.0 u/ml) in patients with metastatic disease (normal range 0-7 u/ml). The median levels of CEA were 2.6 ng/ml (1.7-3.3 ng/ml) for Dukes' stage A, 3.3 ng/ml (1.7-7.2 ng/ml) for Dukes' stage B, 3.7 ng/ml (2.2-7.9 ng/ml) for Dukes' stage C and 34.5 ng/ml (13.3-289.4 ng/ml) for metastatic disease. A rising level of CA 195 or CEA after operation suggested recurrence of the tumour. In none of these patients was the recurrence operable. In patients with pancreatic adenocarcinoma, the level of CA 195 was significantly higher in patients with metastatic disease but it did not discriminate between resectable and unresectable disease. The duration of survival correlated with the initial level of CA 195 (Rs = -0.66, p less than 0.001).     

Bone Marrow SSEA1+ Cells Support the Myocardium in Cardiac Pressure Overload

Rationale

Stage specific embryonic antigen 1+ (SSEA1+) cells have been described as the most primitive mesenchymal progenitor cell in the bone marrow. Cardiac injury mobilizes SSEA1+ cells into the peripheral blood but their in vivo function has not been characterized.

Objective

We generated animals with chimeric bone marrow to determine the fate and function of bone marrow SSEA1+ cells in response to acute cardiac pressure overload.

Methods and Results

Lethally irradiated mice were transplanted with normal bone marrow where the wild-type SSEA1+ cells were replaced with green fluorescent protein (GFP) SSEA1+ cells. Cardiac injury was induced by trans-aortic constriction (TAC). We identified significant GFP+ cell engraftment into the myocardium after TAC. Bone marrow GFP+ SSEA1 derived cells acquired markers of endothelial lineage, but did not express markers of c-kit+ cardiac progenitor cells. The function of bone marrow SSEA1+ cells after TAC was determined by transplanting lethally irradiated mice with bone marrow depleted of SSEA1+ cells (SSEA1-BM). The cardiac function of SSEA1-BM mice declined at a greater rate after TAC compared to their complete bone marrow transplant counterparts and was associated with decreased bone marrow cell engraftment and greater vessel rarefication in the myocardium.

Conclusions

These results provide evidence for the recruitment of endogenous bone marrow SSEA1+ cells to the myocardium after TAC. We demonstrate that, in vivo, bone marrow SSEA1+ cells have the differentiation potential to acquire endothelial lineage markers. We also show that bone marrow SSEA1+ deficiency is associated with a reduced compensatory capacity to cardiac pressure overload, suggesting their importance in cardiac homeostasis. These data demonstrate that bone marrow SSEA1+ cells are critical for sustaining vascular density and cardiac repair to pressure overload.     

R-Spondin potentiates Wnt/β-catenin signaling through orphan receptors LGR4 and LGR5

The Wnt/β-catenin signaling pathbway controls many important biological processes. R-Spondin (RSPO) proteins are a family of secreted molecules that strongly potentiate Wnt/β-catenin signaling, however, the molecular mechanism of RSPO action is not yet fully understood. We performed an unbiased siRNA screen to identify molecules specifically required for RSPO, but not Wnt, induced β-catenin signaling. From this screen, we identified LGR4, then an orphan G protein-coupled receptor (GPCR), as the cognate receptor of RSPO. Depletion of LGR4 completely abolished RSPO-induced β-catenin signaling. The loss of LGR4 could be compensated by overexpression of LGR5, suggesting that LGR4 and LGR5 are functional homologs. We further demonstrated that RSPO binds to the extracellular domain of LGR4 and LGR5, and that overexpression of LGR4 strongly sensitizes cells to RSPO-activated β-catenin signaling. Supporting the physiological significance of RSPO-LGR4 interaction, Lgr4-/- crypt cultures failed to grow in RSPO-containing intestinal crypt culture medium. No coupling between LGR4 and heterotrimeric G proteins could be detected in RSPO-treated cells, suggesting that LGR4 mediates RSPO signaling through a novel mechanism. Identification of LGR4 and its relative LGR5, an adult stem cell marker, as the receptors of RSPO will facilitate the further characterization of these receptor/ligand pairs in regenerative medicine applications.     

Cardiac pressure overload initiates a systemic stem cell response

Background aims. Acute cardiac injury results in the activation and recruitment of resident and non-cardiac stem cells. In this study we sought to define the pattern of peripheral stem cells and resident cardiac stem cell (CSC) activation induced acutely by cardiac pressure overload (PO). Methods. PO was induced in mice by transaortic constriction (TAC). CSC, endothelial progenitor cells (EPC), hematopoietic stem cells (HSC) and stage-specific embryonic antigen (SSEA)-1(+) cells were profiled in the heart, spleen and bone marrow after TAC by flow cytometry. Results. The combination of a systemic and local stem cell response resulted in increases in SSEA-1 (+) cells and EPC in the heart 7 and 14 days post-TAC, respectively. Locally, modest SSEA-1(+) proliferation at 4 days preceded the elevated myocardial stem cell number. We observed no significant proliferation of EPC and CSC in the heart. The systemic stem cell response was characterized by a biphasic loss of splenic SSEA-1(+) cells at 2 and 7 days post-TAC and loss of bone marrow and spleen EPC at 4 and 7 days, respectively. Spleen size changed dynamically after TAC. A negligible response of HSC to TAC was observed. Significant EPC and SSEA-1(+) proliferation in the bone marrow and spleen occurred only after their local levels were decreased. Conclusions. Our results demonstrate that an orchestrated systemic stem cell response (EPC and SSEA-1 (+) ) takes place in response to TAC. The increase of SSEA-1(+) cells and EPC in the heart in response to pressure is likely to be because of a combination of local proliferation and stem cell recruitment.     

Novel DNA Sequences from Natural Strains of the Nitrogen-Fixing Symbiotic Bacterium Sinorhizobium meliloti

Variation in genome size and content is common among bacterial strains. Identifying these naturally occurring differences can accelerate our understanding of bacterial attributes, such as ecological specialization and genome evolution. In this study, we used representational difference analysis to identify potentially novel sequences not present in the sequenced laboratory strain Rm1021 of the nitrogen-fixing bacterium Sinorhizobium meliloti. Using strain Rm1021 as the driver and the type strain of S. meliloti ATCC 9930, which has a genome size ~370 kilobases bigger than that of strain Rm1021, as the tester, we identified several groups of sequences in the ATCC 9930 genome not present in strain Rm1021. Among the 85 novel DNA fragments examined, 55 showed no obvious homologs anywhere in the public databases. Of the remaining 30 sequences, 24 contained homologs to the Rm1021 genome as well as unique segments not found in Rm1021, 3 contained sequences homologous to those published for another S. meliloti strain but absent in Rm1021, 2 contained sequences homologous to other symbiotic nitrogen-fixing bacteria (Rhizobium etli and Bradyrhizobium japonicum), and 1 contained a sequence homologous to a gene in a non-nitrogen-fixing species, Pseudomonas sp. NK87. Using PCR, we assayed the distribution of 12 of the above 85 novel sequences in a collection of 59 natural S. meliloti strains. The distribution varied widely among the 12 novel DNA fragments, from 1.7% to 72.9%. No apparent correlation was found between the distribution of these novel DNA sequences and their genotypes obtained using multilocus enzyme electrophoresis. Our results suggest potentially high rates of gene gain and loss in S. meliloti genomes.     

Human gamma-aminobutyric acid A receptor alpha2 gene moderates the acute effects of alcohol and brain mRNA expression

γ-aminobutyric acid A (GABA_A) receptors moderate several of the behavioral effects of alcohol. In fact, recent studies have shown an association between the gene for the α2-subunit of the GABA_A receptor ( GABRA2 ) and alcoholism. In the present study, we examined the functional relevance of the GABRA2 gene in alcohol dependence by assessing brain GABRA2 mRNA and GABA_Aα2-subunit protein levels in post-mortem prefrontal cortical tissue collected from control and alcohol-dependent individuals. In addition, using an endophenotype approach, we tested whether the GABRA2 gene moderates sensitivity to the acute effects of alcohol in two independent samples from distinct human alcohol challenge studies. Results indicated that GABRA2 mRNA levels significantly differed by GABRA2 genotype. GABRA2 single nucleotide polymorphisms (rs573400, rs279871 and rs279858) were significantly associated with sensitivity to the acute effects of alcohol. Specifically, there was a significant main effect of GABRA2  × breath alcohol concentration on several measures of subjective responses to alcohol, including the hedonic value of alcohol. Importantly, reanalysis of a previous intravenous alcohol administration study confirmed the results of the oral alcohol challenge study. In summary, these results extend previous findings and provide new insights into the putative biobehavioral mechanisms that may moderate the association between the GABRA2 gene, sensitivity to the acute effects of alcohol and ultimately alcohol dependence.     

The immunomodulatory effect of levamisole is influenced by postoperative changes and type of lymphocyte stimulant

The results of both laboratory and clinical research into the immunomodulatory activity of levamisole have shown a considerable degree of inconsistency and sometimes contradiction. This is probably a reflection of the lack of understanding of the mechanism(s) of action of levamisole and it is therefore necessary to base conclusions about its immunomodulatory efficacy in the treatment of disease on experimental assays that take into consideration the in vivo conditions. This investigation was designed to compare the immunomodulatory activity of levamisole under clinically achievable and non-achievable conditions as judged by changes in the perioperative proliferative response of lymphocytes from 30 patients with colorectal cancer. The results obtained showed that proliferation in antigen (purified protein derivative, PPD)-stimulated, but not phytohaemagglutinin(PHA)- or staphylococcal-enterotoxin-B(SEB)-stimulated, lymphocyte cultures was consistently and significantly augmented by levamisole in concentrations of 25 ng–25g/ml. High concentrations of levamisole (25 g/ml and 100 g/ml) were inhibitory to PHA- and SEB-stimulated, but not PPD-stimulated, lymphocyte cultures, especially in the postoperative period. Of particular interest was the observation that, although levamisole temporarily lost its stimulatory activity in the post-operative period (third postoperative day), it did enhance antigen-stimulated lymphocytes at the time of the nadir of the postoperative suppression of lymphocyte proliferation (first postoperative day). Clinically achievable concentrations of levamisole are therefore effective both before and after operation in enhancing the response of lymphocytes to antigens.     

Analysis of a 1600-Kilobase Rhizobium Meliloti Megaplasmid Using Defined Deletions Generated in Vivo

A series of 120-600 kilobase deletions with defined endpoints were made in the 1600-kilobase Rhizobium meliloti megaplasmid pRmeSU47b, by homologous recombination between the IS50 elements of transposon insertions. Utilizing IS 50-mediated homologous recombination we also made defined reductions in deletion size and combined adjacent deletions. Deletion structure was confirmed by phage transduction and Southern hybridization analysis. Collectively these deletions span 1400 kilobases of pRmeSU47b, indicating that the majority of the plasmid is not essential for cell viability. This was further confirmed by the construction of a strain SU47 derivative which carries only 450 kilobases of the pRmeSU47b megaplasmid. Examination of the deletion mutants for phenotype revealed novel loci required for dulcitol, melibiose, raffinose, beta-hydroxybutyrate, acetoacetate, protocatechuate and quinate utilization. Previously unidentified loci required for effective root nodule development and exopolysaccharide synthesis were also found. Various deletion mutants were deficient in dicarboxylate transport, lactose utilization, and thiamine and exopolysaccharide biosynthesis, as predicted from earlier studies of this megaplasmid.