KIR2DL4-HLAG interaction at human NK cell-oligodendrocyte interfaces regulates IFN-γ-mediated effects.

Interactions between germline-encoded natural killer (NK) cell receptors and their respective ligands on tumorigenic or virus-infected cells determine NK cell cytotoxic activity and/or cytokine secretion. NK cell cytokine responses can be augmented in and can potentially contribute to multiple sclerosis (MS), an inflammatory disease of the central nervous system focused upon the oligodendrocytes (OLs). To investigate mechanisms by which NK cells may contribute to MS pathogenesis, we developed an in vitro human model of OL-NK cell interaction. We found that activated, but not resting human NK cells form conjugates with, and mediate cytotoxicity against, human oligodendrocytes. NK cells, when in conjugate with OLs, rapidly synthesize and polarize IFN-γ toward the OLs. IFN-γ is capable of reducing myelin oligodendrocyte and myelin associated glycoproteins (MOG and MAG) content. This activity is independent of MHC class-I mediated inhibition via KIR2DL1, but dependent upon the interaction between NK cell-expressed KIR2DL4 and its oligodendrocyte-expressed ligand, HLA-G. NK cells from patients with MS express higher levels of IFN-γ following conjugation to OLs, more actively promote in vitro reduction of MOG and MAG and have higher frequencies of the KIR2DL4 positive population. These data collectively suggest a mechanism by which NK cells can promote pathogenic effects upon OLs.

Adenoid cystic carcinoma of the maxillary sinus: a clinical-pathological report of 10 years of experience from a single institution.

Adenoid cystic carcinoma (ACC) is the most common malignant salivary gland tumour of the maxillary sinus. The present study describes 24 cases seen over a period of 10 years at the Brazilian National Cancer Institute. Socio-demographic, clinical, pathological, and follow-up data were retrieved from the medical files for the period 1997-2006. The mean age of the patients was 51.1 years. Twenty-one (87.5%) presented advanced tumours. The main signs and symptoms found were a tumour mass (87.5%), pain (50%), nasal obstruction (25%), and epistaxis (20.8%). Most cases (62.5%) were treated with surgery and radiation therapy. Follow-up data showed two patients (8.3%) with residual disease, local recurrences in four (16.7%) patients, and distant metastasis in five (20.8%). The overall 5- and 10-year survival rates were 72.61% and 62.11%, respectively. Maxillary sinus ACC has an aggressive but indolent behaviour, typically presenting at an advanced T stage that reflects a poor prognosis for patients.

Genetic identification of missing persons: DNA analysis of human remains and compromised samples.

Human identification has made great strides over the past 2 decades due to the advent of DNA typing. Forensic DNA typing provides genetic data from a variety of materials and individuals, and is applied to many important issues that confront society. Part of the success of DNA typing is the generation of DNA databases to help identify missing persons and to develop investigative leads to assist law enforcement. DNA databases house DNA profiles from convicted felons (and in some jurisdictions arrestees), forensic evidence, human remains, and direct and family reference samples of missing persons. These databases are essential tools, which are becoming quite large (for example the US Database contains 10 million profiles). The scientific, governmental and private communities continue to work together to standardize genetic markers for more effective worldwide data sharing, to develop and validate robust DNA typing kits that contain the reagents necessary to type core identity genetic markers, to develop technologies that facilitate a number of analytical processes and to develop policies to make human identity testing more effective. Indeed, DNA typing is integral to resolving a number of serious criminal and civil concerns, such as solving missing person cases and identifying victims of mass disasters and children who may have been victims of human trafficking, and provides information for historical studies. As more refined capabilities are still required, novel approaches are being sought, such as genetic testing by next-generation sequencing, mass spectrometry, chip arrays and pyrosequencing. Single nucleotide polymorphisms offer the potential to analyze severely compromised biological samples, to determine the facial phenotype of decomposed human remains and to predict the bioancestry of individuals, a new focus in analyzing this type of markers.

Training of Forensic DNA Scientists - A Commentary.

For the past two decades, forensic DNA analysis has rapidly expanded in both utility and value to criminal investigations. As the number of crime scene and convict/arrestee samples has continued to grow, many forensic DNA laboratories find themselves struggling to test samples in a timely fashion. Agencies employ various methods for calculating their sample intake and processing capacity, yet database and casework sample backlogs continue to present a major challenge. One issue many forensic laboratories face is limited availability of resources for training new analysts. High-quality training enables analysts to effectively perform various aspects of DNA profiling, and as such, it is essential to ensuring consistent, high-quality results. This is well documented in the guidelines established in the FBI's Quality Assurance Standards for Forensic DNA Testing Laboratories in the United States as well as internationally by agencies like INTERPOL. A facility dedicated to training analysts on both theoretical and practical aspects of automated sample processing accelerates the establishment and expansion of high-throughput forensic DNA laboratories. The present article will discuss various aspects of training and agencies that provide such training programs.

Autosomal SNP typing of forensic samples with the GenPlex™ HID System: results of a collaborative study.

The GenPlex™ HID System (Applied Biosystems - AB) offers typing of 48 of the 52 SNPforID SNPs and amelogenin. Previous studies have shown a high reproducibility of the GenPlex™ HID System using 250-500pg DNA of good quality. An international exercise was performed by 14 laboratories (9 in Europe and 5 in the US) in order to test the robustness and reliability of the GenPlex™ HID System on forensic samples. Three samples with partly degraded DNA and 10 samples with low amounts of DNA were analyzed in duplicates using various amounts of DNA. In order to compare the performance of the GenPlex™ HID System with the most commonly used STR kits, 500pg of partly degraded DNA from three samples was typed by the laboratories using one or more STR kits. The median SNP typing success rate was 92.3% with 500pg of partly degraded DNA. Three of the fourteen laboratories counted for more than two thirds of the locus dropouts. The median percentage of discrepant results was 0.2% with 500pg degraded DNA. An increasing percentage of locus dropouts and discrepant results were observed when lower amounts of DNA were used. Different success rates were observed for the various SNPs. The rs763869 SNP was the least successful. With the exception of the MiniFiler™ kit (AB), GenPlex™ HID performed better than five other tested STR kits. When partly degraded DNA was analyzed, GenPlex™ HID showed a very low mean mach probability, while all STR kits except MiniFiler™ had very limited discriminatory power.

Identification of three novel RB1 mutations in Brazilian patients with retinoblastoma by "exon by exon" PCR mediated SSCP analysis.

AIMS: To carry out a retrospective study, screening for mutations of the entire coding region of RB1 and adjacent intronic regions in patients with retinoblastoma. METHODS: Mutation screening in DNA extracts of formalin fixed, paraffin wax embedded tissues of 28 patients using combined "exon by exon" polymerase chain reaction mediated single strand conformational polymorphism analysis, followed by DNA sequencing. RESULTS: Eleven mutations were found in 10 patients. Ten mutations consisted of single base substitutions; 10 were localised in exonic regions (eight nonsense, one missense, and one frameshift) and another one in the intron-exon splicing region. Three novel mutations were identified: a 2 bp insertion in exon 2 (g.5506-5507insAG, R73fsX77), a G to A transition affecting the last invariant nucleotide of intron 13 (g.76429G>A), and a T to C transition in exon 20 (g.156795T>C, L688P). In addition, eight C to T transitions, resulting in stop codons, were found in five different CGA codons (g.64348C>T, g.76430C>T, g.78238C>T, g.78250C>T, and g.150037C>T). Although specific mutation hotspots have not been identified in the literature, eight of the 11 mutations occurred in CGA codons and seven fell within the E1A binding domains (codons 393-572 and 646-772), whereas five were of both types-in CGA codons within E1A binding domains. CONCLUSIONS: CGA codons and E1A binding domains are apparently more frequent mutational targets and should be initially screened in patients with retinoblastoma. Paraffin wax embedded samples proved to be valuable sources of DNA for retrospective studies, providing useful information for genetic counselling.

Single Nucleotide Polymorphisms and Microarray Technology in Forensic Genetics - Development and Application to Mitochondrial DNA.

Variations in the genome, due to base substitutions, insertions, or deletions at single positions, are known as single nucleotide polymorphisms (SNPs). Approximately 85% of human variation is based on such polymorphisms. Therefore, there is an abundance of human SNPs that are available for forensic identity testing purposes. SNP analyses also may be suitable for some forensic identity cases, because they can be detected in smallsized amplicons, allowing for genetic analysis of substantially degraded DNA. While SNP analysis is unlikely to replace short tandem repeat loci typing for routine casework, SNPs may prove useful for certain circumstances, for example, typing mitochondrial DNA (mtDNA). Although sequencing mtDNA enables detection of all SNPs contained within the region of interest, it is currently not a practical approach for simultaneously typing SNPs that reside throughout the entire mtDNA genome. A variety of alternate methods to detect SNPs are available that may facilitate mtDNA analysis. All the methods include amplification, typically by the polymerase chain reaction, of the region containing the SNP of interest. Most assays are based on either hybridization of a probe to amplified product or primer extension chemistry, and multiplexing is possible. Some of these methodologies are: chips, SNaP shot™, Luminex 100™, SNPstream® UHT, and Pyrosequencing™. SNP analysis of mtDNA, both in the noncoding and coding regions, has been demonstrated using a number of these formats.

Control of amphiphilic block copolymer morphologies using solution conditions.

It is well known that the morphology of block copolymer aggregates depends on polymer properties such as the molecular weight, the relative block length, and the chemical nature of the repeat unit. Recently, we have shown that if aggregates are allowed to self-assemble in solution, then in addition to the above factors, a high degree of control over the aggregate architecture can be achieved by adjusting the solution conditions. Factors such as the water content in the solvent mixture, the solvent nature and composition, the presence of additives (ions, surfactants, and homopolymer) and the polymer concentration were successfully employed to control the aggregate shape and size. In this paper, we review a series of studies performed in our group to show how solution properties can control the architecture of aggregates prepared from a given copolymer. The control mechanism is explained in terms of the effect of each property on the forces that govern the formation of any given morphology, namely the core-chain stretching, corona-chain repulsion and interfacial tension.

Block copolymers modify the internalization of micelle-incorporated probes into neural cells.

An important therapeutic concern is rate and extent of internalization of drugs into cells. Hydrophilic agents often internalize poorly and slowly, and highly lipophilic ones too rapidly. The incorporation of drugs into micelles allows regulation of their internalization parameters, and newly-described block copolymers can be selectively tailored to suit specific drugs. This report compares internalization of Cell Tracker CM-DiI (DiI), a highly lipophilic non-cytotoxic fluorescent probe in common use in biology, from the freely-presented (non-micelle-incorporated) and micelle-incorporated states. DiI was effectively incorporated (>60%) into 25-50 nm diameter spherical micelles made from polycaprolactone-b-polyethylene oxide block copolymer. Confocal microscopy was used to evaluate the internalization of DiI into mixed neuron-glia cultures (2-14 days in vitro, 2DIV-14DIV). Incorporation of DiI into micelles strikingly reduced the rate and extent of its internalization in both 2DIV and 14DIV cultures. Both the age of the cultures and the block copolymer employed to construct the micelles significantly influence the internalization of micelle-incorporated probe.

PCL-b-PEO micelles as a delivery vehicle for FK506: assessment of a functional recovery of crushed peripheral nerve.

The aim of this work was to test in vivo a new block copolymer-based delivery system containing lipophilic drug FK506, known as Tacrolimus. Tacrolimus is currently used in clinics as an immunosupressant agent, and more recently it has been shown that it can exert neurotrophic effects. We prepared, characterized, and assessed polycaprolactone-b-polyethylenoxyde (PCL-b-PEO) micelles containing FK506 in vitro and in vivo. By using well-established animal model of peripheral nerve injury (crushed sciatic nerve), we show that the rate of functional recovery of injured nerve is significantly enhanced in rats treated with micellar FK506. These findings support the notion that PCL-b-PEO is a suitable polymer material for FK506 and suggest its wider applicability as a delivery vehicle for other biologically active, poorly soluble therapeutic agents.

Rigid piston approximation for computing the transfer function and angular response of a fiber-optic hydrophone.

The transfer function of a fiber-optic hydrophone (FOH) is computed for various fiber core radii. The hydrophone is modeled as a rigid disk, with plane waves impinging at normal or oblique incidence. The total sound field is written as the sum of the incident field and the field diffracted from the hydrophone. The diffracted field is approximated by the field generated by a vibrating planar piston in an infinite rigid baffle. For normal incidence and a pointlike fiber core, an analytical solution is presented. For finite fiber core radii, and for oblique incidence, the transfer functions are computed numerically. The calculated transfer functions exhibit an oscillatory frequency dependency that is most pronounced for small fiber cores. The solution for a core radius of 2.5 microm can be very well approximated by the analytical solution for a pointlike core at frequencies of up to 30 MHz. The results for normal incidence can be directly employed to deconvolute ultrasonic pressure signals measured with an FOH. From the transfer functions for oblique incidence, the angular response of the hydrophone is calculated. The angular response obtained here differs significantly from the model commonly used for piezoelectric hydrophones. The effective hydrophone radius derived from the angular response shows a strong frequency dependency. For low frequencies, it is found to be larger than the outer fiber radius, whereas it generally lies between the outer radius and the fiber core radius for frequencies above 10 MHz.

Polycaprolactone-b-poly(ethylene oxide) copolymer micelles as a delivery vehicle for dihydrotestosterone.

Block copolymer micelles formed from copolymers of poly(caprolactone)-b-poly(ethylene oxide) (PCL-b-PEO) were investigated as a drug delivery vehicle for dihydrotestosterone (DHT). The physical parameters of the PCL-b-PEO micelle-incorporated DHT were measured, including the loading capacity of the micelles for DHT, the apparent partition coefficient of DHT between the micelles and the external medium and the kinetics of the release of DHT from the micelle solution. The MTT survival assay was used to assess the in vitro biocompatibility of PCL-b-PEO micelles in HeLa cell cultures. The biological activity of the micelle-incorporated DHT was evaluated in HeLa cells which had been co-transfected with the expression vectors for the androgen receptor and the MMTV-LUC reporter gene. The PCL-b-PEO micelles were found to have a high loading capacity for DHT and the release profile of the drug from the micelle solution was found to be a slow steady release which continued over a 1-month period. The biological activity of the micelle-incorporated DHT was found to be fully retained.

5-Cyanovaleramide production using immobilized Pseudomonas chlororaphis B23.

A biocatalytic process for the hydration of adiponitrile to 5-cyanovaleramide has been developed which can be run to higher conversion, produces more product per weight of catalyst, and generates significantly less waste products than alternate chemical processes. The biocatalyst consists of Pseudomonas chlororaphis B23 microbial cells immobilized in calcium alginate beads. The cells contain a nitrile hydratase (EC 4.2.1.84) which catalyzes the hydration of adiponitrile to 5-cyanovaleramide with high regioselectivity, and with less than 5% selectivity to byproduct adipamide. Fifty-eight consecutive batch reactions with biocatalyst recycle were run to convert a total of 12.7 metric tons of adiponitrile to 5-cyanovaleramide. At 97% adiponitrile conversion, the yield of 5-cyanovaleramide was 13.6 metric tons (93% yield, 96% selectivity), and the total weight of 5-cyanovaleramide produced per weight of catalyst was 3150 kg/kg (dry cell weight).

Cellular internalization of PCL(20)-b-PEO(44) block copolymer micelles.

The cellular internalization of polycaprolactone-b-poly(ethylene oxide) (PCL(20)-b-PEO(44)) copolymer micelles were investigated in PC12 cells cultures. The micelles were found to be internalized into PC12 cells when followed over the 4-h incubation period. Also, the internalization process was found to fulfill the basic criteria for endocytotic uptake in that it was time, temperature, pH and energy dependent. In addition, the use of other pharmacological manipulations (hypertonic treatment, Brefeldin A) provide further evidence that the mode of cellular internalization is in fact endocytotic.

Polycaprolactone-b-poly(ethylene oxide) block copolymer micelles as a novel drug delivery vehicle for neurotrophic agents FK506 and L-685,818.

Micelles formed from polycaprolactone-b-poly(ethylene oxide) (PCL-b-PEO) diblock copolymers were investigated as a novel drug delivery system. The affinity of the micelles for hydrophobic solubilizates was assayed by determining the partition coefficient for the lipophilic compound, pyrene, between the micelles and water; the partition coefficient was found to be on the order of 10(2). The Trypan blue and Alamar blue survival assays were used to assess the in vitro biocompatibility of the micelles with PC 12 cells, MCF-7 breast cancer cells, and primary cultures of human microglia, astrocytes, and cortical neurons. The micelles were then studied as a delivery vehicle for the neurotrophic agents FK506 and L-685,818 in PC 12 cell cultures. In both cases, the micelle-incorporated drugs, in the presence of nerve growth factor (5 ng/mL), were able to promote the degree of differentiation of the PC 12 rat pheochromocytoma cells.

Transsphenoidal resection of pituitary adenoma: using a critical pathway.

The use of a transsphenoidal critical pathway can be a vital tool for critical care nurses in anticipating complications and improving patient outcomes. Complications such as diabetes insipidus and cerebrospinal fluid leak associated with posttranssphenoidal patients may result in prolonged hospitalization and worsened functional outcome. Implementing a transsphenoidal critical pathway for these patients can guide their care and alert critical care nurses to potential complications and their prevention and/or treatment.