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Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal neoplasms of the gastrointestinal tract and are often associated with KIT or PDGFRA gene mutations. GIST cells might arise from the interstitial cells of Cajal (ICCs) or from a mesenchymal precursor that is common to ICCs and smooth muscle cells (SMCs). Here, we analyzed the mRNA and protein expression of RNA-Binding Protein with Multiple Splicing-2 (RBPMS2), an early marker of gastrointestinal SMC precursors, in human GISTs (n=23) by in situ hybridization, quantitative RT-PCR analysis and immunohistochemistry. The mean RBPMS2 mRNA level in GISTs was 42-fold higher than in control gastrointestinal samples (p<0.001). RBPMS2 expression was not correlated with KIT and PDGFRA expression levels, but was higher in GISTs harboring KIT mutations than in tumors with wild type KIT and PDGFRA or in GISTs with PDGFRA mutations that were characterized by the lowest RBPMS2 levels. Moreover, RBPMS2 levels were 64-fold higher in GIST samples with high risk of aggressive behavior than in adult control gastrointestinal samples and 6.2-fold higher in high risk than in low risk GIST specimens. RBPMS2 protein level was high in 87% of the studied GISTs independently of their histological classification. Finally, by inhibiting the KIT signaling pathway in GIST882 cells, we show that RBPMS2 expression is independent of KIT activation. In conclusion, RBPMS2 is up-regulated in GISTs compared to normal adult gastrointestinal tissues, indicating that RBPMS2 might represent a new diagnostic marker for GISTs and a potential target for cancer therapy., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The longitudinal prestrain of arteries facilitates their physiological function. Remodeling, adaptation and aging result in an age-dependent magnitude of the pretension. Although the phenomenon is known, detailed statistics, especially for human arteries, are lacking. This study was designed to propose the regression model capable of estimating the prestrain of the human abdominal aorta. The length of the abdominal aorta before, l, and after excision from the body, L, the diameter, heart weight, thickness of left ventricle and degree of atherosclerosis were collected in autopsies of 156 male cadavers of known age. Longitudinal prestrain was quantified by means of the stretch ratio λ=l/L. Statistical analysis revealed significant dependence between age, prestrain, diameter and atherosclerosis, which were best fitted to the power law equation. Longitudinal prestretch reduced with age significantly; λmean=1.30±0.07 for age<30 (n=29), whereas λmean=1.06±0.03 for age>59 (n=31) with p-value<0.0001. Raw data gave linear correlation coefficients as follows: λ-age (R=-0.842); l-age (R=0.023); L-age (R=0.476); (l-L)-age (R=-0.811). It was concluded that longitudinal prestrain decreases nonlinearly with age and both age and diameter are suitable predictors of the prestrain. Data suggests that unloaded length elongates with age in contrast to the elastic retraction., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Unlabelled: Bilateral prophylactic mastectomy without reconstruction is not accepted by the majority of patients. Successful reconstruction is therefore a mandatory condition for prophylactic mastectomy. Of the many options for autologous breast reconstruction, the deep inferior epigastric perforator (DIEP) flap best meets requirements for bilateral reconstruction in selected patients. The goal of this study is to verify the feasibility of the procedure in our conditions and to find out how it is accepted by patients. We present 55 consecutive patients who were scheduled for bilateral DIEP flap reconstruction during a 4-year period. We reviewed medical charts, performed clinical assessments and processed anonymous questionnaires. There were 77 immediate and 33 delayed breast reconstructions. There was 100% flap survival and no microanastomoses revisions. In 11 patients (10%) the surgeon preferred to convert the DIEP into a mini transverse rectus abdominis muscle (miniTRAM) flap in order to provide adequate blood supply., Complications: revision for haematoma under the flap in four patients (7.2%), excessive blood loss in four patients (7.2%) and partial mastectomy skin flap necrosis in 10 immediate breast reconstructions (12.9%). Patients' evaluation of the aesthetic result was good or excellent in 96.2% of cases. In 33.9% of patients the postoperative quality of life was considered unchanged and 50.9% of them it even improved. The DIEP flap is recommended for bilateral breast reconstruction. Occasional conversion into a miniTRAM flap can increase the total flap survival rate. Bilateral prophylactic mastectomy and DIEP flap reconstruction are very well accepted by patients.

An alternative pathway to glucuronic acid-containing di- and trisaccharide thioglycoside building blocks, suitable for the synthesis of Cryptococcus neoformans capsular polysaccharide structures, has been developed. As opposed to our earlier synthesis, this approach features the introduction of the glucuronic acid motif at the di- and trisaccharide level through oxidation of a glucose residue. This approach circumvents problems encountered in glycosylations with glucuronic acid donors and benzylation of glucuronic acid-containing derivatives. Selective protection of primary alcohols was obtained at the di- and trisaccharide stage using TBDMS or trityl protecting groups, respectively. After benzylation of the secondary hydroxyl groups and subsequent removal of the TBDMS or trityl group, oxidation of the free primary alcohols to carboxylic acids was performed in high yield using the TEMPO-BAIB reagent mixture, which does not tend to oxidize thioglycosides. The new approach requires a number of extra steps, but has proven to be more reliable and easily reproducible.

It has previously been shown that human granulocyte-macrophage colony-stimulating factor (GM-CSF) can be fused to a truncated diphtheria toxin (DT) to produce a recombinant fusion toxin that kills GM-CSF receptor-bearing cells. We now report that DT388-GM-CSF induces apoptosis and inhibition of colony formation in semisolid medium in receptor positive cells, and that the induction of apoptosis correlates with GM-CSF-receptor occupancy at low ligand concentrations. Also, the induction of apoptosis correlates with the inhibition of protein synthesis and is inversely related to the amount of intracellular antiapoptotic proteins (Bcl2 and Bc1XL). Nine myeloid leukemia cells lines and four nonmyeloid leukemia cell lines were incubated with 0.7 nmol/L of 125I-GM-CSF in the presence or absence of excess cold GM-CSF and bound label measured. High affinity receptor numbers varied from 0 to 291 molecules per cell. Cells were incubated with varying concentrations of recombinant fusion toxin for 48 hours and incorporation of 3H-leucine (protein synthesis), segmentation of nuclei after DAPI staining (apoptosis), and colony formation in 0.2% agarose (clonogenicity) were measured. DT388-GM-CSF at 4 x 10(-9) mol/L inhibited colony formation 1.5 to 3.0 logs for receptor positive cell lines. Protein synthesis and apoptosis IC50s varied among cell lines from greater than 4 x 10(-9) mol/L to 3 x 10(-13) mol/L. GM-CSF-receptor occupancy at 0.7 nmol/L GM-CSF-ligand concentration correlated with the protein synthesis IC50. Similarly, the protein synthesis inhibition and apoptosis induction correlated well, except in cells overexpressing Bcl2 and BclXL, in which 25- to 150-fold inhibition of apoptosis was observed. We conclude that DT388-GM-CSF can kill acute myeloid leukemia blasts but that apoptotic sensitivities will depend on the presence of at least 100 high affinity GM-CSF receptors/cell and the absence of overexpressed antiapoptotic proteins.

Ricin toxin with genetic or chemical modification of lectin sites has been previously reported to show markedly reduced cytotoxicity to cells following uptake by several receptors including the mannose receptor. Investigators have hypothesized that an intracellular galactoside-binding function was required for optimal intracellular targeting of ricin for these receptors. We have prepared insect-derived mutant ricin toxin B chain (RTB) with modifications of three lectin side domains (1 alpha, 1 beta, and 2 gamma) yielding a 1000-fold reduced galactoside avidity. After reassociation with plant RTA, the recombinant heterodimer and plant ricin were tested for cytotoxicity on mammalian cells expressing (mouse peritoneal macrophages, J774E cells, and MMR61 cells) or not expressing (KB cells) the D-mannose receptor. Receptor expression was confirmed by immunofluorescence microscopy. Lactose was included in the media to block cell-surface galactoside binding, and mannan was added as a control in each experiment to confirm mannose receptor-specific targeting. Plant ricin A chain (RTA) and E. coli-derived RTA were also tested for cytotoxicity on J774E and KB cells. Both wild-type and lectin-deficient ricin displayed mannose-receptor mediated cell cytotoxicity. This is the first report of a genetically modified ricin showing that RTB intracellular galactose binding activity is not required for ricin cytotoxicity. Sensitivity of mannose-receptor bearing cells, but not control cells, to mannosylated RTA, but not unglycosylated RTA, confirmed these observations. These results imply fusion toxins employing ricin can be prepared with maximal reductions in normal tissue binding.