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Biomedical subjects

James L Zehnder

Publications and source records attributed to James L Zehnder.

At least 19 recordsLinked to original sources
PubMed2006

Gene expression profile of idiopathic thrombocytopenic purpura (ITP).

To search for novel mechanisms that contribute to the pathophysiology of idiopathic thrombocytopenic purpura (ITP), we determined the whole blood gene expression profile in five ITP patients and five control samples. Using DNA microarrays that contained 24,473 unique putative genes, we found 176 cDNAs that were strongly correlated with ITP. These included a cluster of interferon-regulated genes and TLR7, as well many less-well characterized genes which are candidates for further study. We believe this approach is likely to yield new insights into our understanding of the molecular pathophysiology of ITP.

Adolescent↗
PubMed2006

The JAK2 V617F mutation occurs in hematopoietic stem cells in polycythemia vera and predisposes toward erythroid differentiation.

Although a large proportion of patients with polycythemia vera (PV) harbor a valine-to-phenylalanine mutation at amino acid 617 (V617F) in the JAK2 signaling molecule, the stage of hematopoiesis at which the mutation arises is unknown. Here we isolated and characterized hematopoietic stem cells (HSC) and myeloid progenitors from 16 PV patient samples and 14 normal individuals, testing whether the JAK2 mutation could be found at the level of stem or progenitor cells and whether the JAK2 V617F-positive cells had altered differentiation potential. In all PV samples analyzed, there were increased numbers of cells with a HSC phenotype (CD34+CD38-CD90+Lin-) compared with normal samples. Hematopoietic progenitor assays demonstrated that the differentiation potential of PV was already skewed toward the erythroid lineage at the HSC level. The JAK2 V617F mutation was detectable within HSC and their progeny in PV. Moreover, the aberrant erythroid potential of PV HSC was potently inhibited with a JAK2 inhibitor, AG490.

ADP-ribosyl Cyclase 1↗
PubMed2006

Gene expression patterns in human placenta.

The placenta is the principal metabolic, respiratory, excretory, and endocrine organ for the first 9 months of fetal life. Its role in fetal and maternal physiology is remarkably diverse. Because of the central role that the placenta has in fetal and maternal physiology and development, the possibility that variation in placental gene expression patterns might be linked to important abnormalities in maternal or fetal health, or even variations in later life, warrants investigation. As an initial step, we used DNA microarrays to analyze gene expression patterns in 72 samples of amnion, chorion, umbilical cord, and sections of villus parenchyma from 19 human placentas from successful full-term pregnancies. The umbilical cord, chorion, amnion, and villus parenchyma samples were readily distinguished by differences in their global gene-expression patterns, many of which seemed to be related to physiology and histology. Differentially expressed genes have roles that include placental trophoblast secretion, signal transduction, metabolism, immune regulation, cell adhesion, and structure. We found interindividual differences in expression patterns in villus parenchyma and systematic differences between the maternal, fetal, and intermediate layers. A group of genes that was expressed in both the maternal and fetal villus parenchyma sections of placenta included genes that may be associated with preeclampsia. We identified sets of genes whose expression in placenta was significantly correlated with the sex of the fetus. This study provides a rich and diverse picture of the molecular variation in the placenta from healthy pregnancies.

Female↗
PubMed2006

Detection of the JAK2 V617F mutation by LightCycler PCR and probe dissociation analysis.

A point mutation in the JAK2 gene, a member of the tyrosine kinase family, was recently identified and shown to be associated with several myeloproliferative disorders. Several studies identified the same JAK2 point mutation (1,849G>T), resulting in the substitution of a valine to phenylalanine at codon 617 (V617F). We developed a simple and sensitive method to detect this mutation via polymerase chain reaction and probe dissociation analysis using the LightCycler platform, and we compared this method to existing restriction fragment-length polymorphism, direct sequencing, and amplification refractory mutation system methods. We found that the LightCycler method offered advantages of speed, reliability, and more straightforward interpretation over the restriction fragment-length polymorphism and sequencing approaches.

DNA Mutational Analysis↗
PubMed2005

A comparison study of different PCR assays in measuring circulating plasma epstein-barr virus DNA levels in patients with nasopharyngeal carcinoma.

PURPOSE: To compare the performance of three PCR assays in measuring circulating Epstein-Barr virus (EBV). DNA levels in nasopharyngeal carcinoma patients and to confirm its prognostic significance. EXPERIMENTAL DESIGN: Plasma from 58 newly diagnosed nasopharyngeal carcinoma patients were collected before, during, and every 3 to 6 months after radiotherapy. EBV DNA levels were determined by real-time quantitative PCR using primer/probe sets for polymerase-1 (Pol-1), latent membrane protein 2 (Lmp2), and BamHI-W. Pretreatment levels from the three assays were correlated with each other and serial measurements from the Pol-1 assay were correlated with clinical variables. RESULTS: Pol-1 was more accurate than BamHI-W in predicting EBV DNA concentrations in cell lines. Of the three assays, BamHI-W yielded the highest concentrations followed by Pol-1 in plasmas (n = 23). The correlation coefficient was 0.99 (P < 0.0001) for Pol-1 and Lmp2, 0.66 (P < 0.0001) for Pol-1 and BamHI-W, and 0.55 (P < 0.0001) for BamHI-W and Lmp2. Elevated pretreatment DNA levels as detected by Pol-1 were correlated with advanced nodal stage (P = 0.04) and overall stage (P = 0.028). There was no correlation between pretreatment EBV DNA levels and freedom-from-relapse or overall survival; however, there was a significant correlation between posttreatment levels and these variables. The 2-year freedom-from-relapse and overall survival rates were 92% and 94% for patients with undetectable, and 37% and 55% for those with detectable, posttreatment levels (P < 0.0001 and P < 0.002). CONCLUSIONS: The three PCR assays yielded similar results in detecting EBV DNA in plasmas. The Pol-1-detected posttreatment EBV DNA level was the strongest predictor for treatment outcomes.

Adolescent↗
PubMed2005

Varicella-zoster virus infection of human dorsal root ganglia in vivo.

Varicella-zoster virus (VZV) causes varicella and establishes latency in sensory ganglia. VZV reactivation results in herpes zoster. We developed a model using human dorsal root ganglion (DRG) xenografts in severe combined immunodeficient (SCID) mice to investigate VZV infection of differentiated neurons and satellite cells in vivo. DRG engrafted under the kidney capsule and contained neurons and satellite cells within a typical DRG architecture. VZV clinical isolates infected the neurons within DRG. At 14 days postinfection, VZ virions were detected by electron microscopy in neuronal cell nuclei and cytoplasm but not in satellite cells. The VZV genome copy number was 7.1 x 10(7) to 8.0 x 10(8) copies per 10(5) cells, and infectious virus was recovered. This initial phase of viral replication was followed within 4-8 weeks by a transition to VZV latency, characterized by the absence of infectious virus release, the cessation of virion assembly, and a reduction in VZV genome copies to 3.7 x 10(5) to 4.7 x 10(6) per 10(5) cells. VZV persistence in DRG was achieved without any requirement for VZV-specific adaptive immunity and was associated with continued transcription of the ORF63 regulatory gene. The live attenuated varicella vaccine virus exhibited the same pattern of short-term replication, persistence of viral DNA, and prominent ORF63 transcription as the clinical isolates. VZV-infected T cells transferred virus from the circulation into DRG, suggesting that VZV lymphotropism facilitates its neurotropism. DRG xenografts may be useful for investigating neuropathogenic mechanisms of other human viruses.

Animals↗
PubMed2004

Granulocyte-macrophage progenitors as candidate leukemic stem cells in blast-crisis CML.

BACKGROUND: The progression of chronic myelogenous leukemia (CML) to blast crisis is supported by self-renewing leukemic stem cells. In normal mouse hematopoietic stem cells, the process of self-renewal involves the beta-catenin-signaling pathway. We investigated whether leukemic stem cells in CML also use the beta-catenin pathway for self-renewal. METHODS: We used fluorescence-activated cell sorting to isolate hematopoietic stem cells, common myeloid progenitors, granulocyte-macrophage progenitors, and megakaryocyte-erythroid progenitors from marrow during several phases of CML and from normal marrow. BCR-ABL, beta-catenin, and LEF-1 transcripts were compared by means of a quantitative reverse-transcriptase-polymerase-chain-reaction assay in normal and CML hematopoietic stem cells and granulocyte-macrophage progenitors. Confocal fluorescence microscopy and a lymphoid enhancer factor/T-cell factor reporter assay were used to detect nuclear beta-catenin in these cells. In vitro replating assays were used to identify self-renewing cells as candidate leukemic stem cells, and the dependence of self-renewal on beta-catenin activation was tested by lentiviral transduction of hematopoietic progenitors with axin, an inhibitor of the beta-catenin pathway. RESULTS: The granulocyte-macrophage progenitor pool from patients with CML in blast crisis and imatinib-resistant CML was expanded, expressed BCR-ABL, and had elevated levels of nuclear beta-catenin as compared with the levels in progenitors from normal marrow. Unlike normal granulocyte-macrophage progenitors, CML granulocyte-macrophage progenitors formed self-renewing, replatable myeloid colonies, and in vitro self-renewal capacity was reduced by enforced expression of axin. CONCLUSIONS: Activation of beta-catenin in CML granulocyte-macrophage progenitors appears to enhance the self-renewal activity and leukemic potential of these cells.

Adult↗
PubMed2004

CyclinD1/CyclinD3 ratio by real-time PCR improves specificity for the diagnosis of mantle cell lymphoma.

We developed a real-time, quantitative, reverse transcription PCR assay for cyclin D1 (CCND1) expression to aid in the diagnosis of mantle cell lymphoma (MCL). The diagnosis of MCL can be problematic, and existing CCND1 expression assays show a lack of specificity, with elevated expression also detected in other lymphoproliferative disorders. We postulated that evaluating CCND1 expression relative to CCND3 expression by quantitative PCR could offer an improved specificity over an evaluation of CCND1 alone. This method quantitates both CCND1 and CCND3, each normalized to a housekeeping gene (GADPH), using the 5'-exonuclease technique. We analyzed 107 clinical specimens: MCL (17), chronic lymphocytic leukemias (CLL) (10), other non-MCL hematolymphoid disorders (41), non-malignant tissues with an epithelial component (7) and other normal samples (32). This method correctly identified 16 of 17 MCLs, and there were no false positives among any of the other diagnostic groups tested including CLL. CLL presents the major diagnostic dilemma at this institution when diagnosing MCL. Sensitivity studies showed that this method could detect an elevated CCND1/CCND3 ratio when the tumor infiltrate is at least 10% of the cells. We compared the specificity of CCND1 expression alone against the CCND1/CCND3 ratio to demonstrate the increased specificity for the latter. We conclude that the CCND1/CCND3 ratio is a sensitive and specific test for the diagnosis of MCL.

Cyclin D1↗
PubMed2004

Rapid combined genotyping assay for four achondroplasia and hypochondroplasia mutations by real-time PCR with multiple detection probes.

Achondroplasia (ACH) and hypochondroplasia (HYCH) are the most prevalent genetic short-stature syndromes. Whereas the diagnosis of ACH can be established on clinical and radiologic grounds alone in the majority of cases, HYCH is more difficult to confirm. Molecular genetic analysis of both skeletal dysplasias can be especially helpful for the purpose of prenatal diagnosis, in early childhood to differentiate definitively between the largely overlapping phenotypes, and in atypical presentations. The two most prevalent mutations for each syndrome cause substitution of a single respective nucleotide. These mutations can be identified by a variety of molecular methods, including PCR with restriction enzyme digestion or direct DNA sequencing. We have developed a single-step, real-time PCR assay in which two detection probes are applied in combination with a single anchor probe at each mutation position. Because the two most prevalent mutations for each syndrome cause substitution of a single respective nucleotide, this approach guarantees optimal differentiation during probe dissociation analysis after amplification. This assay, which is performed on the LightCycler thermocycler, enables the rapid and reliable detection of the two most common FGFR3 mutations associated with ACH (1138G --> A and 1138G --> C; G380R) and HYCH (1620C --> A and 1620 C --> G; N540K) in a single test.

Achondroplasia↗
PubMed2004

Randomized trial of folic acid for prevention of cardiovascular events in end-stage renal disease.

High serum total homocysteine (tHcy) is gaining scrutiny as a risk factor for cardiovascular disease in the general population. The relationship between tHcy and mortality and cardiovascular events in patients with end-stage renal disease (ESRD) is unsettled. This randomized trial evaluates the efficacy of high-dose folic acid in preventing events in ESRD. A total of 510 patients on chronic dialysis were randomized to 1, 5, or 15 mg of folic acid contained in a renal multivitamin with a median follow-up of 24 mo. Mortality, cardiovascular events, and homocysteine levels were assessed. There were 189 deaths, and 121 patients experienced at least one cardiovascular event. Composite rates of mortality and cardiovascular events among the folic acid groups did not differ (at 24 mo: 43.7% in 1 mg group, 38.6% in 5 mg group, 47.1% in 15 mg group; log-rank P = 0.47). Unexpectedly, high baseline tHcy was associated with lower event rates. From lowest to highest quartile, event rates at 24 mo were 54.5% for Q1, 41.8% for Q2, 41.2% for Q3, and 34.7% for Q4 (log-rank P = 0.033). In contrast to some studies describing tHcy as a risk factor for mortality and cardiovascular events, this study found a reverse relationship between tHcy and events in ESRD patients. Administration of high-dose folic acid did not affect event rates.

Cardiovascular Diseases↗
PubMed2004

Congenital and acquired thrombocytopenia.

The diagnosis and management of thrombocytopenia is a growing component in the practice of hematology. The frequency with which hematologists are called in consultation for thrombocytopenia continues to increase with the advent of routine automated platelet determinations and the introduction of new medications. For most patients, such as those with inherited and auto-immune thrombocytopenia, emphasis is focused on efforts to treat or forestall bleeding without excess drug-induced toxicity or burden to the patient. However, in disorders such as heparin-induced thrombocytopenia (HIT), avoidance of thrombotic complications is the key to management. In this chapter, we provide the pediatric and adult hematologist with new insights into the pathogenesis and recognition of congenital inherited thrombocytopenias (CTP), a hitherto difficult to comprehend constellation of clinical entities. We also highlight new approaches to the diagnosis and treatment of two of the more common thrombocytopenic conditions encountered in practice, autoimmune or idiopathic thrombocytopenic purpura (ITP) and HIT. In Section I, Dr. James Bussel discusses CTPs and their distinction from childhood ITP. He emphasizes the clinical features that enable the pediatrician and hematologist to suspect the diagnosis of CTP and those that are of use to subcategorize the various entities, where possible. He also emphasizes newer molecular markers that afford definitive diagnosis in some cases and provide insight into platelet production. This section highlights the characteristic associated findings and differences in the natural history and approaches to management of the various entities. In Section II, Dr. Robert McMillan discusses adult chronic ITP. He revisits the utility of platelet antibody determination in diagnosis and review new insights into pathogenesis. The role of Helicobacter pylori infection and the timing of splenectomy in the management of acute and emergent ITP are examined. New insights into the natural history of ITP post-splenectomy and management strategies for patients with severe, chronic, refractory ITP are discussed. In Section III, Dr. James Zehnder updates us on HIT. He emphasizes new insights into the clinical presentation and pathogenesis of this condition. He critically reviews the utility of laboratory testing for heparin-dependent antibodies. Recent studies on the use of direct thrombin inhibitors are examined and the management of cardiopulmonary bypass surgery in patients with HIT is discussed.

Adult↗
PubMed2003

Prothrombin gene variants in non-Caucasians with fetal loss and intrauterine growth retardation.

Thrombotic predisposition may affect pregnancy outcome, but in non-Caucasians the contributing genetic factors are poorly characterized. Two recently identified prothrombin gene mutations (20209C>T and 20221C>T) have been observed in non-Caucasian patients with thrombosis. The mutations are located near the commonly identified variant 20210G>A and have not been reported in Caucasian patients. The authors report a novel connection with pregnancy complications. The identification of sequence variants other than 20210G>A in the 3'-untranslated region of the prothrombin gene suggests that additional nucleotide substitutions may contribute to the development of thrombotic events and adverse pregnancy outcomes, especially in less well-characterized populations.

Abortion, Spontaneous↗
PubMed2003

Lack of human herpesvirus 8 and Epstein-Barr virus in Kikuchi's histiocytic necrotizing lymphadenitis.

Kikuchi's histiocytic necrotizing lymphadenitis is a self-limited disorder that typically involves the cervical lymph nodes of young women. Although a viral etiology has been postulated, a definitive viral agent has not been identified. Recent reports have suggested that human herpesvirus 8 (HHV 8) or Epstein-Barr virus (EBV) may play an etiologic role. We investigated the presence of HHV 8 and EBV in archival tissue from 34 cases of Kikuchi's histiocytic necrotizing lymphadenitis. We examined 29 cases for HHV 8 using a nested polymerase chain reaction (PCR) on paraffin-embedded or frozen tissue, and 24 cases for EBV RNA using in situ hybridization (ISH) for EBER1. Controls included reactive lymph nodes from 8 adult women presenting with cervical or axillary lymphadenopathy. The study patients included 7 men and 27 women with a mean age of 28 years. All patients were previously healthy without evidence of immunocompromise and presented with cervical, axillary, or inguinal lymphadenopathy. Two cases exhibited EBV RNA by ISH; this was confirmed by PCR for EBV DNA. HHV 8 DNA was not amplified by nested PCR in any of the cases of Kikuchi's histiocytic necrotizing lymphadenitis or reactive lymph nodes; control PCR demonstrated the presence of amplifiable DNA in all cases. These findings suggest that HHV 8 and EBV do not play causative roles in Kikuchi's histiocytic necrotizing lymphadenitis.

Adolescent↗
PubMed2003

Diagnostic single nucleotide polymorphism analysis of factor V Leiden and prothrombin 20210G > A. A comparison of the Nanogen Eelectronic Microarray with restriction enzyme digestion and the Roche LightCycler.

Genetic thrombosis risk factors include a sequence variant in the prothrombin gene (20210G > A) and factor V Leiden (1691G > A). These single nucleotide polymorphisms can be diagnosed with restriction fragment length polymorphism (RFLP) analysis, fluorescent genotyping on the LightCycler (Roche Diagnostics, Indianapolis, IN), and microarray-based testing on the novel NanoChip electronic microarray (NanoChip Molecular Biology Workstation, Nanogen, San Diego, CA). We compared these methods for accuracy, time to results, throughput, and interpretation. Results from 789 of 800 individual amplicons analyzed on the NanoChip were in complete agreement with the other assays. Eleven were "no calls" (uninterpreted by the NanoChip system) resulting from failed polymerase chain reaction amplifications. Although the NanoChip System, when used in a low-throughput setting, requires more overall time than the LightCycler, it is nearly equivalent per genotyping call. Owing to minimal sample handling, assay results are more reliable on the NanoChip platform and on the LightCycler than with RFLP. The NanoChip assay is reliable and may be especially valuable to laboratories with a large volume of thrombophilia test requests.

Factor V↗
PubMed2003

Comprehensive validation of a real-time quantitative bcr-abl assay for clinical laboratory use.

We developed and extensively validated a real-time PCR assay for the quantitation of bcr-abl to determine residual disease in patients with chronic myelogenous leukemia. This method quantitates the p210 and the p190 bcr-abl RNA fusion transcripts with results normalized to a housekeeping gene, using the 5'-exonuclease technique and the ABI PRISM 7700 Sequence Detection System (Applied Biosystems, Foster City, CA). We parallel tested 372 clinical specimens and 50 peripheral blood samples from patients not known to have any myeloproliferative disorders. The results were 100% specific. Sensitivity studies showed that this method can detect bcr-abl in cell lines diluted to 0.0001% and can detect a single bcr-abl plasmid spiked into negative RNA. The between-run reproducibility showed a coefficient of variance (CV) of 12.3%, and within-run reproducibility showed a CV of 13.8%. This method can be used to reliably monitor the disease load in patients with bcr-abl-positive diseases.

Clinical Laboratory Techniques↗