Dichloroacetate reverses the hypoxic adaptation to bevacizumab and enhances its antitumor effects in mouse xenografts.

Inhibition of vascular endothelial growth factor increases response rates to chemotherapy and progression-free survival in glioblastoma. However, resistance invariably occurs, prompting the urgent need for identification of synergizing agents. One possible strategy is to understand tumor adaptation to microenvironmental changes induced by antiangiogenic drugs and test agents that exploit this process. We used an in vivo glioblastoma-derived xenograft model of tumor escape in presence of continuous treatment with bevacizumab. U87-MG or U118-MG cells were subcutaneously implanted into either BALB/c SCID or athymic nude mice. Bevacizumab was given by intraperitoneal injection every 3 days (2.5 mg/kg/dose) and/or dichloroacetate (DCA) was administered by oral gavage twice daily (50 mg/kg/dose) when tumor volumes reached 0.3 cm(3) and continued until tumors reached approximately 1.5-2.0 cm(3). Microarray analysis of resistant U87 tumors revealed coordinated changes at the level of metabolic genes, in particular, a widening gap between glycolysis and mitochondrial respiration. There was a highly significant difference between U87-MG-implanted athymic nude mice 1 week after drug treatment. By 2 weeks of treatment, bevacizumab and DCA together dramatically blocked tumor growth compared to either drug alone. Similar results were seen in athymic nude mice implanted with U118-MG cells. We demonstrate for the first time that reversal of the bevacizumab-induced shift in metabolism using DCA is detrimental to neoplastic growth in vivo. As DCA is viewed as a promising agent targeting tumor metabolism, our data establish the timely proof of concept that combining it with antiangiogenic therapy represents a potent antineoplastic strategy

A clinical and experimental study of the autonomous nervous system in the heart

Abstract Background: The introduction of the arterial switch operation (ASO) made it the procedure of choice for surgical correction of transposition of the great arteries. A majority of the sympathetic nerves innervate the heart alongside the great vessels; these are therefore likely to be damaged during the surgical procedure; imposing new challenges and questions that need to be addressed. The main aim for this thesis was to assess the long-term cardiac consequences on the autonomic nervous system after surgery (paper I and II) and to create an animal model allowing for cardiac physiological studies (paper III and IV). Methods: Long-term follow-up in adolescents who had undergone ASO as neonates (n=17, 1 female, mean fractional shorting 32±5%) was performed. This included sympathetic nervous system function assessed through infusion of tritiated Norepinephrine ([3H]NE) during heart catheterisation (n=8)(controls n=15) and blood samples analysed with high performance liquid chromatography. Samples were obtained both before and after adenosine stimulation as a response to sympathetic excitation. 24-hour heart rate variability (HRV)(n=15 in both groups) was measured both during the day and night using different algorithms. Baroreflex sensitivity and QT variability index (QTVI) (n=17 in both groups) were measured in awake patients. An animal model was developed using complex open heart surgery during cardiopulmonary bypass to mimick the arterial switch operation in piglets 8 weeks of age. The piglets surviving at least 5 to 6 weeks post-operation had follow-up of physiological response to catecholamines and were studied in vivo and in vitro using the Langendorff perfusion system. Results: In both groups the specific activity of [3H]NE decreased from the artery to the coronary sinus, but to a lesser extent in the ASO group. The extraction fraction in the ASO group was 56±10% compared to 82±9% in the healthy subjects (p<0.001). The arterial to coronary sinus plasma concentration of [3H] dihydroxyphenylglycol (DHPG) was significantly increased in the healthy group (70%, p=<0.0001) but was not so in the ASO group (8%, p=0.5). The difference of endogenous DHPG increase from the arterial to the coronary sinus was significantly smaller in the ASO group (p=0.008). After adenosine infusion, the total body NE spillover increased in the ASO group (p=0.002), reflecting major sympathetic activation. [3H]DHPG step-up from the artery to the coronary sinus increased 4-fold following adenosine. HRV frequency-domain at night-time, when cardio-parasympathetic drive is likely to be most pronounced, showed a significant decrease of normalized high frequency in the ASO group (52±20) compared to healthy subjects (68±15)(p=0.018). Time-domain showed no statistical difference between the two groups, neither during day-time nor night-time. Baroreflex sensitivity and QTVI did not show significant differences between groups. The animal model resulted in 14 out of 19 piglets surviving the mimicked ASO. Piglets operated with mimicked ASO had a significantly higher basal heart rate both in vivo (p=0.042) and in vitro (p=0.0056). Conclusion: A disturbed but functioning sympathetic cardiac innervation was found in the ASO patients at long-term follow-up. The vagal tone seemed normal in terms of BRS, however, frequencydomain analysis showed a decreased parasympathetic tone at night time in the ASO group. The surgical challenges due to translocation of the coronary arteries and the consequences of an injured autonomic nervous system impose risks of decreased myocardial perfusion and arrhythmias. Thus, the present data suggest that these patients ought to have follow-up that includes autonomic nervous system assessment

Amino-terminal anchored surface display in insect cells and budded baculovirus using the amino-terminal end of neuraminidase.

Methods currently used for surface display on insect cells and budded baculovirus, all utilize the sequences from class I transmembrane proteins. This gives rise to some problems when handling unknown genes or cDNAs encoding full-length proteins. First, the stop codon from the cloned gene will be located upstream of the sequence for the transmembrane region. Second, the chance of getting the sequences encoding the signal peptide and the transmembrane region in frame with the cloned gene is small. To minimize these problems, we here present a method by which cDNAs or genes of interest can be cloned and fused to the codons for the signal peptide and transmembrane region of neuraminidase (NA), a class II transmembrane protein of the influenza virus. By placing both the signal peptide and transmembrane region at the amino-terminal, potential problems regarding stop codons are eliminated and errors in frame-shift minimized. To obtain proof of principle, the gene encoding enhanced green fluorescent protein, EGFP, was subcloned into a shuttle vector downstream of the neuraminidase sequence and the fusion product was then transferred to a baculovirus vector and transfected into insect cells (Sf9). Using this method, EGFP was found to be expressed on the surface of both infected cells and budded virus in an accessible manner

Expression of the AMBP gene transcript and its two protein products, alpha(1)-microglobulin and bikunin, in mouse embryogenesis

The expression pattern of the alpha(1)-microglobulin/bikunin precursor (AMBP) gene, and its two protein products were studied in mouse embryos of 8.5-15.5 days of embryonic development by in situ hybridization and immunohistochemistry. AMBP mRNA is strongly transcribed in liver parenchyma, pancreas, and intestine epithelium. Sites of weaker expression are the vessels of the umbilical cord, the developing vertebral bodies, and kidney. The alpha(1)-microglobulin and bikunin proteins are accordingly present in developing hepatocytes, pancreas, kidney, and gut. However, additional sites of protein distribution were found that do not correlate to mRNA localization: alpha(1)-microglobulin was found in myocytes and bikunin in cardiac muscle, nervous system microvasculature, and connective tissue. Both proteins were found in brain mesenchyme and meninges. Thus, a restricted expression of the AMBP mRNA in a few organs contrasts to a widespread and unique distribution of each of the two proteins

Expression of the AMBP gene transcript and its two protein products, α1-microglobulin and bikunin, in mouse embryogenesis

The expression pattern of the α1-microglobulin/bikunin precursor (AMBP) gene, and its two protein products were studied in mouse embryos of 8.5–15.5 days of embryonic development by in situ hybridization and immunohistochemistry. AMBP mRNA is strongly transcribed in liver parenchyma, pancreas, and intestine epithelium. Sites of weaker expression are the vessels of the umbilical cord, the developing vertebral bodies, and kidney. The α1-microglobulin and bikunin proteins are accordingly present in developing hepatocytes, pancreas, kidney, and gut. However, additional sites of protein distribution were found that do not correlate to mRNA localization: α1-microglobulin was found in myocytes and bikunin in cardiac muscle, nervous system microvasculature, and connective tissue. Both proteins were found in brain mesenchyme and meninges. Thus, a restricted expression of the AMBP mRNA in a few organs contrasts to a widespread and unique distribution of each of the two proteins.This work was supported by DGICYT (PM1998-0056) and European Commission grants to S.M. (QLG3-CT-2000-01556), the Swedish Research Council (project no. 07144), Foundations of Magnus Bergvall, Alfred Österlund and Greta and Johan Kock, King Gustav V:s 80-year foundation and the Royal Physiographic Society in Lund. D.S. was a visiting researcher supported by the MEC (Spain).Peer reviewe

Expression of a functional proteinase inhibitor capable of accepting xylose: bikunin

Bikunin is a Kunitz-type proteinase inhibitor, which is cross-linked to heavy chains via a chondroitin sulfate chain, forming inter-alpha-inhibitor and related molecules. Rat bikunin was produced by baculovirus-infected insect cells. The protein could be purified with a total yield of 20 mg/liter medium. Unlike naturally occuring bikunin the recombinant protein had no galactosaminoglycan chain. Endoglycosidase digestion also suggested that the recombinant form lacked N-linked oligosaccharides. Bikunin is translated as a part of a precursor, alpha1-microglobulin/bikunin, but the functional significance of the cotranslation is unknown. Our results indicate that the proteinase inhibitory function of bikunin is not regulated by the alpha1-microglobulin-part of the alpha1-microglobulin/bikunin precursor since recombinant bikunin had the same trypsin inhibitory activity as the recombinant precursor. Both free bikunin and the precursor were also functional as a substrate in an in vitro xylosylation system. This demonstrates that the alpha1-microglobulin-part is not necessary for the first step of galactosaminoglycan assembly

Increase of bikunin and alpha1-microglobulin concentrations in urine of rats during pregnancy is due to decreased tubular reabsorption

Bikunin and alpha1-microglobulin are two plasma proteins of about 25 kDa which are made in the liver from a common precursor. The concentration of bikunin in human urine has been shown to increase several fold during various conditions of stress. The mechanism behind this increase is unknown. We have studied pregnant rats and found that the bikunin and alpha1-microglobulin levels in their urine increased 3-fold towards the end of the pregnancy, whereas those of albumin and orosomucoid did not. There were no significant changes in either the bikunin/alpha1-microglobulin mRNA level or the concentrations of the two proteins in serum. These findings imply that the synthesis and the clearance rates of bikunin and alpha1-microglobulin are normal during pregnancy but that the tubular reabsorption of these proteins is decreased

alpha 1-Microglobulin destroys the proteinase inhibitory activity of alpha 1-inhibitor-3 by complex formation

The immunoregulatory plasma protein alpha 1-microglobulin (alpha 1-m) and the proteinase inhibitor alpha 1-inhibitor-3 (alpha 1I3) form a complex in rat plasma. In the present work, it was demonstrated that the alpha 1I3.alpha 1-m complex has no inhibitory activity, the bait region was not cleaved by low amounts of proteinases, and it was unable to covalently incorporate proteinases. The results also indicated that the thiolester bond of the alpha 1I3.alpha 1-m complex was broken. The alpha 1I3.alpha 1-m complex was cleared from the circulation much faster than native alpha 1I3, with a half-life of approximately 7 min. Structurally, however, the alpha 1I3.alpha 1-m complex was similar to native alpha 1I3 rather than alpha 1I3 cleaved by proteinases. It is speculated that the role of alpha 1-m is to destroy the function of alpha 1I3 by blocking the bait region and breaking the thiolester and causing its physical elimination by rapid clearing from the blood circulation. It is also possible that the formation of complexes between alpha 1-m and alpha 1I3 may serve as a mean to regulate the function of alpha 1-m since its complex with alpha 1I3 is taken up rapidly by cellular receptors for alpha-macroglobulins