Identifying a newly discovered HLA-C allele, HLA-C*02:138.

A new allele, now named HLA-C*02:138, was discovered during testing of a registry donor for possible stem cell transplantation.

Comparison Between Total IgG, C1q, and C3d Single Antigen Bead Assays in Detecting Class I Complement-Binding Anti-HLA Antibodies.

BACKGROUND: Complement-binding donor-specific antibodies (DSAs) are associated with antibody-mediated rejection and allograft loss. Novel single antigen bead (SAB) assays-that is, complement component 1q (C1q) and complement component 3d (C3d) assays-have been developed to specifically detect complement-binding DSA, but it remains unclear whether these assays have an improved ability to detect complement-binding DSA as compared with using the total IgG SAB assay with a high mean fluorescence intensity (MFI) cutoff. The aim of this study was to compare the ability of the total IgG, C1q, and C3d SAB assays in detecting complement-binding anti-HLA antibodies. METHODS: Twenty sera known to have complement-binding anti-HLA antibodies (serologic class I HLA typing by complement-dependent cytotoxicity method) were tested with 3 different SAB assays: total IgG (undiluted and 1:8 dilution), C1q, and C3d. Serologic anti-HLA specificities were compared with those obtained by IgG, C1q, and C3d SAB assays. RESULTS: IgG SAB was more sensitive in detecting complement-binding antibodies (sensitivity 24 of 24 = 1, odds ratio infinity). Pearson correlation showed the association between (1) C1q and IgG SAB assays (cutoff C1q SAB 1000 MFI, cutoff IgG SAB 5000 MFI: r = 0.347, P < .0001) and (2) C3d and IgG SAB assays (cutoff 500 MFI C3d SAB, 5000 MFI for IgG SAB: r = -0.173, P = .279). CONCLUSIONS: For class I anti-HLA antibodies, IgG SAB (cutoff MFI > 5000) was more sensitive in detecting complement-binding antibodies when compared with C1q and C3d SAB assays.

Targeted diagnostic magnetic nanoparticles for medical imaging of pancreatic cancer.

Highly aggressive cancer types such as pancreatic cancer possess a mortality rate of up to 80% within the first 6months after diagnosis. To reduce this high mortality rate, more sensitive diagnostic tools allowing an early stage medical imaging of even very small tumours are needed. For this purpose, magnetic, biodegradable nanoparticles prepared using recombinant human serum albumin (rHSA) and incorporated iron oxide (maghemite, γ-Fe2O3) nanoparticles were developed. Galectin-1 has been chosen as target receptor as this protein is upregulated in pancreatic cancer and its precursor lesions but not in healthy pancreatic tissue nor in pancreatitis. Tissue plasminogen activator derived peptides (t-PA-ligands), that have a high affinity to galectin-1 have been chosen as target moieties and were covalently attached onto the nanoparticle surface. Improved targeting and imaging properties were shown in mice using single photon emission computed tomography-computer tomography (SPECT-CT), a handheld gamma camera, and magnetic resonance imaging (MRI).

Arylsulfatase A bound to poly(butyl cyanoacrylate) nanoparticles for enzyme replacement therapy--physicochemical evaluation.

Arylsulfatase A (ASA) deficiency is the cause of metachromatic leucodystrophy (MLD), a lysosomal storage disease associated with severe neurological disorders. Poly(butyl cyanoacrylate) (PBCA) nanoparticles overcoated with polysorbate 80 enabled the delivery of several drugs across the blood-brain barrier to the brain suggesting that these nanoparticles also may transport ASA across this barrier. The objective of this research, therefore, was to evaluate the feasibility of loading ASA onto PBCA nanoparticles. A stable ASA-loaded PBCA nanoparticle formulation was developed that could be easily freeze-dried and stored over a period of more than 8 weeks. The maximum loading capacity for this enzyme was -59 microg per 1 mg of PBCA. In the presence of 3% sucrose as a lyoprotector the activity of freeze-dried ASA was found to be 100% recoverable.

Development of nanoparticle-bound arylsulfatase B for enzyme replacement therapy of mucopolysaccharidosis VI.

Lysosomal storage disorders like mucopolysaccharidosis (MPS) VI are rare diseases with a lack of well-suited treatments. Even though an enzyme replacement therapy (ERT) of recombinant arylsulfatase B (ASB) is available for MPS VI, the administration cannot positively affect the neurologic manifestations such as spinal cord compression. Since nanoparticles (NP) have shown to be effective drug carriers, the feasibility of arylsulfatase B adsorption onto poly(butyl cyanoacrylate) (PBCA) nanoparticles was investigated in this study. In order to advance the ERT of ASB, the adsorption of the latter on the surface of PBCA NP as well as in vitro release in serum was investigated. With alteration of parameters like temperature, incubation time, pH, and enzyme amount, the adsorption process revealed to be stable with a maximum capacity of 67 microg/mg NP at a pH of 6.3. In vitro release experiments demonstrated that the adsorption is stable for at least 60 minutes in human blood serum, indicating that the ASB-loaded PBCA nanoparticles represent a promising candidate for ERT of MPS VI.

Efficient systemic therapy of rat glioblastoma by nanoparticle-bound doxorubicin is due to antiangiogenic effects.

The objective of this study was to investigate the therapeutic effects of doxorubicin bound to polysorbate-coated nanoparticles that had previously been shown to significantly enhance survival in the orthotopic rat 101/8 glioblastoma model. Tumor-bearing animals were subjected to chemotherapy using doxorubicin in solution (Dox-sol) or doxorubicin bound to polysorbate 80-coated poly(butyl cyanoacrylate) nanoparticles (Dox-np) injected intravenously on Days 2, 5 and 8 post tumor implantation. The antitumor effect was assessed on Days 10, 14 and 18 post tumor implantation. Tumors showed signs of malignancy including invasion of brain tissue, brisk mitotic activity, microvascular proliferation, necrosis and increased proliferation resembling human glioblastoma. Dox-np produced a considerably more pronounced antitumor effect exhibited as a reduced tumor size, lower proliferation, and a decreased necrotic area compared to Dox-sol and to untreated control groups. A drastic effect of Dox-np on vascularization indicated an antiangiogenic mode of action.

Antiparkinsonian effect of nerve growth factor adsorbed on polybutylcyanoacrylate nanoparticles coated with polysorbate-80.

The study examined the antiparkinsonian effect of nerve growth factor adsorbed on the surface of polybutylcyanoacrylate nanoparticles coated with polysorbate-80 surfactant. The parkinsonian syndrome in C57B1/6 mice was provoked by intraperitoneal injection of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. The basic symptoms of the parkinsonian syndrome decreased under the action of the nerve growth factor adsorbed on nanoparticles coated with polysorbate-80, which was seen from decreased rigidity and increased locomotor activity compared to control mice receiving with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine alone. This effect of nerve growth factor on nanoparticles persisted after 7 and 21 days after single injection of the neurotoxin. These data attest to the possibility of using nanoparticles prepared from amphiphilic polymers and coated with polysorbate-80 for the delivery of nerve growth factor into the brain during systemic treatment.

Intravenous tolerance of a nanoparticle-based formulation of doxorubicin in healthy rats.

A comparative toxicological study of doxorubicin bound to poly(butyl cyanoacrylate) nanoparticles coated with polysorbate 80 was performed in male and female Wistar rats. The drug substance was used as a reference formulation. The formulations were injected intravenously at a therapeutic dose of 6 mg/kg administered either as a single injection or in form of four weekly injections. The animals were followed up for 4 and 40 days (single injection) or 25 and 61 days (multiple injections) for assessment of the dynamics of body weight, hematological parameters, blood biochemical parameters, and urinalysis. Pathomorphological evaluation included macroscopic evaluation and weight measurement of the internal organs. The heart, lung, spleen, testes, and liver were also subjected to the histological evaluation. The overall result of this study suggests that the surfactant-coated nanoparticle formulation of doxorubicin has a favorable toxicological profile. Specifically, this formulation displays a considerably reduced cardio- and testicular toxicity, as compared to a free drug.

Radiation sterilisation of doxorubicin bound to poly(butyl cyanoacrylate) nanoparticles.

Doxorubicin-loaded poly(butyl cyanoacrylate) (PBCA) nanoparticles were prepared by anionic polymerisation under non-aseptic conditions. The feasibility of sterilisation of this formulation using either gamma-irradiation or electron beam irradiation was investigated. The irradiation doses ranged from 10 to 35 kGy. Bacillus pumilus was used as the official test microorganism. The bioburden of the untreated formulation was found to be 100 CFU/g. Microbiological monitoring revealed that at this level of the bioburden the irradiation dose of 15 kGy was sufficient for sterilisation of the nanoparticles. The formulation showed excellent stability with both types of irradiation in the investigated dose range. The irradiation did not influence the physicochemical parameters of the drug-loaded and empty nanoparticles, such as the mean particle size, polydispersity, and aggregation stability. The molecular weights of the PBCA polymer as well as the polydispersity indices (M(w)/M(n)) remained nearly unchanged. The drug substance was stable to radiolysis. Additionally, the presence of irradiation-induced radicals was evaluated by ESR spectroscopy after storage of the particles at ambient temperature. The paramagnetic species found in the formulation were mainly produced by irradiation of mannitol and dextran used as excipients.

T cells for suicide gene therapy: activation, functionality and clinical relevance.

In order to control graft-versus-host disease after donor lymphocyte infusion, T cells can be retrovirally transduced with a suicide gene. However, the immune competence of activated T cells appears compromised, responsible for reduced alloreactivity. The present study compared different activation protocols using soluble or bead-coupled antibodies regarding T-cell subtype expansion capacity and functionality. T cells were purified on a laboratory and clinical scale using both CD3 and CD4/CD8 antibodies for selection, leading to a mean purity of 96%. Transductions were performed with a GMP-grade CD34/HSV-TK vector. Activation with soluble CD3/CD28-antibodies +1000 U/ml IL-2 induced a 50-fold expansion of T cells over 14 days, whereas T cells activated with bead-coupled antibodies only expanded 2-4-fold restricted to the first week. Apart from using soluble antibodies, proliferation was highly IL-2 dependent. Expansion of CMV-specific T cells coincided with the expansion of whole CD3(+) cells. Soluble antibodies and higher IL-2 concentrations preferentially stimulated CD8(+) T cells, while bead-coupled antibodies +20 U/ml IL-2 preserved the CD4/CD8 ratio. Irrespective of the activation protocol, there was a shift from a naive to memory phenotype. When activated with soluble antibodies, mainly CD8(+) T cells were transduced. Furthermore, Th1/Th2 cytokine secretion was reduced. In contrast, CD4(+)/CD8(+) T cells activated with bead-coupled antibodies were rather homogenously transduced and cytokine secretion did not appear to be affected.

Preparation, characterisation and maintenance of drug efficacy of doxorubicin-loaded human serum albumin (HSA) nanoparticles.

Human serum albumin (HSA) nanoparticles represent promising drug carrier systems. Binding of cytostatics to HSA nanoparticles may diminish their toxicity, optimise their body distribution and/or may overcome multidrug resistance. In the present study, doxorubicin-loaded HSA nanoparticle preparations were prepared. Doxorubicin was loaded to the HSA nanoparticles either by adsorption to the nanoparticles' surfaces or by incorporation into the particle matrix. Both loading strategies resulted in HSA nanoparticles of a size range between 150nm and 500nm with a loading efficiency of 70-95%. The influence on cell viability of the resulting nanoparticles was investigated in two different neuroblastoma cell lines. The anti-cancer effects of the drug-loaded nanoparticles were increased in comparison to doxorubicin solution. Based on these result a standard protocol for the preparation of doxorubicin-loaded HSA nanoparticles for further antitumoural studies was established.

Influence of the formulation on the tolerance profile of nanoparticle-bound doxorubicin in healthy rats: focus on cardio- and testicular toxicity.

A toxicological study of doxorubicin bound to poly(butyl cyanoacrylate) or human serum albumin nanoparticles coated with polysorbate 80 was performed in healthy rats. The doxorubicin formulations were injected at a therapeutic dose regimen (3 x 1.5 mg/kg with a 72 h interval), and the animals were followed up for 15 or 30 days. The overall result of this study suggests that the surfactant-coated nanoparticle formulations of doxorubicin have favorable toxicological profiles. Specifically, these formulations display a considerably reduced cardio- and testicular toxicity, as compared to a free drug.

Pharmacology of intracellular cytosine-arabinoside-5'-triphosphate in malignant cells of pediatric patients with initial or relapsed leukemia and in normal lymphocytes.

PURPOSE: The prodrug cytosinearabinoside (ara-C) is widely used in the treatment of acute leukemias. The active drug is the intracellular metabolite cytosine-arabinoside-5'-triphosphate (ara-CTP). The purpose of the present study was to investigate the relation between sensitivity and pharmacokinetic parameters Cmax, t1/2 and AUC of ara-CTP. The obtained results were compared to previous studies. EXPERIMENTAL DESIGN: Cmax, t1/2 and AUC of ara-CTP were assessed in leukemic cells of 17 pediatric patients with acute lymphoblastic leukemia (ALL) and in 6 lymphoblastic cell lines and compared with normal lymphocytes of 9 healthy donors by high pressure liquid chromatography (HPLC). The sensitivity of the cells against ara-C was determined by the MTT assay. RESULTS: The intracellular accumulation of ara-CTP was significantly lower in normal lymphocytes (Cmax 47.7-60.9 pmol/10(6) cells) compared to leukemic cell lines (Cmax 11-1128 pmol/10(6) cells) and leukemic cells of our patients (Cmax 85.9-631 pmol/10(6) cells). Similar results were found for the AUC. There was no significant difference between initial and relapsed leukemias in our small cohort. A correlation between sensitivity in terms of IC50 values and the intracellular ara-CTP accumulation was observed in cell lines, but not in leukemic cells and normal lymphocytes from healthy donors. CONCLUSIONS: Pharmacokinetic parameters varied tremendously in leukemic cells in contrast to normal lymphocytes without a difference in sensitivity. It is worthwhile to compare literature data to assess an optimal dosage of ara-C in pediatric patients.

Chemotherapy of brain tumour using doxorubicin bound to surfactant-coated poly(butyl cyanoacrylate) nanoparticles: revisiting the role of surfactants.

Poly(butyl cyanoacrylate) nanoparticles coated with poloxamer 188 (Pluronic) F68) and also, as shown previously, polysorbate 80 (Tween 80) considerably enhance the anti-tumour effect of doxorubicin against an intracranial glioblastoma in rats. The investigation of plasma protein adsorption on the surface of the drug-loaded nanoparticles by two-dimensional electrophoresis (2-D PAGE) revealed that both surfactants, besides other plasma components, induced a considerable adsorption of apolipoprotein A-I (ApoA-I). It is hypothesized that delivery of doxorubicin to the brain by means of nanoparticles may be augmented by the interaction of apolipoprotein A-I that is anchored on the surface of the nanoparticles with the scavenger receptor class B type I (SR-BI) located at the blood-brain barrier. This is the first study that shows a correlation between the adsorption of apolipoprotein A-I on the nanoparticle surface and the delivery of the drug across the blood-brain barrier.

Dihydroxythiophenes are novel potent inhibitors of human immunodeficiency virus integrase with a diketo acid-like pharmacophore.

We have identified dihydroxythiophenes (DHT) as a novel series of human immunodeficiency virus type 1 (HIV-1) integrase inhibitors with broad antiviral activities against different HIV isolates in vitro. DHT were discovered in a biochemical integrase high-throughput screen searching for inhibitors of the strand transfer reaction of HIV-1 integrase. DHT are selective inhibitors of integrase that do not interfere with virus entry, as shown by the inhibition of a vesicular stomatitis virus G-pseudotyped retroviral system. Moreover, in quantitative real-time PCR experiments, no effect on the synthesis of viral cDNA could be detected but rather an increase in the accumulation of 2-long-terminal-repeat cycles was detected. This suggests that the integration of viral cDNA is blocked. Molecular modeling and the structure activity relationship of DHT demonstrate that our compound fits into a two-metal-binding motif that has been suggested as the essential pharmacophore for diketo acid (DKA)-like strand transfer inhibitors (Grobler et al., Proc. Natl. Acad. Sci. USA 99:6661-6666, 2002.). This notion is supported by the profiling of DHT on retroviral vectors carrying published resistance mutations for DKA-like inhibitors where DHT showed partial cross-resistance. This suggests that DHT bind to a common site in the catalytic center of integrase, albeit with an altered binding mode. Taken together, our findings indicate that DHT are novel selective strand transfer inhibitors of integrase with a pharmacophore homologous to DKA-like inhibitors.

Covalent linkage of apolipoprotein e to albumin nanoparticles strongly enhances drug transport into the brain.

Drug delivery to the brain is becoming more and more important but is severely restricted by the blood-brain barrier. Nanoparticles coated with polysorbates have previously been shown to enable the transport of several drugs across the blood-brain barrier, which under normal circumstances is impermeable to these compounds. Apolipoprotein E was suggested to mediate this drug transport across the blood-brain barrier. In the present study, apolipoprotein E was coupled by chemical methods to nanoparticles made of human serum albumin (HSA-NP). Loperamide, which does not cross the blood-brain barrier but exerts antinociceptive effects after direct injection into the brain, was used as model drug. Apolipoprotein E was chemically bound via linkers to loperamide-loaded HSA-NP. This preparation induced antinociceptive effects in the tail-flick test in ICR mice after i.v. injection. In contrast, nanoparticles linked to apolipoprotein E variants that do not recognize lipoprotein receptors failed to induce these effects. These results indicate that apolipoprotein E attached to the surface of nanoparticles facilitates transport of drugs across the blood-brain barrier, probably after interaction with lipoprotein receptors on the brain capillary endothelial cell membranes.

Cocaine-induced Fos expression in rat striatum is blocked by chloral hydrate or urethane.

Anesthetics used in electrophysiological studies alter the effects of cocaine and amphetamine on neural activity in the striatum. However, the mechanism underlying this alteration has not been established. In the present study, we examined the effects of anesthetics on cocaine-induced neural activity in the striatum. We first assayed the ability of 20 mg/kg cocaine to induce Fos expression in the striatum following pretreatment with 400 mg/kg chloral hydrate or 1.3 g/kg urethane, two of the most commonly used anesthetics for in vivo electrophysiology. Chloral hydrate blocked, while urethane strongly attenuated cocaine-induced Fos expression without affecting basal levels of expression. We then examined dopaminergic and glutamatergic mechanisms for anesthetic effects on cocaine-induced Fos expression. Chloral hydrate and urethane did not attenuate basal or cocaine-induced increases of dopamine levels as assessed by microdialysis in dorsal striatum. In contrast, chloral hydrate attenuated glutamatergic neurotransmission as assessed by microdialysis in the presence of the glutamate transport blocker L-trans-pyrrolidone-2,4-dicarboxylic acid. Chloral hydrate attenuated basal levels of glutamate by 70%, while cocaine had no effect on glutamate levels. Since glutamate levels were tetrodotoxin-sensitive, the majority of glutamate measured in our assay was by synaptic release. To assess a causal role for a reduction of glutamatergic neurotransmission in anesthetic effects on cocaine-induced Fos expression, we injected the glutamate receptor agonists AMPA and NMDA into the dorsal striatum of chloral hydrate-anesthetized rats. The glutamate receptor agonists partially reinstated cocaine-induced Fos expression in anesthetized rats. We conclude anesthetics attenuate cocaine-induced neuronal activity by reducing glutamatergic neurotransmission.

Physicochemical characterization of protamine-phosphorothioate nanoparticles.

Protamine-oligonucleotide nanoparticles represent effective colloidal drug carriers for antisense phosphorothioate oligonucleotides (PTO). This study describes improvements in particle preparation and the physicochemical properties of the complexes prepared. The influence of component concentrations, length of the PTO chain and the PTO/protamine weight ratio on particle formation and size, shape and surface charge of the particles were studied in detail. Nanoparticles with diameters of 90-200nm were obtained, using protamine free base (PFB) and phosphorothioate in water. The chemical composition of the nanoparticles was analysed. More than 90% of the PTO could be assembled in the particle matrix using a > or = 1:2 ratio (w/w) of PTO and PFB. About 53-68% of the PFB was incorporated in the particle matrix. The complexes had a zetapotential of -19 up to +32 mV, depending on the PTO/PFB ratio. The kinetics of the assembly of this binary system were observed by dynamic light scattering (DLS) measurements and by sedimentation velocity analysis in the analytical ultracentrifuge (AUC). In addition, scanning electron microscopy (SEM) and atomic force microscopy (AFM) were applied to verify the results of DLS and the ultracentrifuge measurements. According to sedimentation velocity analysis, the particles were only moderately stable in water and unstable in salt solutions. However, the colloidal solution in water could be stabilized by polyethylenglycol 20000 (PEG), which also led to an increase of stability in cell medium.

[Drug delivery to the brain with nanoparticles]. / Dostavka lekarstvennykh preparatov v mozg s pomoshch'iu nanochastits.

Drugs can be delivered to brain with the aid of poly(butylcyanoacrylate) (PBCA) nanoparticles coated with polysorbate 80. These carriers can penetrate BBB and deliver drugs of various structures, including peptides, hydrophilic compounds, and lipophilic compounds eliminated from brain with P-glycoprotein. When a suspension of polysorbate-coated PBCA nanoparticles is introduced into blood, apolipoproteins of the blood plasma adsorb on the particle surface and then interact with receptors of low-density lipoproteins situated in endothelial cells of cerebral vessels, thus stimulating endocytosis.