Ten patient stories illustrating the extraordinarily diverse clinical features of patients with thrombotic thrombocytopenic purpura and severe ADAMTS13 deficiency.

Patients with thrombotic thrombocytopenic purpura (TTP) and severe ADAMTS13 deficiency are often considered to have typical clinical features. However, our experience is that there is extraordinary diversity of the presenting features and the clinical courses of these patients. This diversity is illustrated by descriptions of 10 patients. The patients illustrate that ADAMTS13 activity may be normal initially but severely deficient in subsequent episodes. Patients with established diagnoses of systemic infection as the cause of their clinical features may have undetectable ADAMTS13 activity. Patients may have a prolonged prodrome of mild symptoms with only microangiopathic hemolytic anemia and thrombocytopenia or they may have the sudden onset of critical illness with multiple organ involvement. Patients may die rapidly or recover rapidly; they may require minimal treatment or extensive and prolonged treatment. Patients may have acute and severe neurologic abnormalities before microangiopathic hemolytic anemia and thrombocytopenia occur. Patients may have concurrent TTP and systemic lupus erythematosus. Patients may have hereditary ADAMTS13 deficiency as the etiology of their TTP rather than acquired autoimmune ADAMTS13 deficiency. These patients' stories illustrate the clinical spectrum of TTP with ADAMTS13 deficiency and emphasize the difficulties of clinical diagnosis.

Amplification refractory mutation system, a highly sensitive and simple polymerase chain reaction assay, for the detection of JAK2 V617F mutation in chronic myeloproliferative disorders.

An acquired mutation in Janus kinase 2 (JAK2), V617F, has recently been identified in human myeloproliferative disorders. Detection of the mutation is helpful in differential diagnosis, prognosis, and predication of therapeutic response. Because the mutation can be present in a small proportion of granulocytic populations in myeloproliferative disorder patients, a highly sensitive detection method is required. In this study, we systematically optimized the reaction conditions of a published amplification refractory mutation system-polymerase chain reaction research protocol to make it a robust clinical diagnostic test. The modifications led to a clear demonstration of the V617F mutation in a patient who would have been easily missed by the original amplification refractory mutation system-polymerase chain reaction assay. The test detects the V617F mutation not only with a high analytic sensitivity of 0.05 to 0.1% but also with a high diagnostic specificity of 99%. In addition, the assay has the ability to distinguish cases with only mutant alleles from cases with mixed normal and mutant alleles. The assay is fast and easy to perform, and no special equipment other than thermocyclers is required. All these features make the assay readily and broadly applicable in clinical molecular diagnostic laboratories.

[Expression level of TRF1 protein in human acute leukemia and its relationship with activity of telomerase].

The study was aimed to investigate the expression level of TRF1 protein in human acute leukemia and relationship between expression level of TRF1 protein and activity of telomerase. A quantitative Western blot technique was developed using anti-TRF1(33 - 277) monoclonal antibody and GST-TRF1 fusion protein as a standard to further determine the expression level of TRF1 protein in total proteins extracted from clinical specimens. 20 cases of acute leukemias were studied when 11 normal volunteer's bone marrow was used as control. The results showed that the expression level of TRF1 protein in normal bone marrow (2.217 +/- 0.461 microg/microl) was significantly higher than that in bone marrow of acute leukemia patients (0.754 +/- 0.343 microg/microl) (P < 0.01). There was no remarkable difference of expression level of TRF1 protein between ALL and ANLL (0.628 +/- 0.281 microg/microl vs 0.844 +/- 0.360 microg/microl, P > 0.05). After chemotherapy, TRF1 expression level of patients with complete remission raised (0.772 +/- 0.307 microg/microl vs 1.683 +/- 0.344 microg/microl, P < 0.01), but lower than that of normal (2.217 +/- 0.461 microg/microl, P < 0.01). TRF1 expression level of patients without complete remission was not remarkable different after chemotherapy (0.726 +/- 0.443 microg/microl vs 0.894 +/- 0.338 microg/microl, P > 0.05). TRF1 expression level of patients with complete remission was higher than that in patients without complete remession (1.683 +/- 0.344 microg/microl vs 0.894 +/- 0.338 microg/microl, P < 0.01). For all sample the telomerase activity was determined. It was confirmed that the activity of telomerase in normal bone marrow was lower than that in bone marrow of acute leukemia patients (0.125 +/- 0.078 microg/microl vs 0.765 +/- 0.284 microg/microl, P < 0.01). There was no significantly difference of expression level of TRF1 protein between ALL and ANLL (0.897 +/- 0.290 microg/microl vs 0.677 +/- 0.268 microg/microl, P > 0.05). After chemotherapy, telomerase activity of patients with complete remission reduced (0.393 +/- 0.125 microg/microl), but higher than that of normal (0.125 +/- 0.078 microg/microl, P < 0.01). It is concluded that expression level of TRF1 protein in AL patients is significantly decrese and associated with therapeutic efficaciousness and the activity of telomerase (P < 0.001).

beta-Glucuronidase is an optimal normalization control gene for molecular monitoring of chronic myelogenous leukemia.

Quantitative monitoring of breakpoint cluster region (BCR)-Abelson kinase (ABL) transcripts has become indispensable in the clinical care of patients with chronic myelogenous leukemia. Because quantity and quality of RNA in clinical samples are highly variable, a suitable internal normalization control is required for accurate BCR-ABL quantification. However, few studies have examined suitability of the control genes using criteria relevant to residual disease testing. In this study, we evaluated a number of control genes with the application of several novel criteria, including control gene performance on serial patient sample testing and in a residual disease model. We also examined expression of the control genes in BCR-ABL-positive K562 cells in response to Gleevec treatment. We found that beta-glucuronidase is the best control gene among those studied. Importantly, ABL, a widely used control gene, generates misleading BCR-ABL changes that potentially affect the clinical management of chronic myelogenous leukemia patients.

A study of the relationship between expression level of TRF1 protein and telomerase activity in human acute leukemia.

OBJECTIVE: To study the expression level of TRF1 (telomeric repeat binding factor 1) protein in human acute leukemia and relationship between expression level of TRF1 protein and telomerase. METHODS: A quantitative Western-Blot technique was developed using anti-TRF1(33-277) monoclonal antibody and GST-TRF1 purity protein as a standard to further determine the expression level of TRF1 protein in total proteins extracted from clinical specimens. RESULTS: Bone marrow tissues of 20 acute leukemia patients were studied, 11 healthy donors' bone marrows were taken as a control. The expression level of TRF1 protein was significantly higher (P<0.01) in normal bone marrow ((2.217+/-0.462) microg/microl) than that of acute leukemia patients ((0.754+/-0.343) microg/microl). But there was no remarkable difference between ALL and ANLL patients ((0.618+/-0.285) microg/microl vs (0.845+/-0.359) microg/microl, P>0.05). After chemotherapy, TRF1 expression level of patients with complete remission elevated ((0.772+/-0.307) microg/microl vs (1.683+/-0.344) microg/microl, P<0.01), but lower than that of normal ((2.217+/-0.462) microg/microl, P<0.01). There was no significantly difference after chemotherapy ((0.726+/-0.411) microg/microl vs (0.895+/-0.339) microg/microl, P>0.05). TRF1 expression level of patients with complete remission is higher than that of patients without complete remission ((1.683+/-0.344) microg/microl vs (0.895+/-0.339) microg/microl, P<0.01). All samples were determined for telomerase activity. It was confirmed that the activity of telomerase in normal bone marrow was lower than that of acute leukemia patients ((0.125+/-0.078) microg/microl vs (0.765+/-0.284) microg/microl, P<0.01). There was no significant difference of expression level of TRF1 protein between ALL and ANLL patients ((0.897+/-0.290) microg/microl vs (0.677+/-0.268) microg/microl, P>0.05). After chemotherapy, telomerase activity of patients with complete remission decreased ((0.393+/-0.125) microg/microl), but was still higher than that of normal ((0.125+/-0.078) microg/microl, P<0.01). CONCLUSION: The expression level of TRF1 protein has correlativity to the activity of telomerase (P<0.001).

Gene expression and phenotypic characterization of mouse heart after chronic constant or intermittent hypoxia.

Chronic constant hypoxia (CCH), such as in pulmonary diseases or high altitude, and chronic intermittent hypoxia (CIH), such as in sleep apnea, can lead to major changes in the heart. Molecular mechanisms underlying these cardiac alterations are not well understood. We hypothesized that changes in gene expression could help to delineate such mechanisms. The current study used a neonatal mouse model in CCH or CIH combined with cDNA microarrays to determine changes in gene expression in the CCH or CIH mouse heart. Both CCH and CIH induced substantial alterations in gene expression. In addition, a robust right ventricular hypertrophy and cardiac enlargement was found in CCH- but not in CIH-treated mouse heart. On one hand, upregulation in RNA and protein levels of eukaryotic translation initiation factor-2alpha and -4E (eIF-2alpha and eIF-4E) was found in CCH, whereas eIF-4E was downregulated in 1- and 2-wk CIH, suggesting that eIF-4E is likely to play an important role in the cardiac hypertrophy observed in CCH-treated mice. On the other hand, the specific downregulation of heart development-related genes (e.g., notch gene homolog-1, MAD homolog-4) and the upregulation of proteolysis genes (e.g., calpain-5) in the CIH heart can explain the lack of hypertrophy in CIH. Interestingly, apoptosis was enhanced in CCH but not CIH, and this was correlated with an upregulation of proapoptotic genes and downregulation of anti-apoptotic genes in CCH. In summary, our results indicate that 1) the pattern of gene response to CCH is different from that of CIH in mouse heart, and 2) the identified expression differences in certain gene groups are helpful in dissecting mechanisms responsible for phenotypes observed.

Role of trehalose phosphate synthase and trehalose during hypoxia: from flies to mammals.

Trehalose is a nonreducing disaccharide in which the two glucose units are linked in an alpha,alpha-1,1-glycosidic linkage. The best known and most widely distributed pathway of trehalose synthesis involves the transfer of glucose from UDP-glucose to glucose 6-phosphate to form trehalose-6-phosphate and UDP via the trehalose-6-phosphate synthase (TPS1). Trehalose-6-phosphate phosphatase (TPS2) then converts trehalose-6-phosphate to free trehalose. This sugar is present in a wide variety of organisms, including bacteria, yeast, fungi, insects, invertebrates and plants, and because of its particular physical features, trehalose is able to protect the integrity of cells against a variety of environmental stresses such as desiccation, dehydration, heat, cold and oxidation. Our current studies described here indicate that trehalose protects Drosophila and mammalian cells from hypoxic and anoxic injury. The mechanism of this protection is probably related to a decrease in protein denaturation through protein-trehalose interactions.

Expression of Drosophila trehalose-phosphate synthase in HEK-293 cells increases hypoxia tolerance.

Increasing hypoxia tolerance in mammalian cells is potentially of major importance, but it has not been feasible thus far. The disaccharide trehalose, which accumulates dramatically during heat shock, enhances thermotolerance and reduces aggregation of denatured proteins. Previous studies from our laboratory showed that over-expression of Drosophila trehalose-phosphate synthase (dtps1) increases the trehalose level and anoxia tolerance in flies. To determine whether trehalose can protect against anoxic injury in mammalian cells, we transfected the dtps1 gene into human HEK-293 cells using the recombinant plasmid pcDNA3.1(-)-dtps1 and obtained more than 20 stable cell strains. Glucose starvation in culture showed that HEK-293 cells transfected with pcDNA3.1(-)-dtps1 (HEK-dtps1) do not metabolize intracellular trehalose, and, interestingly, these cells accumulated intracellular trehalose during hypoxic exposure. In contrast to HEK-293 cells transfected with pcDNA3.1(-) (HEK-v), cells with trehalose were more resistant to low oxygen stress (1% O2). To elucidate how trehalose protects cells from anoxic injury, we assayed protein solubility and the amount of ubiquitinated proteins. There was three times more insoluble protein in HEK-v than in HEK-dtps1 after 3 days of exposure to low O2. The amount of Na+-K+ ATPase present in the insoluble proteins dramatically increased in HEK-v cells after 2 and 3 days of exposure, whereas there was no significant change in HEK-dtps1 cells. Ubiquitinated proteins increased dramatically in HEK-v cells after 2 and 3 days of exposure but not in HEK-dtps1 cells over the same period. Our results indicate that increased trehalose in mammalian cells following transfection by the Drosophila tps1 gene protects cells from hypoxic injury. The mechanism of this protection is likely related to a decrease in protein denaturation, through protein-trehalose interactions, resulting in enhanced cellular recovery from hypoxic stress.

Developmental expression and enzymatic activity of pre-mRNA deaminase in Drosophila melanogaster.

Pre-mRNA editing, conducted by adenosine deaminase acting on RNA (ADAR), plays an important role in many biological/physiological processes. This post-transcriptional event creates protein diversity that stems from a single gene via alteration of either genetic codons or alternative splicing sites. Our data demonstrate that both expression of Drosophila ADAR (dADAR) gene and dADAR editing activity are highly regulated during development. The lack of editing activity during embryonic development and the CNS-limited expression of dADAR in the adult may specify its important role in maintaining neuronal function in the Drosophila CNS.

Role of trehalose phosphate synthase in anoxia tolerance and development in Drosophila melanogaster.

Recent studies have shown that trehalose plays a protective role in yeast in a variety of stresses, including heat, freezing and thawing, dehydration, hyperosmotic shock, and oxidant injury. Because (a) heat shock and anoxia share mechanisms that allow organisms to survive, (b) Drosophila melanogaster is tolerant to anoxia, and (c) trehalose is present in flies and is metabolically active, we asked whether trehalose can protect against anoxic stress. Here we report on a new role of trehalose in anoxia resistance in Drosophila. We first cloned the gene trehalose-6-phosphate synthase (tps1), which synthesizes trehalose, and examined the effect of tps1 overexpression as well as mutation on the resistance of Drosophila to anoxia. Upon induction of tps1, trehalose increased, and this was associated with increased tolerance to anoxia. Furthermore, in vitro experiments showed that trehalose reduced protein aggregation caused by anoxia. Homozygous tps1 mutant (P-element insertion into the third intron of the gene) leads to lethality at an early larval stage, and excision of the P-element rescues totally the phenotype. We conclude that trehalose contributes to anoxia tolerance in flies; this protection is likely to be due to a reduction of protein aggregation.

[Preparation and characterization of monoclonal antibody against human telomeric repeat binding factor 1].

OBJECTIVE: To prepare a monoclonal antibody against human telomeric repeat binding factor 1 (TRF1) protein and explore its biological characteristics. METHODS: BALB/c mice were immunized with GST-TRF1(33-277) fusion protein for the preparation of monoclonal antibody by hybridoma technique. The obtained antibody was used for clinical assay by Western-blot and immunohistochemical staining. RESULTS: One strain of hybridoma was obtained. It was confirmed by Western-blot that the antibody specifically recognized the 60 kD TRF1 protein. Immunohistochemical staining of the antibody showed that TRF1 protein located in the cytoplasm of epithelial cells and bone marrow cells. CONCLUSION: A TRF1 monoclonal antibody, with high specificity was developed. It is useful for detection of TRF1 protein in tissue specimens.