Fatal fat embolism following amphotericin B lipid complex injection

A case of amphotericin B lipid complex induced fatal fat embolism is described. A 41-year-old Caucasian man with AIDS was undergoing treatment for cryptococcal meningitis with amphotericin B. His course was complicated by renal failure necessitating a change in therapy to amphotericin B lipid complex (Abelcet). At approximately 48 h, the patient developed tachycardia, tachypnea, respiratory failure, decline in hematocrit, thrombocytopenia, and alteration in mental status. Autopsy findings included fat emboli involving heart, lungs, kidney, and brain. To our knowledge, this is the first case report of a fatal fat embolism caused by intravenous liposome drug delivery.     

Enhanced molecular volume of conservatively pegylated Hb: (SP-PEG5K)6-HbA is non-hypertensive

Recent studies have suggested that the "pressor effect" of acellular Hb is a consequence of perturbation of the macro-and microcirculatory system in multiple ways, and that PEGylation is an effective approach for controlling the same. In an attempt to confirm this concept, a new and simple thiolation mediated, maleimide chemistry-based conservative PEGylation protocol has been developed to conjugate multiple copies of PEG-chains to Hb. This approach combines the high reactivity of maleimides towards thiols with the propensity of iminothiolane to derivatize the epsilon-amino groups of proteins into reactive thiol groups, with conservation of their positive charge. One of the PEGylated products, namely (SP-PEG5K)6-HbA, that carries on an average six copies of PEG5000 chains per Hb, is non-hypertensive in hamster top load and in rat 50% exchange transfusion models. This hexa-PEGylated-Hb has (i) a hydrodynamic volume corresponding to that of an oligomerized Hb of 256kDa, (ii) a molecular radius of approximately 6.8 nm, (iii) high oxygen affinity, (iv) lowered Bohr effect, and (v) increased viscosity and colloidal osmotic pressure. These properties of (SP-PEG5K)6-HbA are consistent with the emerging new paradigms for the design of Hb based oxygen carriers and confirm the concept that the "pressor effect" of Hb is a multifactorial event. The thiolation mediated maleimide chemistry-based PEGylation protocol described here for the generation of (SP-PEG5K)6-Hb is simple, highly efficient, and is carried out under oxy conditions. The results demonstrate that a non-hypertensive PEG-Hb can be generated by conjugation of a lower number of PEG chains than previously reported.     

Sequence variation within botulinum neurotoxin serotypes impacts antibody binding and neutralization

The botulinum neurotoxins (BoNTs) are category A biothreat agents which have been the focus of intensive efforts to develop vaccines and antibody-based prophylaxis and treatment. Such approaches must take into account the extensive BoNT sequence variability; the seven BoNT serotypes differ by up to 70% at the amino acid level. Here, we have analyzed 49 complete published sequences of BoNTs and show that all toxins also exhibit variability within serotypes ranging between 2.6 and 31.6%. To determine the impact of such sequence differences on immune recognition, we studied the binding and neutralization capacity of six BoNT serotype A (BoNT/A) monoclonal antibodies (MAbs) to BoNT/A1 and BoNT/A2, which differ by 10% at the amino acid level. While all six MAbs bound BoNT/A1 with high affinity, three of the six MAbs showed a marked reduction in binding affinity of 500- to more than 1,000-fold to BoNT/A2 toxin. Binding results predicted in vivo toxin neutralization; MAbs or MAb combinations that potently neutralized A1 toxin but did not bind A2 toxin had minimal neutralizing capacity for A2 toxin. This was most striking for a combination of three binding domain MAbs which together neutralized >40,000 mouse 50% lethal doses (LD(50)s) of A1 toxin but less than 500 LD(50)s of A2 toxin. Combining three MAbs which bound both A1 and A2 toxins potently neutralized both toxins. We conclude that sequence variability exists within all toxin serotypes, and this impacts monoclonal antibody binding and neutralization. Such subtype sequence variability must be accounted for when generating and evaluating diagnostic and therapeutic antibodies.     

Improvement of Schwann cell attachment and proliferation on modified hyaluronic acid strands by polylysine

Hyaluronic acid (HyA) has the intrinsic ability to promote cell proliferation and reduce scar formation. However, the clinical use of HyA has so far been limited because of its water solubility and nonadhesive characteristics. Increasing interest in HyA as a clinically useful biomaterial has prompted our study of altering HyA's physical properties to render it a potential component of nerve grafts. In this study, strands of HyA were cross-linked by glutaraldehyde (Glut), coated with polylysine, and then inoculated with Schwann cells (SCs). Results in vivo and in vitro demonstrated that cross-linked HyA strands were water insoluble and thus less biodegradable. Poly-D-lysine-resurfaced strands showed significant SC attachment of 350-400 cells/mm(2), compared to uncoated controls (0-10 cells/mm(2), p < 0.01). Fibroblast control groups showed an attachment of 40-100 cells/mm(2) on coated strands. Immunostaining for proliferating cells showed SCs as and fibroblasts as +. Cells neither adhered to nor proliferated on the modified HyA strands that were not resurfaced. The results suggest that polylysine promotes SC attachment and proliferation to glutaraldehyde-cross-linked HyA strands, the product being a three-dimensional composite with low solubility that may have potential application in nerve grafts.     

Alterations of plasma antioxidants and mitochondrial DNA mutation in hair follicles of smokers

The effects of long-term smoking on mitochondrial DNA (mtDNA) deletions in hair follicles were investigated in subjects with different antioxidant capacity. Twenty-two male smokers with a smoking index of greater than 5 pack-years and without any known systemic diseases were recruited for this study. Forty healthy nonsmoking males were included as controls. We found that the concentrations of ascorbate and alpha-tocopherol and the activities of glutathione S-transferase (GST) and glutathione peroxidase in blood plasma were significantly decreased in smokers. The levels of glutathione and protein thiols in whole blood and the incidence of a 4,977 bp deletion of mtDNA (dmtDNA) in hair follicles were significantly increased in smokers. A significantly higher incidence of the 4,977 bp dmtDNA was found in smokers with plasma GST activity less than 5.66 U/l (OR = 7.2, P = 0.020). Using multiple covariate ANOVA and logistic regression, we found that age and low plasma GST activity were the only two risk factors for the 4,977 bp dmtDNA. These results suggest that smoking depletes antioxidants and causes mtDNA deletions and that plasma GST may play an important role in the preservation of the mitochondrial genome in tissue cells of smokers.     

Production and characterization of monoclonal antibodies to type 8 lipooligosaccharide of Neisseria meningitidis.

Eight monoclonal antibodies (MAbs) to lipooligosaccharides (LOSs) of Neisseria meningitidis were produced by immunizing mice with purified LOS from group A meningococcal strain A1. The specificities of the MAbs were examined by enzyme-linked immunosorbent assay (ELISA), immunodot assay, and ELISA inhibition by using the homologous A1 LOS, 12 immunotype LOSs of N. meningitidis (L1 through L12), and LOSs or lipopolysaccharides from other gram-negative bacteria. Two of the MAbs, 4385G7 (immunoglobulin G2b [IgG2b]) and 4387A5 (IgG2a), had the strongest reactivities with the homologous A1 LOS, moderate reactivities with the M978 (L8) LOS, but no reactivity with other LOSs. The other six MAbs (4 IgM and 2 IgG3) reacted with the A1 LOS and with several or many of the 12 LOSs. ELISA inhibition at 50% showed that the inhibitory activities of the LOSs from strains A1 and BB431 (a group B strain) to the specific MAb 4387A5 were about 10 to 20 times greater than that of the M978 (L8) LOS. When compared with MAb 2-1-L8 (L8) by Western blot (immunoblot) analysis and ELISA inhibition, the two specific MAbs recognized a different epitope in the 3.6-kDa LOSs of strains A1 and BB431. We propose that the new epitope is L8a, since the MAbs also reacted with the M978 (L8) LOS. The expression of the L8a epitope in the A1 LOS requires a few monosaccharide residues in its oligosaccharide moiety, and the fatty acid residues in its lipid A moiety also play a role. In a whole-cell ELISA, the two specific MAbs bound specifically to the homologous strain A1 and the L8 prototype strain M978 but not to any other LOS prototype strains. These results suggest that the two specific MAbs can be used for LOS typing of N. meningitidis.     

Dissemination of CTX-M-3 and CMY-2 beta-lactamases among clinical isolates of Escherichia coli in southern Taiwan

A total of 1,210 clinical isolates of Escherichia coli collected from a university hospital in southern Taiwan were screened for production of extended-spectrum beta-lactamases (ESBLs). Expression of classical ESBLs (resistant to extended-spectrum beta-lactam agents and susceptible to beta-lactam inhibitors) was inferred in 18 isolates by the phenotypic confirmatory test. These included 10 isolates producing CTX-M-3, 2 strains carrying SHV-12, 1 strain harboring SHV-5, 1 strain expressing TEM-10, and 4 strains producing unidentifiable ESBLs with a pI of 8.05, 8.0, or 7.4. Eighteen isolates that showed decreased susceptibilities to ceftazidime and/or cefotaxime, negative results for the confirmatory test, and high-level resistance to cefoxitin (MICs of >/=128 microg/ml) were also investigated. Five isolates were found to produce CMY-2 AmpC enzymes, one isolate carried both CTX-M-3 and CMY-2, and the remaining three and nine isolates expressed putative AmpC beta-lactamases with pIs of >9.0 and 8.9, respectively. Thus, together with the isolate producing CTX-M-3 and CMY-2, 19 (1.6%) isolates produced classical ESBLs. Pulsed-field gel electrophoresis revealed that all isolates carrying CTX-M-3 and/or CMY-2 were genetically unrelated, indicating that dissemination of resistance plasmids was responsible for the spread of these two enzymes among E. coli in this area. Among the 16 isolates expressing CTX-M-3 and/or CMY-2, 5 might have colonized outside the hospital environment. Our data indicate that CTX-M-3 and CMY-2, two beta-lactamases initially identified in Europe, have been disseminated to and are prevalent in Taiwan.     

A formalism for independent checking of Gamma Knife dose calculations

For stereotactic radiosurgery using the Leksell Gamma Knife system, it is important to perform a pre-treatment verification of the maximum dose calculated with the Leksell GammaPlan (DLGP) stereotactic radiosurgery system. This verification can be incorporated as part of a routine quality assurance (QA) procedure to minimize the chance of a hazardous overdose. To implement this procedure, a formalism has been developed to calculate the dose DCAL(X,Y,Z,dav,t) using the following parameters: average target depth (dav), coordinates (X,Y,Z) of the maximum dose location or any other dose point(s) to be verified, 3-dimensional (3-dim) beam profiles or off-centerratios (OCR) of the four helmets, helmet size i, output factor Oi, plug factor Pi, each shot j coordinates (x,y,z)i,j, and shot treatment time (ti,j). The average depth of the target dav was obtained either from MRI/CT images or ruler measurements of the Gamma Knife Bubble Head Frame. DCAL and DLGP were then compared to evaluate the accuracy of this independent calculation. The proposed calculation for an independent check of DLGP has been demonstrated to be accurate and reliable, and thus serves as a QA tool for Gamma Knife stereotactic radiosurgery.