Isolation of human lymphatic malformation endothelial cells, their in vitro characterization and in vivo survival in a mouse xenograft model

Human lymphatic vascular malformations (LMs), also known as cystic hygromas or lymphangioma, consist of multiple lymphatic endothelial cell-lined lymph-containing cysts. No animal model of this disease exists. To develop a mouse xenograft model of human LM, CD34^NegCD31^Pos LM lymphatic endothelial cells (LM-LEC) were isolated from surgical specimens and compared to foreskin CD34^NegCD31^Pos lymphatic endothelial cells (LECs). Cells were implanted into a mouse tissue engineering model for 1, 2 and 4 weeks. In vitro LM-LECs showed increased proliferation and survival under starvation conditions (P < 0.0005 at 48 h, two-way ANOVA), increased migration (P < 0.001, two-way ANOVA) and formed fewer (P = 0.029, independent samples t test), shorter tubes (P = 0.029, independent samples t test) than foreskin LECs. In vivo LM-LECs implanted into a Matrigel?-containing mouse chamber model assembled to develop vessels with dilated cystic lumens lined with flat endothelium, morphology similar to that of clinical LMs. Human foreskin LECs failed to survive implantation. In LM-LEC implanted chambers the percent volume of podoplanin^Pos vessels was 1.18 ± 2.24 % at 1 week, 6.34 ± 2.68 % at 2 weeks and increasing to 7.67 ± 3.60 % at 4 weeks. In conclusion, the significantly increased proliferation, migration, resistance to apoptosis and decreased tubulogenesis of LM-LECs observed in vitro is likely to account for their survival and assembly into stable LM-like structures when implanted into a mouse vascularised chamber model. This in vivo xenograft model will provide the basis of future studies of LM biology and testing of potential pharmacological interventions for patients with lymphatic malformations.     

Comparison of the clinical courses of the opticospinal and conventional forms of multiple sclerosis in Japan

We evaluated the clinical courses of 216 patients with multiple sclerosis (MS) diagnosed according to the recommended diagnostic criteria of McDonald et al (10). Sixty-five patients clinically displaying selective involvement of the optic nerves and spinal cord were classified as opticospinal MS (OS-MS), while the other 151 showing disseminated involvement of the central nervous system were classified as conventional MS (C-MS). The disease duration did not differ significantly between the two subtypes (11.2 years vs. 11.5 years). In addition to a higher age of onset, female preponderance and higher Kurtzke's expanded disability status scale (EDSS) scores, the OS-MS patients showed a markedly lower frequency of secondary progressive MS than the C-MS patients (4.6% vs. 29.1%, p=0.0001). The EDSS scores of the C-MS patients were significantly correlated with the disease duration, while those of the OS-MS patients were not. Among the C-MS patients, the frequency of secondary progressive MS was significantly more common in patients with a disease duration of more than 10 years than in those with a shorter duration. These results suggest that the irreversible disability in OS-MS is determined by relapses, rather than by chronic progression, whereas C-MS has a similar clinical course to MS in Westerners.     

Hyperglycemia per se can reduce plasma free fatty acid and glycerol levels in the acutely insulin-deficient dog

To evaluate the in vivo effect of hyperglycemia per se on plasma free fatty acid (FFA) and glycerol concentrations, euglycemic and hyperglycemic clamp studies were performed in six overnight fasted dogs in the state of insulin deficiency produced by somatostatin (SRIF) infusion. The mean blood glucose concentrations during the steady-state (the second hour of each study) averaged 4.65 +/- 0.10 mmol/L in euglycemic clamp and 14.11 +/- 0.10 mmol/L in hyperglycemic clamp. During the SRIF infusion, plasma FFA concentrations increased from 0.32 +/- 0.05 mumol/mL at the basal state to 0.76 +/- 0.04 mumol/mL at the steady-state in euglycemic clamp and from 0.26 +/- 0.04 mumol/mL to 0.43 +/- 0.02 mumol/mL in hyperglycemic clamp. Plasma glycerol concentrations increased from the basal value of 0.07 +/- 0.01 mumol/mL to 0.15 +/- 0.01 mumol/mL during the steady-state in euglycemic clamp and from 0.06 +/- 0.01 mumol/mL to 0.08 +/- 0.01 mumol/mL in hyperglycemic clamp. The steady-state concentrations of plasma FFA and glycerol in hyperglycemic clamp were significantly lower than those in euglycemic clamp (P less than .001; respectively). These results suggest that hyperglycemia per se might decrease plasma FFA and glycerol concentrations at least in part by decreasing lipolysis in the acutely insulin-deficient dog.     

Designer broad-spectrum polyimidazolium antibiotics

For a myriad of different reasons most antimicrobial peptides (AMPs) have failed to reach clinical application. Different AMPs have different shortcomings including but not limited to toxicity issues, potency, limited spectrum of activity, or reduced activity in situ. We synthesized several cationic peptide mimics, main-chain cationic polyimidazoliums (PIMs), and discovered that, although select PIMs show little acute mammalian cell toxicity, they are potent broad-spectrum antibiotics with activity against even pan-antibiotic-resistant gram-positive and gram-negative bacteria, and mycobacteria. We selected PIM1, a particularly potent PIM, for mechanistic studies. Our experiments indicate PIM1 binds bacterial cell membranes by hydrophobic and electrostatic interactions, enters cells, and ultimately kills bacteria. Unlike cationic AMPs, such as colistin (CST), PIM1 does not permeabilize cell membranes. We show that a membrane electric potential is required for PIM1 activity. In laboratory evolution experiments with the gram-positive Staphylococcus aureus we obtained PIM1-resistant isolates most of which had menaquinone mutations, and we found that a site-directed menaquinone mutation also conferred PIM1 resistance. In similar experiments with the gram-negative pathogen Pseudomonas aeruginosa, PIM1-resistant mutants did not emerge. Although PIM1 was efficacious as a topical agent, intraperitoneal administration of PIM1 in mice showed some toxicity. We synthesized a PIM1 derivative, PIM1D, which is less hydrophobic than PIM1. PIM1D did not show evidence of toxicity but retained antibacterial activity and showed efficacy in murine sepsis infections. Our evidence indicates the PIMs have potential as candidates for development of new drugs for treatment of pan-resistant bacterial infections.

AMPs and AMP mimics have attracted considerable attention as candidates for therapeutic development (1). The basic design elements include a region of charged residues, generally cationic residues, enabling interaction with bacterial cell surfaces, combined with a hydrophobic nature in AMPs (2). Unfortunately, AMPs and related polymers, in general, have one or more issues that limit their use as broad-spectrum antibiotics. Some are quite toxic to human cells, the potency of some is not adequate for human administration, others are sensitive to salt at levels present in human fluids, and some are too difficult and expensive to synthesize (3, 4). One broad-spectrum antimicrobial peptide, CST has seen increased recent use as a last resort antibiotic. CST is believed to kill bacteria by virtue of its ability to disrupt membrane integrity (5). This antibiotic requires intravenous administration and is nephrotoxic (6). The emergence of CST-resistant pathogens has also become a significant problem (7). We are unaware of any new broad-spectrum AMPs that have advanced to clinical trials.Imidazolium (IM) salts are antimicrobials (8), and there is an emerging literature on antimicrobial activity of side-chain and main-chain polyimidazolium (PIM) salts with chemical structures that are in some ways similar to those we describe. Although PIMs are potent antimicrobials, there are biocompatibility problems hindering their development, and some have somewhat limited activity spectra. As with other AMPs, there have been toxicity issues, potency issues, and delivery issues as many have large molecular masses, and there is little known about mammalian cell toxicity or mechanism of action (9–12).Here we show that members of a series of PIMs we designed and synthesized are potent broad-spectrum antibacterial compounds. We selected two for further analysis and showed they retain activity even against pan-antibiotic-resistant bacteria. Unlike CST and many other AMPs, which disrupt bacterial membranes, our model PIM is bactericidal without disrupting bacterial membranes. Our experiments provide insights about mechanism of action, the potential for the emergence of PIM resistance, and indicate PIMs are effective against a model gram-negative and a model gram-positive pathogen in murine infection models.     

Plasma levels of cell adhesion molecules during hyperinsulinemia and modulation of vasoactive mediators

Endothelial expression of cell adhesion molecules (CAMs) plays an important role in atherosclerosis. Atherosclerosis is increased in hyperinsulinemic states, but whether insulin per se is proatherogenic remains unclear. To investigate the effects of hyperinsulinemia on CAM expression, plasma levels of ICAM-1, VCAM-1 and E-selectin were measured before and after forearm infusion of insulin in healthy subjects. Insulin administration for 2h resulted in significant hyperinsulinemia, whereas no significant change was observed in soluble CAMs (all p > 0.05). Because insulin stimulates endothelial release of both endothelin-1 (ET-1) and nitric oxide (NO), which may modulate the expression of CAMs, we also investigated the response of CAMs to ET-1 receptor blockade, alone and in combination with NO synthesis inhibition. ET-1 receptor blockade during hyperinsulinemia resulted in a vasodilator response, but did not affect soluble CAMs (all p > 0.05). Superimposition of NO inhibition by L-NMMA reversed the vasodilator effect of ET-1 blockade, without affecting soluble CAMs (all p > 0.05). In conclusion, acute hyperinsulinemia, alone or during ET-1 and NO pathway blockade, does not affect soluble CAMs. These results do not support a direct effect of insulin on endothelial cells to affect leukocyte adhesiveness to the vascular wall.