Kuwait's marine biodiversity: Qualitative assessment of indicator habitats and species.

The tropical waters of the Northern Arabian Gulf have a long history of maritime resource richness. High levels of biodiversity result from the complex matrix of coastal habitats, coral reefs and sea grass beds that characterise the region. Insight into the ongoing health of such habitats and the broader Kuwait maritime environment can be gauged by the status of indicator species found within these habitats. Here we review information on the occurrence, distribution and threats to key marine habitats and associated indicator species to provide an updated assessment of the state of the Kuwait's marine biodiversity. Critical evaluation of historic data highlights knowledge gaps needed inform the focus of future monitoring and conservation efforts. This assessment is designed to evaluate performance against environmental policy commitments, while providing a solid foundation for the design of comprehensive marine ecosystem management strategies.

Importance of reference gene selection for articular cartilage mechanobiology studies.

OBJECTIVE: Identification of genes differentially expressed in mechano-biological pathways in articular cartilage provides insight into the molecular mechanisms behind initiation and/or progression of osteoarthritis (OA). Quantitative PCR (qPCR) is commonly used to measure gene expression, and is reliant on the use of reference genes for normalisation. Appropriate validation of reference gene stability is imperative for accurate data analysis and interpretation. This study determined in vitro reference gene stability in articular cartilage explants and primary chondrocytes subjected to different compressive loads and tensile strain, respectively. DESIGN: The expression of eight commonly used reference genes (18s, ACTB, GAPDH, HPRT1, PPIA, RPL4, SDHA and YWHAZ) was determined by qPCR and data compared using four software packages (comparative delta-Ct method, geNorm, NormFinder and BestKeeper). Calculation of geometric means of the ranked weightings was carried out using RefFinder. RESULTS: Appropriate reference gene(s) for normalisation of mechanically-regulated transcript levels in articular cartilage tissue or isolated chondrocytes were dependent on experimental set-up. SDHA, YWHAZ and RPL4 were the most stable genes whilst glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and to a lesser extent Hypoxanthine-guanine phosphoribosyltransferase (HPRT), showed variable expression in response to load, demonstrating their unsuitability in such in vitro studies. The effect of using unstable reference genes to normalise the expression of aggrecan (ACAN) and matrix metalloproteinase 3 (MMP3) resulted in inaccurate quantification of these mechano-sensitive genes and erroneous interpretation/conclusions. CONCLUSION: This study demonstrates that commonly used 'reference genes' may be unsuitable for in vitro cartilage chondrocyte mechanobiology studies, reinforcing the principle that careful validation of reference genes is essential prior to each experiment to obtain robust and reproducible qPCR data for analysis/interpretation.

Protein kinase R plays a pivotal role in oncostatin M and interleukin-1 signalling in bovine articular cartilage chondrocytes.

This study investigated whether treatment of articular cartilage chondrocytes with a combination of oncostatin M (OSM) and interleukin-1 (IL-1) could induce a degradative phenotype that was mediated through the protein kinase R (PKR) signalling pathway. High-density monolayer cultures of full depth, bovine chondrocytes were treated with a combination of OSM and IL-1 (OSM+IL-1) for 7 days. To inhibit the activation of PKR, a pharmacological inhibitor of PKR was added to duplicate cultures. Pro- and active matrix metalloproteinase-9 (MMP9) and MMP9 mRNA were significantly upregulated by OSM+IL-1 through a PKR dependent mechanism. ADAMTS4 and ADAMTS5 mRNA were also upregulated by OSM+IL-1. The upregulation of ADAMTS4 and ADAMTS5 were, in part, mediated through PKR. OSM+IL-1 resulted in a loss of type II collagen, which could not be rescued by PKR inhibition. OSM+IL-1 reduced the expression of COL2A1 (type II collagen), COL9A1 (type IX collagen), COL11A1 (type XI collagen), and ACAN (aggrecan) mRNAs. Expression of type II and XI collagen and aggrecan was reduced further when PKR was inhibited. OSM+IL-1 resulted in an 11-fold increase in TNFa mRNA which was, in part, mediated through the PKR pathway. This study demonstrates, for the first time, that a number of catabolic and pro-inflammatory effects known to be important in human arthritis and induced by OSM and IL-1, are mediated by the PKR signalling pathway.

Human-immunodeficiency-virus-negative, human-herpes-virus-8-negative abdominal cavity primary effusion lymphoma.

Primary effusion lymphoma (PEL) is an unusual class of non-Hodgkin's lymphoma, which is characterised by lymphomatous effusions in body cavities, but no associated mass lesions. It is usually associated with an immunodeficient state most often with the human immunodeficiency virus (HIV). We describe a case of a man with HIV-negative, human-herpes-virus-8 (HHV8)-negative PEL, with a history of heavy alcohol intake. The abdominal cavity was the only area involved; no solid tumour masses were observed on scanning, and the bone-marrow investigations were normal. The ascites contained numerous pleomorphic lymphoid, lymphoplasmacytoid cells of B-cell origin. The immunophenotyping was moderately positive for CD 38 and 138, and strongly positive for Ki 67. It is postulated that the immunosuppressed state in this patient may have been caused by the long history of heavy alcohol intake.

Subacute thyroiditis in an immunosuppressed patient.

Subacute thyroiditis is a well-recognized cause of transient thyrotoxicosis, resulting from a destruction injury to the thyroid. The pathogenesis of this condition is not completely understood and there is debate regarding the extent of the contribution of autoimmunity and external agents, such as infections, to this process. We present the first reported case of subacute thyroiditis in a patient who had been on chronic lithium therapy as well as long-term immunosuppression, with cyclosporin and prednisolone, following an allogeneic bone marrow transplant. We speculate that this case suggests a minimal role of autoimmunity in the development of subacute thyroiditis.

H RAS mutations in haematologically normal individuals.

INTRODUCTION: Point mutations in N, K and H RAS have been found in adverse haematological malignancies. The background frequency of RAS mutations in the normal population has yet to be determined. Here we report the results of a screen for RAS mutations from normal individuals. MATERIALS AND METHODS: DNA from peripheral blood or bone marrow from 115 haematologically normal individuals was screened for point mutations in N, K and H RAS, at amino acid positions 12, 13 and 61. The screening was done using polymerase chain reaction and oligonucleotide hybridisation and candidate mutations were subsequently confirmed by cloning and sequencing. RESULTS AND CONCLUSION: Point mutations were identified in DNA from two of the 115 individuals. Both mutations resulted in an amino acid substitution at position 12 in H RAS. Both individuals with detectable H RAS mutations remain haematologically normal.

Four new mutations in the erythroid-specific 5-aminolevulinate synthase (ALAS2) gene causing X-linked sideroblastic anemia: increased pyridoxine responsiveness after removal of iron overload by phlebotomy and coinheritance of hereditary hemochromatosis.

X-linked sideroblastic anemia (XLSA) in four unrelated male probands was caused by missense mutations in the erythroid-specific 5-aminolevulinate synthase gene (ALAS2). All were new mutations: T647C, C1283T, G1395A, and C1406T predicting amino acid substitutions Y199H, R411C, R448Q, and R452C. All probands were clinically pyridoxine-responsive. The mutation Y199H was shown to be the first de novo XLSA mutation and occurred in a gamete of the proband's maternal grandfather. There was a significantly higher frequency of coinheritance of the hereditary hemochromatosis (HH) HFE mutant allele C282Y in 18 unrelated XLSA hemizygotes than found in the normal population, indicating a role for coinheritance of HFE alleles in the expression of this disorder. One proband (Y199H) with severe and early iron loading coinherited HH as a C282Y homozygote. The clinical and hematologic histories of two XLSA probands suggest that iron overload suppresses pyridoxine responsiveness. Notably, reversal of the iron overload in the Y199H proband by phlebotomy resulted in higher hemoglobin concentrations during pyridoxine supplementation. The proband with the R452C mutation was symptom-free on occasional phlebotomy and daily pyridoxine. These studies indicate the value of combined phlebotomy and pyridoxine supplementation in the management of XLSA probands in order to prevent a downward spiral of iron toxicity and refractory anemia.

RAS, FMS and p53 mutations and poor clinical outcome in myelodysplasias: a 10-year follow-up.

The molecular mechanisms underlying the development and evolution of myelodysplastic syndrome (MDS) are largely unknown. The increasing number of blast cells in the bone marrow correlate with poor prognosis and risk of developing acute leukemia. Such progression is frequently associated with increasing chromosomal abnormalities and genetic mutations. A cohort of 75 MDS patients were investigated for RAS, FMS and p53 mutations, and these molecular findings were related to cytogenetics, clinical status, transformation to acute leukemia, prognostic scores and survival. A mutation incidence of 57% (43/75) was found, with 48% (36/75) RAS mutations, 12% (9/75) FMS mutations and 8% (4/50) p53 mutations. The mutation status for RAS and FMS was related to MDS subgroup, increasing with poor-risk disease. The highest incidence was in the chronic myelomonocytic leukemia (CMML) subgroup. The most frequent RAS mutations were of codon 12 and a predominance of FMS codon 969 mutations was observed. A statistically significant increased frequency of transformation to AML was observed in MDS patients harboring RAS or FMS mutations (P < 0.02). Patients with oncogene mutations had a significantly poorer survival compared with those without mutations at 2 years and at the end of the period of follow-up (P < 0.02). Multivariate analysis including mutation, age, gender, diagnosis (FAB), cytogenetics and International score shows that the International score and mutation and age is the best predictive model of a poor outcome, (P < 0.0001). When the analysis was undertaken without the International score, mutation and gender was the best predictor of poor survival (P = 0.005). This study shows that oncogene mutation, indicative of genetic instability, is associated with disease progression and poor survival in MDS.

Oncogenic RAS genes impair erythroid differentiation of erythroleukaemia cells.

RAS mutations occur frequently in acute myeloid leukaemia and myelodysplasia, suggesting a functional role for this oncogene in leukaemogenesis. We show here, for the first time, that both N-RAS and H-RAS can impair erythroid differentiation of erythroleukaemia cells induced with hexamethylene bisacetamide. Transformation by RAS allowed extended proliferation in the presence of inducer and also inhibited maturation as measured by impaired haemoglobinization and reduction in cell size. These data provide an interesting counterpoint to the effect of mutant RAS on monocytic cells, where it has a potentiating effect on differentiation and may indicate a causal link between the activation of RAS and erythroid lineage dysplasia in preleukaemia.

Covert poisoning with difenacoum: clinical and toxicological observations.

1. The coumarin anticoagulant difenacoum was detected by high performance liquid chromatography (HPLC) with multi-wavelength UV detection in plasma from a 41 years old man who presented with a severe deficiency of vitamin K-dependent clotting factors of unknown aetiology. A longitudinal toxicological study of the consequent coagulopathy is described. 2. Plasma concentrations of difenacoum declined from 0.97 to 0.11 mgl-1 in 47 days with a terminal half life of 11.7 days. Rifampacin (300 mg bd) had no apparent effect on the terminal half life of the drug. Subsequently plasma concentrations of difenacoum and descarboxyprothrombin (DCP) unexpectedly increased. 3. Seven months after exposure clotting times were prolonged. The patient continued to have episodes of epistaxis, haematoma, purpurae and bruising and he required frequent treatment with Fresh Frozen Plasma in additional to oral phylloquinone (200 mg day-1). 4. Intermittent and unexpected increases in plasma concentrations of difenacoum and descarboxypro-thrombin suggested that covert, repeated ingestion of the anticoagulant was the most likely cause of the poisoning. The measurement of low concentrations of plasma phylloquinone except following supervised ingestion of the vitamin indicated that as an outpatient, the subject was not compliant with treatment despite his protestations to the contrary. He continued to deny this even when confronted by laboratory findings and at no time did he ever admit to self-poisoning.

A lesson from persistently elevated aspartate transaminases (AST) in a patient with severe haemophilia A.

Prior to the availability of test for hepatitis 'C' in 1989, any elevated AST in a pooled blood product recipient was presumed to be from non-A-non-B hepatitis. We report a patient who received pooled factor VIII in 1984 and had persistently elevated AST which proved to be of nonhepatic origin.

Aberrant expression of p21RAS but not p120GAP is a common feature of myelodysplasia.

The RAS gene family encodes signal transducing proteins which are involved in the regulation of cell growth and differentiation. Constitutively 'activating' point mutations of RAS have been detected at high frequency in preleukaemia and acute leukaemia, however, the distribution of expression of p21RAS in normal or preleukaemic primary haematopoietic cells has not been studied. We have examined the expression of p21RAS and its negative regulator/downstream effector, p120GAP, in combination with lineage-specific cluster of differentiation markers in normal and preleukaemic myeloid bone marrow cells using flow cytometry. Normal bone marrow was characterized by low and uniform levels of p21RAS expression throughout all lineages analysed. In contrast, 28% (9/32) of patients with myelodysplastic syndrome (MDS) over-expressed p21RAS. In three of these patients a single over-expressing peak of p21RAS expression was observed, with no evidence of a population exhibiting expression within the normal range. In 6/32 MDS patients over-expression of p21RAS was observed only in a subpopulation of the myeloid cells. Follow-up samples were analysed in three of these six patients; over-expression was confirmed in each patient and in two patients a relative expansion of the over-expressing cell population was observed. Eight out of nine of the patients with aberrant p21RAS expression were diagnosed with low-risk MDS. From 21 MDS patients screened for p120GAP expression, no reduction or loss of expression was observed, however, one AML patient demonstrated a heterogeneous pattern of expression. p120GAP expression was lower (P < 0.05) in the AML group than in normals. The results of the study suggest that over-expression of the RAS gene product, p21RAS, may represent an alternative or additional mechanism of activation of the RAS signalling pathway and that this may play a role in leukaemogenesis, however, there is no evidence from this study that loss of p120GAP expression is a feature of myelodysplasia.

Late-onset X-linked sideroblastic anemia. Missense mutations in the erythroid delta-aminolevulinate synthase (ALAS2) gene in two pyridoxine-responsive patients initially diagnosed with acquired refractory anemia and ringed sideroblasts.

X-linked sideroblastic anemia (XLSA) is caused by mutations of the erythroid-specific delta-aminolevulinate synthase gene (ALAS2) resulting in deficient heme synthesis. The characteristic hypochromic, microcytic anemia typically becomes manifest in the first three decades of life. Hematologic response to pyridoxine is variable and rarely complete. We report two unrelated cases of highly pyridoxine-responsive XLSA in geriatric patients previously diagnosed with refractory anemia and ringed sideroblasts. A previously unaffected 77-yr-old male and an 81-yr-old female were each found to have developed severe hypochromic, microcytic anemia with ringed sideroblasts in the bone marrow, which responded dramatically to pyridoxine with normalization of hemoglobin values. Sequence analysis identified an A to C transversion in exon 7 (K299Q) of the ALAS2 gene in the male proband and his daughter. In the female proband a G to A transition was identified in exon 5 (A172T). This mutation resulted in decreased in vitro stability of bone marrow delta-aminolevulinate synthase activity. Each patient's recombinant mutant ALAS2 enzyme had marked thermolability. Addition of pyridoxal 5'-phosphate in vitro stabilized the mutant enzymes, consistent with the observed dramatic response to pyridoxine in vivo. This late-onset form of XLSA can be distinguished from refractory anemia and ringed sideroblasts by microcytosis, pyridoxine-responsiveness, and ALAS2 mutations. These findings emphasize the need to consider all elderly patients with microcytic sideroblastic anemia as candidates for XLSA, especially if pyridoxine responsiveness is demonstrated.

Elevated levels of p53 protein in the neutrophils and monocytes of a patient with chronic idiopathic thrombocytopenic purpura or possible early myelodysplasia?

A 68 year old female who presented with long-term thrombocytopenia was clinically diagnosed as having chronic idiopathic thrombocytopenia purpura (ITP). Increased levels of the tumour suppressor p53 protein were detected by immunohistochemistry in the neutrophils and some monocytes of the peripheral blood preparation using the antibody DO-1, recognizing mutant and wild type p53 protein conformations. However, no positive staining in the peripheral blood samples from 41 myelodysplasias (MDS) and six normal individuals was observed. Single-stranded conformational polymorphism analysis performed on DNA extracted from the cytospin preparations from this patient indicated no mutations in exons 5-8 of the p53 gene. This report describes the unusual detection of elevated p53 protein in a non-neoplastic condition by immunohistochemistry using the antibody DO-1. This unexpected finding raises the possibility of classifying such patients as early MDS on the basis of their p53 status.

In vitro drug resistance in acute myeloid and chronic B-lymphocytic leukaemic blasts and in normal blood and marrow populations.

The sensitivities of AML and BCLL blasts to daunorubicin have been determined, using an in vitro (MTT) assay of resistance, and compared with the sensitivities of normal haemopoietic populations and cells of the multidrug-resistant, T-lymphoid line CEM VLB100; The role of the drug-efflux pump, P-glycoprotein, was determined by adding the 'modifier' cyclosporin and by measuring numbers of P-glycoprotein positive cells by immunofluorescence. ID50s for 17 cases of de novo AML varied from 5 to 300 ng/ml giving a median of 105 ng/ml which was similar to the median of 11 normal marrow mononuclear cell preparations (80 ng/ml) but considerably less than the median ID50 of eight blood lymphocyte samples (3500 ng/ml). ID50s for five relapsed and two refractory AML samples ranged from 27 to 240 ng/ml, well within the de novo range: we had obtained presentation samples for two of these and, in both cases, ID50s were lower at relapse. ID50s, however, were raised in seven marrow mononuclear cell populations taken soon after remission induction (ID50 for remission MNC and normal MNC = 200 and 80 ng/ml, respectively); this may reflect either a property of regenerating populations, or an activation of cellular resistance mechanisms following chemotherapy. ID50s for 17 cases of BCLL ranged from 7 to 200 ng/ml with a median of 48 ng/ml which was significantly lower than the ID50 of AML blasts or of blood lymphocytes. Cyclosporin induced less than two-fold reductions in ID50s of blood lymphocytes, marrow mononuclear cells and de novo AML and BCLL blasts despite giving log reversals in resistance in the CEM VLB100 line. This reflected numbers of P-glycoprotein positive cells in our samples, which were high in CEM VLB100 but low in fresh normal or leukaemic cell suspensions. For both de novo AML and BCLL groups, however, the change in ID50, on addition of cyclosporin, was significant. These data imply a minor role for P-glycoprotein in drug resistance of leukaemic blasts. Nevertheless, there was a positive correlation between daunorubicin ID50s in de novo AML and time to remission which confirms that in vitro chemosensitivity assays can provide a useful measure of in vivo resistance.