Canine DLA-79 gene: an improved typing method, identification of new alleles and its role in graft rejection and graft-versus-host disease.

Developing a preclinical canine model that predicts outcomes for hematopoietic cell transplantation in humans requires a model that mimics the degree of matching between human donor and recipient major histocompatibility complex (MHC) genes. The polymorphic class I and class II genes in mammals are typically located in a single chromosome as part of the MHC complex. However, a divergent class I gene in dogs, designated dog leukocyte antigen-79 (DLA-79), is located on chromosome 18 while other MHC genes are on chromosome 12. This gene is not taken into account while DLA matching for transplantation. Though divergent, this gene shares significant similarity in sequence and exon-intron architecture with other class I genes, and is transcribed. Little is known about the polymorphisms of DLA-79 and their potential role in transplantation. This study was aimed at exploring the reason for high rate of rejection seen in DLA-matched dogs given reduced intensity conditioning, in particular, the possibility that DLA-79 allele mismatches may be the cause. We found that about 82% of 407 dogs typed were homozygous for a single, reference allele. Owing to the high prevalence of a single allele, 87 of the 108 dogs (∼80%) transplanted were matched for DLA-79 with their donor. In conclusion, we have developed an efficient method to type alleles of a divergent MHC gene in dogs and identified two new alleles. We did not find any statistical correlation between DLA-79 allele disparity and graft rejection or graft-versus-host disease, among our transplant dogs.

Canine bone marrow-derived mesenchymal stromal cells suppress alloreactive lymphocyte proliferation in vitro but fail to enhance engraftment in canine bone marrow transplantation.

Stable mixed hematopoietic chimerism has been consistently established in dogs who were mildly immunosuppressed by 200 cGy of total body irradiation (TBI) before undergoing dog leukocyte antigen (DLA)-identical bone marrow (BM) transplantation and who received a brief course of immunosuppression with mycophenolate mofetil (28 days) and cyclosporine (35 days) after transplantation. However, when TBI was reduced from 200 to 100 cGy, grafts were nearly uniformly rejected within 3-12 weeks. Here, we asked whether stable engraftment could be accomplished after a suboptimal dose of 100 cGy TBI with host immunosuppression enhanced by donor-derived mesenchymal stromal cells (MSCs) given after transplantation. MSCs were cultured from BM cells and evaluated in vitro for antigen expression. They showed profound immunosuppressive properties in mixed lymphocyte reactions (MLRs) in a cell dose-dependent manner not restricted by DLA. MSC and lymphocyte contact was not required, indicating that immunosuppression was mediated by soluble factors. Prostaglandin E2 was increased in culture supernatant when MSCs were cocultured in MLRs. The addition of indomethacin restored lymphocyte proliferation in cultures containing MSCs. MSCs expressed CD10, CD13, CD29, CD44, CD73/SH-3, CD90/Thy-1, and CD106/VCAM-1. For in vivo studies, MSCs were injected on the day of BM grafting and on day 35, the day of discontinuation of posttransplantation cyclosporine. MSCs derived from the respective BM donors failed to avert BM graft rejection in 4 dogs who received DLA-identical grafts after nonmyeloablative conditioning with 100 cGy TBI in a time course not significantly different from that of control dogs not given MSCs. Although the MSCs displayed in vitro characteristics similar to those reported for MSCs from other species, their immunosuppressive qualities failed to sustain stable BM engraftment in vivo in this canine model.

Sequencing complex polysaccharides.

Although rapid sequencing of polynucleotides and polypeptides has become commonplace, it has not been possible to rapidly sequence femto- to picomole amounts of tissue-derived complex polysaccharides. Heparin-like glycosaminoglycans (HLGAGs) were readily sequenced by a combination of matrix-assisted laser desorption ionization mass spectrometry and a notation system for representation of polysaccharide sequences. This will enable identification of sequences that are critical to HLGAG biological activities in anticoagulation, cell growth, and differentiation.

Renal FNA-based typing of renal masses remains a useful adjunctive modality: evaluation of 31 renal masses with correlative histology.

OBJECTIVES: Given the advances in renal imaging modalities in the recent years, a greater number of renal cell carcinomas (RCCs) with tumour size of <3 cm are being detected radiologically. Consequently, there is a pressing need for accurate typing of RCCs which, in turn, will aid in selection of cases of nephron-sparing surgery. METHODS: A total of 31 cases of renal masses with available fine needle aspiration (FNA) material and concomitant histopathology details were retrieved. They included 27 RCCs (17 clear cells, eight papillary and two chromophobe), one oncocytoma, one liposarcoma and two benign lesions - one xanthogranulomatous pyelonephritis (XPN) and one benign cyst. Two investigators reviewed all FNA material. The degree of concordance between cytological typing and histological typing was assessed. RESULTS: There was excellent agreement between the FNA typing and the final diagnosis, with correct classification in 28 of 31 cases. Among the three discordant cases, two were RCCs. The first was a papillary RCC (PRCC) that was misdiagnosed on FNA as clear cell RCC. Another case that was typed as a PRCC on final histopathology was diagnosed 'suspicious cells' on FNA. The third case was an XPN that was misdiagnosed on FNA as RCC with necrosis. CONCLUSIONS: There is an excellent concordance (90.3%) between the FNA diagnosis and the final histological diagnosis, especially in RCCs. There is a tendency for misdiagnosis with PRCC. Lesions with extensive necrosis and relatively insufficient diagnostic material on FNA specimens must be interpreted with caution. Better concordance might be observed with more extensive sampling.

Development of risk assessment tool for foundry workers.

UNLABELLED: Occupational ill-health and work-related disorders are predominant in manufacturing industries due to the inevitable presence of manual work even after several waves of industrial automation and technological advancements. Ergonomic risk factors and musculoskeletal disorders like low-back symptoms have been noted amongst foundry workers. OBJECTIVE: The purpose of this study was to formulate and develop a Physical Effort Index to assess risk factor. SCOPE: The questionnaire tool applicable to foundry environment has been designed and validated. The data recorded through survey across the foundries has been subjected to regression analysis to correlate between proposed physical effort index and the standard Borg's Ratings of Perceived Exertion (RPE) scale. RESULTS: The physical efforts of sixty seven workers in various foundry shop floors were assessed subjectively. The 'Job factors' and 'Work environment' were the two major parameters considered in assessing the worker discomfort level at workplace. A relation between Borg's RPE scale and the above two parameters were arrived at, through regression analysis. CONCLUSIONS: The study demonstrates the prevalence of risk factors amongst foundry workers and the effectiveness of the proposed index in estimating the risk factor levels. RELEVANCE TO THE INDUSTRY: The proposed tool will assist foundry supervisors and managers to assess the risk factors and helps in better understanding of the workplace to avoid work-related disorders, ensuring better output.

Thirteen novel canine dog leukocyte antigen-88 alleles identified by sequence-based typing.

Major histocompatibility complex (MHC) genes in mammals include highly polymorphic class I and class II genes that are critical for donor-recipient matching for transplantation. Dogs have served as an effective, directly translatable model for stem/progenitor cell transplantation. Previous analyses of MHC class I genes in dogs point to a single highly polymorphic gene, dog leukocyte antigen (DLA)-88, as an important factor in the success or failure of hematopoietic stem cell transplants. Fifty-nine DLA-88 alleles have been identified and reported so far. Here, we extend this list by presenting 13 novel DLA-88 alleles found in domestic dogs.

Analysis of expression of heat shock protein-90 (HSP90) and the effects of HSP90 inhibitor (17-AAG) in multiple myeloma.

Heat shock protein 90 (HSP90) is required for structural folding and maintenance of conformational integrity of various proteins, including several associated with cellular signaling. Recent studies utilizing 17-allylamino-17-demethoxygeldanamycin (17-AAG), an inhibitor of HSP90, demonstrated an antitumor effect in solid tumors. To test whether HSP90 could be targeted in multiple myeloma (MM) patients, we first investigated expression of HSP90 by immunofluorescence and flow cytometric analysis in a myeloma cell line (U266) and primary myeloma cells. Following demonstration of HSP90 expression in myeloma cells, archival samples of 32 MM patients were analysed by immunoperoxidase staining. Myeloma cells in all patients showed strong cytoplasmic expression of HSP90 in all samples and 55% also demonstrated concurrent nuclear immunopositivity. Treatment of U266 and primary MM cells with 17AAG resulted in significantly increased apoptosis compared to untreated control cells. Analysis of anti-apoptotic BCL2 family proteins and akt in MM cells incubated with 17-AAG revealed down-regulation of BCL-2, BCL-XL, MCL-1 and akt. Furthermore, although a low concentration of bortezomib resulted in no cell death, a combination of 17AAG and bortezomib treatment revealed a synergistic apoptotic effect on the U266 cell line. These data suggest that targeted inhibition of HSP90 may prove to be a valid and innovative strategy for the development of future therapeutic options for MM patients.

Heparin and heparan sulfate: biosynthesis, structure and function.

Heparin and heparan sulfate glycosaminoglycans are acidic complex polysaccharides found on the cell surface and in the extracellular matrix. Recent progress has uncovered a virtual explosion of important roles of these biopolymers in fundamental biological processes. Advances in the understanding of biosynthesis and structure and the development of novel analytical methods for composition and sequence analysis have provided remarkable insights into structure/function relationships of these complex and once elusive polysaccharides.

Somatostatin receptor subtype expression in human thyroid and thyroid carcinoma cell lines.

Somatostatin (SRIH) analogs can suppress the proliferation of human differentiated thyroid carcinoma cell lines that express SRIH receptors (SSTRs) demonstrated by radioligand binding analysis. Five distinct human SSTR subtypes (hSSTR1-5) that bind native SRIH exhibit diverse affinities to a wide range of SRIH analogs. Reverse transcriptase-PCR amplification of ribonucleic acids (RNAs) obtained from normal thyroid tissues and nine human thyroid carcinoma cell lines, grown as monolayer cultures and xenograft tumors in nude mice, were used to discriminate expression of SSTR subtype messenger RNAs (mRNAs). The cell lines were derived from a follicular adenoma (KAK-1), two follicular carcinomas (MRO-87 and WRO-82), two papillary carcinomas (NPA87 and KAT-10), and four anaplastic thyroid carcinomas (DRO-90, ARO-81, KAT-4, and KAT-18). Most thyroid cancer cell line monolayers and xenografts expressed SSTR3 and SSTR5 mRNAs. SSTR1 expression was more varied between monolayers and xenografts, whereas SSTR2 mRNA was only faintly detectable at the most extreme resolution. SSTR4 mRNA was faintly positive in only one anaplastic carcinoma xenograft. Normal thyroid also expressed SSTR3 and SSTR5 mRNAs, with only faint expression of SSTR1 and SSTR2 mRNAs (in one of five and three of five samples, respectively). SSTR mRNA expression was dependent upon in vitro culture conditions, as xenograft SSTR mRNA expression tended to decrease compared to that in each respective monolayer culture. Characterization of SSTR subtype expression in human thyroid carcinomas may permit targeting of specific SRIH analogs to inhibit proliferation of differentiated and anaplastic thyroid carcinomas in patients.

Restoration of iodide uptake in dedifferentiated thyroid carcinoma: relationship to human Na+/I-symporter gene methylation status.

Disseminated dedifferentiated thyroid epithelial carcinoma, which cannot sufficiently concentrate therapeutic radioiodide, is a terminal disease without any effective systemic treatment or chemotherapy. This is a likely consequence of loss of human sodium-iodide symporter (hNIS) function. We hypothesized that hNIS transcriptional failure in thyroid carcinoma could be consequent to methylation of DNA in critical regulatory regions and could be reversed with chemical demethylation treatment. Analysis of hNIS messenger ribonucleic acid (mRNA) expression in 23 tumor samples revealed that although loss of this expression corresponded to loss of clinical radioiodide uptake, some thyroid carcinomas with hNIS mRNA expression did not concentrate iodide, suggesting additional posttranscriptional mechanisms for loss of hNIS function. In addition, analysis of DNA methylation in CpG-rich regions of the hNIS promoter extending to the first intron failed to define specific methylation patterns associated with transcriptional failure in human thyroid tumor samples. In seven human thyroid carcinoma cell lines lacking hNIS mRNA, treatment with 5-azacytidine or sodium butyrate was able to restore hNIS mRNA expression in four cell lines and iodide transport in two cell lines. Investigation of methylation patterns in these cell lines revealed that successful restoration of hNIS transcription was associated with demethylation of hNIS DNA in the untranslated region within the first exon. This was also associated with restoration of expression of thyroid transcription factor-1. These results suggest a role for DNA methylation in loss of hNIS expression in thyroid carcinomas as well as a potential application for chemical demethylation therapy in restoring responsiveness to therapeutic radioiodide.

A frequently amplified region in Leishmania contains a gene conserved in prokaryotes and eukaryotes.

A 27.5-kb sequence that is present in an approx. 2-Mb chromosome in Leishmania also occurs as an inverted dimer in a multicopy, 55-kb circular molecule (LD1) in Leishmania infantum ITMAP263. Sequence analysis of a 7100-bp cloned segment from the circular molecule revealed three open reading frames (ORFs). The ORFs are likely to have protein coding function by a number of criteria, including Northern blot analyses. The amino acid (aa) sequences deduced from two ORFs showed no similarity to other sequences in the databases. The C-terminal aa sequence from the third ORF is related (22-29% identity, 57-71% similarity) to a family of genes conserved in bacteria and humans. One member (sfhB) of the gene family in Escherichia coli appears to have a role in regulation of cell growth.

On the regulation of fibroblast growth factor activity by heparin-like glycosaminoglycans.

The activity of several angiogenic factors, like fibroblast growth factors (FGF), is modulated by heparin-like glycosaminoglycans (HLGAGs), which are acidic polysaccharides present in the extracellular matrix and at the cell surface. FGF binds to HLGAG in the matrix, where it is sequestered in a protected and inactive form, and at the cell surface, where it activates its cognate signaling receptor. Here we review recent progress in elucidating how HLGAG regulates FGF-induced signal transduction. Data from crystal structures of FGF complexed to active and inactive oligosaccharides is analyzed in the context of current models for HLGAG modulation of FGF activity. We propose that FGF can dimerize in several different modes, stabilized by HLGAGs. Individual dimer modes may represent active or inactive FGF and it is possible that different HLGAGs preferentially stabilize different FGF dimer modes. Understanding HLGAG-FGF interactions can provide leverage for new approaches to therapeutic control of angiogenesis.

Radiation hazards from 241Am sources used in thyroid studies.

The neutron and gamma photon doses corresponding to the neck and eye level from an 241AmO2 source used in thyroid studies have been theoretically estimated. The radiation hazard to the patient is found to be not significant.

Cloning of the human sodium-iodide symporter promoter and characterization in a differentiated human thyroid cell line, KAT-50.

Elucidation of the regulation of human sodium-iodide symporter (hNIS) gene expression is critical to understanding its effects on iodide concentration abilities of thyroid and thyroid carcinomas. To explore this issue, a 1.2-kb portion of the 5'-flanking region of the hNIS gene was isolated and characterized. Transient transfections with chimeric luciferase-reporter constructs into a differentiated human thyroid cell line, KAT-50, as well as non-thyroidal cells, defined an active promoter with tissue-specificity. Reverse-transcriptase polymerase chain reaction analysis for hNIS mRNA expression in normal human tissues was positive in thyroid, salivary gland, omentum, and gallbladder. KAT-50 cells expressed hNIS mRNA and were capable of thyrotropin-responsive iodide uptake in vitro. Despite the failure to exhibit iodide concentration in clinical anaplastic carcinoma tumors, 4 of 5 cell lines from this cancer phenotype expressed hNIS mRNA. Definition of the active promoter provides further insights and tools to uncover new approaches to use of radioiodine for therapy of thyroid carcinomas.

Polyamine synthesis and transport inhibition in a human anaplastic thyroid carcinoma cell line in vitro and as xenograft tumors.

Polyamines are essential cellular components for neoplastic transformation and cell proliferation. Antineoplastic efforts that inhibit polyamine synthesis are insufficient to induce cytotoxicity, due to compensatory induction of polyamine transport. Treatment of an anaplastic human thyroid carcinoma cell line (DRO90-1) with a novel polymeric spermine conjugate (polyspermine; PSpm) caused in vitro cytotoxicity and inhibited the growth of xenograft tumors at low concentrations. Similar in vitro antineoplastic effects were noted with two other human anaplastic thyroid carcinoma cell lines. This coincided with inhibition of polyamine uptake and synthetic enzyme activities, with reduced ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (SAM-DC) but increased spermidine/spermine N1-acetyltransferase (SSAT) activities, as measured in DRO90-1 cells. In subsequent studies using these cells, PSpm was effective in reducing the intracellular levels of all polyamines in vitro, resulting in cytotoxicity that was not reversed by administration of extracellular polyamines. Low-dose PSpm inhibited tumor growth in vivo, but high doses of PSpm potentiated xenograft tumor growth. PSpm degradation products produced with in vivo treatment may be produced that function as substrates for polyamine biosynthesis. These studies suggest that polyamine metabolism inhibition is a viable target for antineoplastic therapy of anaplastic thyroid carcinoma, although the in vivo response to PSpm suggests that this agent will have limited clinical utility.

Single-stranded DNA and RNA targeted triplex-formation: UV, CD and molecular modeling studies of foldback triplexes containing different RNA, 2'-OMe-RNA and DNA strand combinations.

We studied the influence of different 2'-OMe-RNA and DNA strand combinations on single strand targeted foldback triplex formation in the Py.Pu:Py motif using ultraviolet (UV) and circular dichroism (CD) spectroscopy, and molecular modeling. The study of eight combinations of triplexes (D.D:D, R*.D:D, D.D:R*, R*.D:R*, D.R:D, R*.R:D, D.R:R*, and R*.R:R*; where the first, middle, and last letters stand for the Hoogsteen Pyrimidine, Watson-Crick [WC] purine and WC pyrimidine strands, respectively, and D, R and R* stand for DNA, RNA and 2'-OMe-RNA strands, respectively) indicate more stable foldback triplex formation with a DNA purine strand than with an RNA purine strand. Of the four possible WC duplexes with RNA/DNA combinations, the duplex with a DNA purine strand and a 2'-O-Me-RNA pyrimidine strand forms the most thermally stable triplex, although its thermal stability is the lowest of all four duplexes. Irrespective of the duplex combination, a 2'-OMe-RNA Hoogsteen pyrimidine strand forms a stable foldback triplex over a DNA Hoogsteen pyrimidine strand confirming the earlier reports with conventional and circular triplexes. The CD studies suggest a B-type conformation for an all DNA homo-foldback triplex (D.D:D), while hetero-foldback triplex spectra suggest intermediate conformation to both A-type and B-type structures. A novel molecular modeling study has been carried out to understand the stereochemical feasibility of all the combinations of foldback triplexes using a geometric approach. The new approach allows use of different combinations of chain geometries depending on the nature of the chain (RNA vs. DNA).

Californium-252 line sources: experimental verification of depth dose data using NTA films and dosage tables based on Paterson and Parker rules.

Californium-252 promises to be an effective radium substitute in brachytherapy. In certain situations, such as cancer of the uterus, the dose rate near a linear source may be of interest. Paterson and Parker have tabulated the number of milligram hours for various active lengths of radium sources to give 1000 rad at different distances from the centre of the source. This paper presents similar results for Cf-252 (microgram hours to give 1000 rad neutron dose). The paper also reports the results of experimental measurements of neutron depth dose distributions for both single line sources and arrays. Kodak NTA films and a phantom were used for the study. The experimental results are found to agree well with the theoretically predicted results.