Carbohydrate scaffolds as glycosyltransferase inhibitors with in vivo antibacterial activity.

The rapid rise of multi-drug-resistant bacteria is a global healthcare crisis, and new antibiotics are urgently required, especially those with modes of action that have low-resistance potential. One promising lead is the liposaccharide antibiotic moenomycin that inhibits bacterial glycosyltransferases, which are essential for peptidoglycan polymerization, while displaying a low rate of resistance. Unfortunately, the lipophilicity of moenomycin leads to unfavourable pharmacokinetic properties that render it unsuitable for systemic administration. In this study, we show that using moenomycin and other glycosyltransferase inhibitors as templates, we were able to synthesize compound libraries based on novel pyranose scaffold chemistry, with moenomycin-like activity, but with improved drug-like properties. The novel compounds exhibit in vitro inhibition comparable to moenomycin, with low toxicity and good efficacy in several in vivo models of infection. This approach based on non-planar carbohydrate scaffolds provides a new opportunity to develop new antibiotics with low propensity for resistance induction.

Synthesis of multiantennary complex type N-glycans by use of modular building blocks.

A modular set of oligosaccharide building blocks was developed for the synthesis of multiantennary N-glycans of the complex type, which are commonly found on glycoproteins. The donor building blocks were laid out for the elongation of a core trisaccharide acceptor (beta-mannosyl chitobiose) conveniently protected with a single benzylidene moiety at the beta-mannoside. Through two consecutive regio- and stereoselective couplings the donors gave N-glycans with three to five antennae in high yields. Due to the consistent protection group pattern of the donors the deprotection of the final products can be performed by using a general reaction sequence.

Reduction of ischemia reperfusion injury after liver resection and hepatic inflow occlusion by alpha-lipoic acid in humans.

AIM: To evaluate the protective effects of preconditioning by alpha-lipoic acid (LA) in patients undergoing hepatic resection under inflow occlusion of the liver. METHODS: Twenty-four patients undergoing liver resection for various reasons either received 600 mg LA or NaCl 15 min before transection performed under inflow occlusion of the liver. Blood samples and liver wedge biopsy samples were obtained after opening of the abdomen immediately after inflow occlusion of the liver, and 30 min after the end of inflow occlusion of the liver. RESULTS: Serum levels of aspartate transferase and alanine transferase were reduced at all time points in patients who received LA in comparison to those who received NaCL. This was accompanied by reduced histomorphological features of oncosis. We observed TUNEL-positive hepatocytes in the livers of the untreated patients, especially after 30 min of ischemia. LA attenuated this increase of TUNEL-positive hepatocytes. Under preconditioning with LA, ATP content was significantly enhanced after 30 min of ischemia and after 30 min of reperfusion. CONCLUSION: This is the first report on the potential for LA reducing ischemia/reperfusion injury (IRI) of the liver in humans who were undergoing liver surgery. Beside its simple and rapid application, side effects did not occur. LA might therefore represent a new strategy against hepatic IRI in humans.

Strong expression of chemokine receptor CXCR4 by pancreatic cancer correlates with advanced disease.

Certain chemokines have been proposed to distinctly contribute to tumor growth, dissemination and local immune escape. Expression of the chemokine receptor CXCR4 has been linked to tumor progression in diverse tumor entities. The aim of this study was to evaluate if the expression of CXCR4 influences progression of human pancreatic cancer. CXCR4 expression of pancreatic cancer was retrospectively assessed by immunohistochemistry in 103 patients with pancreatic cancer. Intensity of CXCR4 expression was correlated with both tumor and patient characteristics. Human pancreatic cancer revealed variable intensities of CXCR4 expression. Strong CXCR4 expression was significantly associated with advanced UICC stages (P=0.03) and revealed a trend for hematogenous metastasis (P=0.09) and progressed local tumor stages (P=0.15). In summary, strong expression of CXCR4 was significantly associated with advanced pancreatic cancer.

Liver resection or cryotherapy for colorectal liver metastases: a prospective case control study.

BACKGROUND AND AIMS: While there is promising survival data for cryosurgery of colorectal liver metastases, local recurrence following cryoablation remains a problem. We aimed to compare morbidity and mortality, as well as the recurrence pattern and survival after liver resection and cryotherapy (alone or in combination with resection) for liver metastases. PATIENTS AND METHODS: Between 1996 and 2002, 168 patients underwent liver resection alone and 55 patients had cryotherapy (25 in combination with liver resection) for colorectal liver metastases. The patient, tumour and operative details were recorded prospectively and the two patient groups were compared regarding morbidity, survival and recurrence. RESULTS: More patients had a prior liver resection, liver metastases were smaller and less frequently synchronous, morbidity was significantly lower and hepatic recurrence was significantly more frequent in the cryotherapy group. Five-year survival rates following resection and cryotherapy were comparable (23 and 26% respectively), while overall and hepatic recurrence-free survival was inferior following cryotherapy. CONCLUSION: Cryotherapy is a valuable treatment option for some patients with non-resectable colorectal liver metastases. While survival is comparable to that after resection, higher hepatic recurrence rates following cryotherapy should caution against the use of cryotherapy for resectable disease until the results of randomized controlled trials are available.

Determination of modulation of ligand properties of synthetic complex-type biantennary N-glycans by introduction of bisecting GlcNAc in silico, in vitro and in vivo.

We have investigated the consequences of introducing a bisecting GlcNAc moiety into biantennary N-glycans. Computational analysis of glycan conformation with prolonged simulation periods in vacuo and in a solvent box revealed two main effects: backfolding of the alpha1-6 arm and stacking of the bisecting GlcNAc and the neighboring Man/GlcNAc residues of both antennae. Chemoenzymatic synthesis produced the bisecting biantennary decasaccharide N-glycan and its alpha2-3(6)-sialylated variants. They were conjugated to BSA to probe the ligand properties of N-glycans with bisecting GlcNAc. To assess affinity alterations in glycan binding to receptors, testing was performed with purified lectins, cultured cells, tissue sections and animals. The panel of lectins, including an adhesion/growth-regulatory galectin, revealed up to a sixfold difference in affinity constants for these neoglycoproteins relative to data on the unsubstituted glycans reported previously [André, S., Unverzagt, C., Kojima, S., Dong, X., Fink, C., Kayser, K. & Gabius, H.-J. (1997) Bioconjugate Chem. 8, 845-855]. The enhanced affinity for galectin-1 is in accord with the increased percentage of cell positivity in cytofluorimetric and histochemical analysis of carbohydrate-dependent binding of labeled neoglycoproteins to cultured tumor cells and routinely processed lung cancer sections. Intravenous injection of iodinated neoglycoproteins carrying galactose-terminated N-glycans into mice revealed the highest uptake in liver and spleen for the bisecting compound compared with the unsubstituted or core-fucosylated N-glycans. Thus, this substitution modulates ligand properties in interactions with lectins, a key finding of this report. Synthetic glycan tailoring provides a versatile approach to the preparation of newly substituted glycans with favorable ligand properties for medical applications.

A pig model of hepatic cryotherapy. In vivo temperature distribution during freezing and histopathological changes.

We aimed to assess the temperature distribution in the cryolesion during hepatic cryotherapy and the association with postoperative histological changes to optimise the technique and allow better preoperative planning. Hepatic cryolesions were produced in 22 pigs following laparotomy using a CMS-cryosystem and 8mm-AccuProbe-Cryoprobes. The temperature was measured in 1 min intervals at different distances from the probe during freezing. The animals were treated in 5 groups: (i) single freezing of 20 min; (ii) double freezing of 20 min each; (iii) single freezing of 40 min; (iv) single freezing of 20 min (n=4), histology at 1 week p.o., and (v) single freezing of 20 min and Pringle manoeuvre; [(i)-(iii) and (v): histology at 24 h p.o.]. The mean diameter of the -38 degrees C isotherm, i.e., the zone of effective treatment for colorectal metastases was 37 mm for group (i) with a mean iceball diameter of 59 mm and about 46 mm for groups (ii, iii, and v) with mean iceball diameters of 78, 75, and 75 mm, respectively. At 7 days postoperatively secondary necrosis was seen in the largest central part of the lesion, wherever temperatures of -15 degrees C or lower were achieved during cryosurgery. Under the hypothesis that -38 degrees C is the effective temperature for the destruction of colorectal liver metastases, a lesion of 37-mm diameter may be effectively treated with a single 8mm-AccuProbe-Cryoprobe and a 20 min single freeze cycle and a lesion of 46 mm may be effectively treated when a double freeze-thaw cycle of 20 min each, a single freeze cycle of 40 min, or a 20 min single freeze cycle with additional Pringle manoeuvre is used, when it is perfectly placed in the lesion.

Large volume hepatic freezing: association with significant release of the cytokines interleukin-6 and tumor necrosis factor a in a rat model.

Although cryotherapy of liver tumors is generally considered a safe procedure, a syndrome of coagulopathy and fatal multiorgan failure has been observed in some patients and is called the cryoshock phenomenon. Our aim was to establish an animal model of this phenomenon and examine the effects of the basic parameters of freezing or cryotherapy on it. A group of 75 female Sprague-Dawley rats were allocated randomly to five groups: (1) sham laparotomy (n = 15); (2) small (25% liver volume) single freeze (n = 15); (3) small (25% liver volume) double freeze (n = 15); (4) large (50% liver volume) single freeze (n = 15); (5) large (50% liver volume) double freeze (n = 15). Blood samples were collected at different postoperative times, and organs were harvested for histopathology. There was a significant release of tumor necrosis factor-a (TNFa) and interleukin 6 (IL-6) following hepatic freezing, which was greatest in group 5. Postoperative serum cytokine levels were significantly associated with hepatocellular injury, as measured by postoperative serum aspartate transaminase (AST) concentrations. Severe hemoglobinuria and renal injury, as demonstrated by the serum creatinine level and the glomerular neutrophil count, were observed and were greatest in group 5. Hepatic cryosurgery is associated with release of IL-6 and TNFa and renal injury in a rat model. It is likely that the cryoshock phenomenon is another form of the systemic inflammatory response syndrome. Based on the results of this study, it is possibly mediated by cytokines released from the frozen liver tissue. We therefore caution against cryotherapy of large tumor volumes.

Structure-activity profiles of complex biantennary glycans with core fucosylation and with/without additional alpha 2,3/alpha 2,6 sialylation: synthesis of neoglycoproteins and their properties in lectin assays, cell binding, and organ uptake.

The consideration of oligosaccharides and glycoconjugates as biopharmaceuticals is an emerging topic in drug design. Chemoenzymatic synthesis of N-glycans was performed to examine the influence of N-glycan core fucosylation on lectin-binding properties and biodistribution. As a first step in a systematic comparison of N-glycans, the core fucose moiety was chemically introduced into a complex-type biantennary heptasaccharide azide. After deprotection and attachment of a spacer, the terminal sections of the N-glycan were elongated enzymatically. Conversion of the amino group in the spacer to an isothiocyanate gave derivatives allowing convenient ligand attachment to bovine serum albumin (BSA). The resulting neoglycoproteins contained an average of 2.9-4.6 chains per carrier molecule. Relative to unsubstituted biantennary complex-type N-glycans, the core fucosylation appears to favor the extended orientation of the alpha 1,6-arm. This was deduced from an up to 5-fold alteration of affinity for lectins in solid-phase assays. Marked differences were also found for cell surface binding of cultured tumor cells, for staining of tumor cells in lung sections, and in organ distribution. In vivo, the alpha 2,6-sialylated neoglycoproteins showed a reduced serum half-life in mice relative to the alpha 2,3-sialylated isomer and the non-fucosylated congeners. These results support the notion that changing the shape of a glycan provides a promising strategy to optimize the affinity of protein-carbohydrate interactions. Overall, our study underscores the importance of chemoenzymatic synthesis to define the effect of chain orientation on the ligand properties of N-glycans.