Analysis Of Peripheral Blood Leukocytes Research Articles

Telomere Shortening At Diagnosis Of Chronic Phase Chronic Myelogenous Leukemia (CML-CP) Is Significantly More Pronounced In Patients With Less-Than-Optimal Versus Optimal Molecular Response (ELN criteria) After 12 Months Of Treatment With Nilotinib

IntroductionChronic myeloid leukemia (CML) is a clonal stem cell disorder characterized by the BCR-ABL translocation. Telomere length (TL) reflects the replicative history of eukaryotic cells and progressive telomere shortening is associated with genetic instability. Clinically, accelerated telomere shortening has been demonstrated in patients with CML and was found to correlate with disease progression and clinical risk score in the pre-tyrosine kinase inhibitor (TKI) era. The aim of the current study was to investigate whether telomere length (TL) at diagnosis might predict response to treatment in patients receiving nilotinib as first line treatment of chronic phase CML on the ENEST1st study (NCT01061177) Methods and PatientsTL analysis of peripheral blood leukocytes was analyzed using monochrome multiplex quantitative PCR in blood samples from 93 newly diagnosed CML patients enrolled on study in Germany. One extreme outlier was excluded from the analysis. 89 healthy controls were used for age-adaption of TL. Median age of the analyzed CML patients was 49.6 years (range: 19-83), Sokal (32 low, 31 intermediate, 13 high risk) and Euro (34 low, 36 intermediate, 6 high risk) scores were available in 76 patients. Response to treatment according to standard criteria was available at 3 month (mo, n=79 patients), 6 mo (n=75), 12 mo (n=71) and 18 mo (n=55) after treatment start. ResultsMean age-adjusted TL in CML patients was significantly shortened compared to normal individuals (ΔT/S ratio: -0.30 +/- 0.67, p=<0.001). Interestingly, whereas TL followed an expected linear decline over time in the control population of healthy individuals, this age-correlation was no longer detectable in CML patients pointing to a significantly more pronounced TL deficit in younger as opposed to older patients with CML. In univariate analysis, no significant correlation between age-adjusted TL at diagnosis and Euro or Sokal risk score nor with any standard individual prognostic parameters was detected with the exception of the peripheral blast count which was shown to be inversely correlated with age adjusted telomere (ΔT/S ratio) length by linear regression (p=0.01).When ΔT/S ratio measured at diagnosis was correlated with response to treatment at defined time points according to the ELN criteria 2013 (i.e. <10% BCR-ABL/ABL ratio at 3 mo, <1% at 6 mo, and <0.1% at 12 mo), we could demonstrate that while all cohorts had shortened telomere length compared to age-adjusted controls, less-than-optimal responders had more accelerated telomere shortening compared to optimal responders, i.e. ΔT/S -0.66 (n=4) vs. -0.25 (n=75) at 3 mo; ΔT/S -0.37 (n=10) vs. -0.28 (n=65) at 6 mo and ΔT/S -0.67 (n=15) vs. -0.21 (n=56) at 12 mo. While this pattern seems rather consistent, only the difference at the 12 mo time point reached statistical significance (p=0.028), potentially due to the imbalance of the groups induced by the low number of less-than-optimal responders to treatment according to ELN criteria observed under nilotinib first line treatment. ConclusionsThese data were generated on the first prospective study of the role of telomere length as a potential biomarker for TKI treatment in CML. We could confirm that TL is significantly shortened in CML patients at diagnosis. Even more so, telomere shortening seems to be significantly more accelerated in younger as opposed to older patients pointing to a threshold value of telomere length in CML similar to observations in patients with bone marrow failure syndromes. Furthermore, telomere shortening tends to be more pronounced in patients not meeting the criteria for optimal response according to ELN 2013. Further follow up will reveal whether TL has the potential to serve as a long-term predictive biomarker for frequency and durability of response as well as for the sustainability of treatment cessation in CML patients responding well to TKI treatment. Disclosures:Frank:Novartis: Employment. Walasek:Novartis: Employment. Hochhaus:Novartis: Consultancy, Honoraria, Research Funding, Travel Other; BMS: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Ariad: Consultancy, Honoraria. Giles:Novartis: Consultancy, Research Funding. Koschmieder:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Brümmendorf:Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Bristol Myer Squibb: Consultancy, Honoraria; Pfizer: Consultancy, Honoraria; Ariad: Consultancy.

SAT0173 Molecular signatures in SLE: untangling flare from infection

BackgroundInability to distinguish between infection from increased SLE disease activity based on clinical judgment often compromises timely and effective treatment. Gene expression profiling of circulating leukocytes has been used to...

Characterization of novel germline c-kit gene mutation, KIT-Tyr553Cys, observed in a family with multiple gastrointestinal stromal tumors

We found a novel type germline mutation at exon 11 of the c-kit gene, which results in a substitution of Tyr to Cys at codon 553 of the c-kit gene product (KIT-Tyr553Cys), in a 68-year-old female patient with multiple gastrointestinal stromal tumors (GISTs). In the present study, we carried out mutational analysis in her family members to determine the carriers and characterized the mutation by introducing the corresponding mutation (murine KIT-Tyr552Cys) into expression vector possessing murine c-kit cDNA. Mutational analysis of peripheral blood leukocytes of her family members revealed that a 44-year-old son had the same mutation, but at present he had neither apparent symptoms nor images of multiple GISTs. By transfection with the expression vector possessing the murine mutant c-kit cDNA, interleukin-3-dependent Ba/F3 murine lymphoid cells started growing autonomously without any growth factors, indicating that the mutation was considered to be of gain-of-function. Imatinib, a small molecule of tyrosine kinase inhibitor, effectively inhibited autophosphorylation of KIT-Tyr552Cys. Nilotinib, another small molecule of the KIT inhibitor, also effectively inhibited autophosphorylation of KIT-Tyr552Cys. In fact, proliferation of Ba/F3 cells expressing KIT-Tyr552Cys was effectively inhibited by both imatinib and nilotinib. These findings indicate that the novel type human KIT-Tyr553Cys mutation is the cause of the present familial and multiple GISTs, and that both imatinib and nilotinib might effectively inhibit the growth of GISTs developing in the patients of this family.

Identification of Gene Networks and Pathways Associated with Guillain-Barré Syndrome

BackgroundThe underlying change of gene network expression of Guillain-Barré syndrome (GBS) remains elusive. We sought to identify GBS-associated gene networks and signaling pathways by analyzing the transcriptional profile of leukocytes in the patients with GBS.Methods and FindingsQuantitative global gene expression microarray analysis of peripheral blood leukocytes was performed on 7 patients with GBS and 7 healthy controls. Gene expression profiles were compared between patients and controls after standardization. The set of genes that significantly correlated with GBS was further analyzed by Ingenuity Pathways Analyses.256 genes and 18 gene networks were significantly associated with GBS (fold change ≥2, P<0.05). FOS, PTGS2, HMGB2 and MMP9 are the top four of 246 significantly up-regulated genes. The most significant disease and altered biological function genes associated with GBS were those involved in inflammatory response, infectious disease, and respiratory disease. Cell death, cellular development and cellular movement were the top significant molecular and cellular functions involved in GBS. Hematological system development and function, immune cell trafficking and organismal survival were the most significant GBS-associated function in physiological development and system category. Several hub genes, such as MMP9, PTGS2 and CREB1 were identified in the associated gene networks. Canonical pathway analysis showed that GnRH, corticotrophin-releasing hormone and ERK/MAPK signaling were the most significant pathways in the up-regulated gene set in GBS.ConclusionsThis study reveals the gene networks and canonical pathways associated with GBS. These data provide not only networks between the genes for understanding the pathogenic properties of GBS but also map significant pathways for the future development of novel therapeutic strategies.

Association of an increased frequency of CD14+HLA‐DRlo/neg monocytes with decreased time to progression in chronic lymphocytic leukaemia (CLL)

Association of an increased frequency of CD14⁺HLA‐DR^lo/neg monocytes with decreased time to progression in chronic lymphocytic leukaemia (CLL)

Parkinson’s disease is a TH17 dominant autoimmune disorder against accumulated alpha-synuclein

AbstractParkinson’s disease is a very common neurodegenerative disorder. Patients usually undergo destruction of substantia nigra to develop typical symptoms such as resting tremor, hypokinesia, and rigidity. However, the exact mechanism of Parkinson’s disease is still unknown, so it is called idiopathic Parkinsonism. According to my microarray analysis of peripheral blood leukocytes and substantia nigra brain tissue, I propose that Parkinson’s disease is actually a TH17 dominant autoimmune disease. The autoantigen is mainly alpha-synuclein. After knowing the exact disease pathophysiology, we can develop better drugs to prevent or control the detrimental disorder.

BCR‐ABL translocation in a dog with chronic monocytic leukemia

A 9-year-old female spayed mixed breed dog was evaluated at the University of Florida Small Animal Hospital for marked leukocytosis with no associated clinical signs. CBC abnormalities included marked leukocytosis (106,000/μL), marked monocytosis (78,000/μL), and the presence of 13% blast cells (13,832/μL), supporting a diagnosis of leukemia. Cytopenias and dysplastic changes in other cell lines were not present. Microscopic examination of bone marrow showed hypercellular uniparticles with a marginal increase in frequency of unclassified blast cells (2%), but was otherwise unremarkable. Flow cytometric immunophenotyping of blood cells determined that leukemic cells were CD45(+) , CD14(+) , and CD34(-) , and based on side scatter and CD45 reactivity the marrow contained 19% monoblasts. By immunocytochemical staining, the leukemic cells in the bone marrow were CD11b(+) , CD11c(+) , CD11d(+) , MHC-II(+) , MPO(+) , and CD34(-) . Fluorescence in situ hybridization (FISH) analysis of peripheral blood leukocytes documented a chromosomal translocation producing a BCR-ABL gene hybrid, similar to the "Philadelphia" chromosome abnormality recognized in human chronic myelogenous leukemia, as well as a phosphatase and tensin homolog (PTEN) gene deletion. Hydroxyurea therapy was attempted, but was ineffective; the dog died 7 months after initial presentation. Clinical and laboratory findings and the protracted course supported a diagnosis of chronic monocytic leukemia (CMoL) and, to our knowledge, this is the first case of CMoL with a BCR-ABL chromosomal abnormalitiy described in dogs. This may have clinical implications for treatment of dogs with chronic leukemias associated with particular genetic mutations. However, more case studies are needed to further characterize this disease.

The JAK2 46/1 Haplotype Predisposes to Myeloproliferative Neoplasms Characterized by Diverse Mutations.

Abstract 433A common constitutional JAK2 haplotype termed 46/1 (also known as GGCC) predisposes to V617F JAK2-positive myeloproliferative neoplasms (MPN) but the underlying mechanism is obscure. Two hypotheses have been postulated: (i) ‘hypermutability' of JAK2 on 46/1 compared to other haplotypes and (ii) a functional difference of JAK2 on 46/1 that positively interacts with V617F and thus provides ‘fertile ground' for development of an MPN. To investigate these possibilities we analyzed patients with essential thrombocythemia entered into the PT-1 studies. As expected, 46/1 was highly overrepresented in V617F positive cases (n=404) compared to population controls (n=1492; P=3.9×10-11) and in informative individuals V617F preferentially arose on the 46/1 chromosome (P<0.0001). This haplotype was also overrepresented in cases without V617F (n=347, P=0.009), with an excess seen for individuals with MPL exon 10 mutations as well as those who were V617F and MPL exon 10 negative. Analysis of further MPL-positive, V617F negative cases confirmed an excess of 46/1 alleles (n=176, P=0.004) however we found no association between MPL mutations and MPL haplotype. An excess of 46/1 was also seen in cases with JAK2 exon 12 mutations (n=69, P=0.002) and in informative individuals these mutations preferentially arose on the 46/1 chromosome (P=0.029). No association between 46/1 and clinical or laboratory features was seen in the PT-1 cohort either with or without V617F. An excess of 46/1 in JAK2 exon 12 mutated cases is compatible with both the hypermutability and fertile ground hypotheses, but the excess in MPL mutated cases argues against the former. However, on analysis of peripheral blood leukocytes we detected no difference in sequence, splicing or expression of JAK2 on 46/1 compared to JAK2 on other haplotypes suggesting that any functional difference of JAK2 on 46/1, if it exists, must be relatively subtle. Finally, since 46/1 is seen at comparable frequencies in different MPN subtypes it does not explain the phenotypic diversity associated with V617F JAK2. In an analysis of 138 MPL mutated cases (ET, n=99; MF, n=36; other MPN, n=3) we found that the median W515K/L mutation levels as determined by pyrosequencing in ET (21%) were significantly lower than those seen in MF (46%; P<0.0001). However we were unable to confirm previously reported differences in SNP frequencies (rs1524395 at 7p11, rs2279784 at 3q21 and rs12500918 at 4q31) between cases with ET (n=763) and PV (n=163). In summary, our data provide further evidence for the fertile ground hypothesis whereby a proposed subtle functional difference of JAK2 on 46/1 makes the development of an MPN more likely when a JAK2 mutation arises on this haplotype, or when JAK2 46/1 is present with a MPL exon 10 mutation. JAK2 and MPL mutation levels, but not 46/1 status, are related to disease phenotype. Disclosures:Schnittger:MLL Munich Leukemia Laboratory: Equity Ownership. Green:Bristol Myers Squibb: Consultancy; Shire: Consultancy; Incyte: Consultancy; Astex Therapeutics: Consultancy, Research Funding.

Detection of Growth Hormone Doping by Gene Expression Profiling of Peripheral Blood

GH abuse is a significant problem in many sports, and there is currently no robust test that allows detection of doping beyond a short window after administration. Our objective was to evaluate gene expression profiling in peripheral blood leukocytes in-vivo as a test for GH doping in humans. Seven men and thirteen women were administered GH, 2 mg/d sc for 8 wk. Blood was collected at baseline and at 8 wk. RNA was extracted from the white cell fraction. Microarray analysis was undertaken using Agilent 44K G4112F arrays using a two-color design. Quantitative RT-PCR using TaqMan gene expression assays was performed for validation of selected differentially expressed genes. GH induced an approximately 2-fold increase in circulating IGF-I that was maintained throughout the 8 wk of the study. GH induced significant changes in gene expression with 353 in women and 41 in men detected with a false discovery rate of less than 5%. None of the differentially expressed genes were common between men and women. The maximal changes were a doubling for up-regulated or halving for down-regulated genes, similar in magnitude to the variation between individuals. Quantitative RT-PCR for seven target genes showed good concordance between microarray and quantitative PCR data in women but not in men. Gene expression analysis of peripheral blood leukocytes is unlikely to be a viable approach for the detection of GH doping.

Micro-Array Analysis of Peripheral Blood Leukocytes in Early Pregnant or Bred, Non-Pregnant Dairy Cows.

Micro-Array Analysis of Peripheral Blood Leukocytes in Early Pregnant or Bred, Non-Pregnant Dairy Cows.

DNA Polymerases η and θ Function in the Same Genetic Pathway to Generate Mutations at A/T during Somatic Hypermutation of Ig Genes

Somatic hypermutation of the Ig genes requires the activity of multiple DNA polymerases to ultimately introduce mutations at both A/T and C/G base pairs. Mice deficient for DNA polymerase eta (POLH) exhibited an approximately 80% reduction of the mutations at A/T, whereas absence of polymerase (POLQ) resulted in approximately 20% reduction of both A/T and C/G mutations. To investigate whether the residual A/T mutations observed in the absence of POLH are generated by POLQ and how these two polymerases might cooperate or compete with each other to generate A/T mutations, here we have established mice deficient for both POLH and POLQ. Polq(-/-)Polh(-/-) mice, however, did not show a further decrease of A/T mutations as compared with Polh(-/-) mice, suggesting that POLH and POLQ function in the same genetic pathway in the generation of these mutations. Frequent misincorporation of nucleotides, in particular opposite template T, is a known feature of POLH, but the efficiency of extension beyond the misincorporation differs significantly depending on the nature of the mispairing. Remarkably, we found that POLQ catalyzed extension more efficiently than POLH from all types of mispaired termini opposite A or T. Moreover, POLQ was able to extend mispaired termini generated by POLH albeit at a relatively low efficiency. These results reveal genetic and biochemical interactions between POLH and POLQ and suggest that POLQ might cooperate with POLH to generate some of the A/T mutations during the somatic hypermutation of Ig genes.

Effects of Cord Blood Cell Subset Populations in the Development of the Dominant Cord Blood Unit in Non-Myeloablative Sequential Double Cord Blood Transplantation (DCBT).

Umbilical cord blood has become an established alternative hematopoietic stem cell source for patients without suitable donors. However, single cord blood transplantation in adults has been associated with high mortality rates due to graft failure, delayed engraftment, and poor immune reconstitution. In order to improve engraftment, sequential cord blood transplantation has been employed. One consistent observation with this strategy has been the eventual dominance of one cord graft over the other. However, the factors that determine the fate of one cord over the other have not been well elucidated. Our previous work demonstrated that the dominant cord tends to have an initial higher CD34+ cell dose. This study attempted to identify other cell populations in the cord units that may determine which unit becomes the ultimate source of hematopoiesis. Forty patients with hematologic malignancies underwent non-myeloablative conditioning with fludarabine, melphalan, and ATG followed by two sequential cord blood infusions. All cord units were at least a 4/6 HLA match with the recipient and each other. GVHD prophylaxis consisted of cyclosporine & mycophenolate mofetil or FK-506 & rapamycin. Chimerism analysis of peripheral blood leukocytes was performed by PCR of short tandem repeat loci. Flow cytometry for T-cells (CD4 and CD8), B-cells (CD19), and NK cells (CD3−CD56+CD16− & CD3−CD56+CD16+) were done using standard immunofluorescence methods. A total of 10 patients were available for this analysis. The Wilcoxon signed rank test was used to evaluate the significance of the difference in cell populations between the transplanted cords on graft outcome. This analysis demonstrated that 80% (8 of 10) of the dominant cords had higher NK cells when compared to the rejected unit with average total NK cells of 4.15 x 106 vs 2.30 x 106 per kg of recipient, respectively. Total NK cells included both CD16− and CD16+ cells, but the difference was much higher for CD16+ NK cells, which represent a more mature NK phenotype. B-cells, CD4+ T-cells, and CD8+ T-cells were similar between the two cords. The mean total cell/kg of recipient for each subpopulation between cord units are summarized in Table 1. A trend towards a difference in NK cells (p=0.1) was observed; the small number of samples, however, limited the interpretation of the analysis. Future studies involving additional DCBT recipients are ongoing to better define the influence of NK cells as well as other cell populations including mesenchymal stem cells and T-reg cells on cord graft outcome.Mean Subpopulation Cell Count/kg of recipient between Cord UnitsCordCD3+CD4+ T-CellsCD3+CD8+ T-CellsCD3−CD56+CD16− NK CellsCD3−CD56+CD16+ NK CellsTotal NK CellsCD19− B-CellsDominant Cells/kg3.92E+06 (1.35–7.30E+06)1.76E+06 (0.5–3.22E+06)4.64E+05 (0.06–1.52E+06)3.68E+06 (0.4–9.65E+06)4.15E+06 (0.7–9.97E+06)1.68E+06 (0.3–3.96E+06)Rejected Cells/kg4.34E+06 (2.04–7.83E+06)1.71E+06 (0.7–4.86E+06)3.60E+05 (0.0–0.9E+06)1.94E+06 (0.3–3.55E+06)2.30E+06 (0.3–4.10E+06)1.80E+06 (0.9–3.15E+06)

Pancreatic Endocrine Tumors are a Rare Manifestation of the Neurofibromatosis Type 1 Phenotype

The tumorigenesis of sporadic endocrine tumors is still not fully understood. It is well known that patients with von Recklinghausen syndrome (NF-1) (OMIM 162200) carrying NF1 germline mutations are predisposed to endocrine tumors including pheochromocytomas and duodenal somatostatinomas. It is unclear, however, whether the rarely reported occurrence of pancreatic insulinomas in NF-1 patients represents a coincidental finding or whether insulinomas are a rare manifestation of the NF-1 syndrome. To determine the potential association between the NF-1 syndrome and pancreatic endocrine tumors, we analyzed a NF-1 patient with a well-differentiated pancreatic endocrine carcinoma for NF1 mutation, allelic loss of the NF1 gene and its expression in peripheral blood and tumor cells. The germline mutation c. 499 del TGTT known in the family was confirmed by polymerase chain reaction (PCR) and direct sequencing of exon 4 in DNA extracted from peripheral blood. Loss of heterozygosity (LOH) analysis of the NF1 gene was carried out using 3 intragenic microsatellite markers on 17q11.2. RNA expression was examined by reverse transcription and a consecutive PCR spanning intron 3 of the NF1 gene including the mutated site in exon 4. Immunohistochemistry was used to analyze NF-1 protein expression. Mutation analysis of peripheral blood leukocytes confirmed the 4 base pair deletion in exon 4 starting at codon 167 (499 del TGTT). LOH analysis of tumor tissue revealed retention of both NF1 alleles. While reverse transcriptase-PCR of peripheral blood showed bi-allelic expression of both the wild-type NF1 and the mutated form, reverse transcriptase-PCR of tumor extracts demonstrated expression of the mutated but not the wild-type NF1 allele. Additionally, neurofibromin, the NF1 gene product, was absent in the tumor tissue of the NF-1 patient. These results show that the wild-type NF1 transcrips and protein are reduced, in the reported insulinoma, supposedly by epigenetic mechanisms. This provides strong evidence that there is a relationship between von Recklinghausen disease and the patient's insulinoma. In this line, insulinomas may be viewed as a rare manifestation of the NF-1 syndrome. Furthermore, the NF1 gene must be considered as a candidate tumor suppressor gene for sporadic insulinomas and probably other pancreatic endocrine tumors.

Elevated cerebrospinal fluid lactate levels and the pathomechanism of calcification in Fahr's disease

In this study, we report the case of a 68-year-old man complaining of involuntary movement of his left shoulder and lower jaw plus dyspnea. On cranial computed tomography and magnetic resonance imaging, marked and symmetrical calcification at the basal ganglia and dentate nuclei was documented. An elevated cerebrospinal fluid (CSF) lactate level was confirmed by spinal tap examination and magnetic resonance spectroscopy. The raised CSF lactate level, clinical characteristics such as diabetes, bilateral hearing loss and symmetrical cerebral calcification strongly suggested some kinds of mitochondrial disease. However, gene analysis of peripheral blood leukocytes revealed no typical or known mutations. Under the diagnosis of Fahr's disease, we treated him with haloperidol, which completely abolished his symptoms. In Ellsworth-Howard test, he showed markedly decreased phosphaturic response to parathyroid hormone with same pattern as type 2 pseudohypoparathyroidism. This abnormal response in our patient, probably due to respiratory alkalosis reflecting chronic hyperventilation, might in part explain similar mechanism of ectopic calcification underlying these two diseases.

Pre-Infusion Characteristics of the Predominant Cord Blood Unit Correlate with Hematopoietic Engraftment in the Setting of Non-Myeloablative Double Cord Blood Transplant (DCBT).

It is well established that delayed engraftment following transplant of single cord blood unit is mainly due to low CD34 cell dose. Protocols utilizing sequential infusion of two cord blood units increase the total dose of CD34 cells and lead to more rapid hematopoietic engraftment. One of the two cord bloods usually predominates. We assessed the effect of the pre-infusion variables of each cord blood unit on engraftment. Twenty-one patients with hematologic malignancies underwent non-myeloablative conditioning with fludarabine, mephalan and antithymocyte globulin. Two cord bloods, each a minimum 4/6 HLA match with the recipient and each other, were then infused sequentially 1 to 6 hours apart. GVHD prophylaxis consisted of cyclosporine and mycophenolate mofetil. Chimerism analysis of peripheral blood leukocytes was performed by PCR amplification of short tandem repeat loci. Four patients with less than 4 weeks of chimerism data due to death or failed engraftment were excluded. The logrank test, using median values as the cutoff, was used to determine the relationship between pre-infusion variables and time to engraftment. All exact p-values are based on two-sided tests. In all patients, a single cord blood predominated by 3 months post-infusion. In 13 out of 17 transplants (76%), the first cord blood infused predominated (p=.05). The median times for engraftment were 41 days (range: 21–55 days) for platelets >20000 (P20), 65 days (range: 34–91 days) for platelets > 100000 (P100) and 18 days (range: 15–34 days) for ANC>500. Total (cord1 + cord2) CD34 cells/kg (median: 1.9 x 105; range: 6.3 x 104 to 1.0 x 106) and total nucleated cells/kg (median: 4.02 x 107; range 2.98 x 107 to 5.27 x 107) infused did not affect engraftment. However, patients receiving greater than 1.1x 105 CD34/kg in the predominant unit (median: 1.1 x 105; range: 3.0x104 to 8.7x105) but not losing unit (median: 6.92 x 104; range: 8.3.0x103 to 7.4x105) had faster P100 (p=.042 versus p=0.54). The median storage time of the cord blood units infused was 1374 days (range: 272–3047 days). Faster P100 was observed when the predominant unit had a shorter storage time relative to the losing unit (median difference between units: 392 days, p=.03). Our study demonstrated that in the setting of non-myeloablative sequential DCBT, the majority of predominant units engrafted is the first one infused. The CD34 dose and storage time of the predominant unit are factors that can affect engraftment. Although only a small number of patients were studied, these results suggest that the capacity of stem cell niche may be limited and the quality of the cord blood unit occupying this niche can affect the speed of hematopoietic engraftment.

Gene expression analysis of peripheral blood leukocytes from discordant sib-pairs with schizophrenia and bipolar disorder reveals points of convergence between genetic and functional genomic approaches

We performed global RNA transcript analysis and comprehensive gene group analysis of peripheral blood leukocyte (PBL) RNA from two groups of matched sib-pairs that were discordant for either schizophrenia (n = 33 sib-pairs) or bipolar disorder (n = 5 sib-pairs). The pairs chosen for these analyses were selected from families with known patterns of genetic linkage (5q for schizophrenia and 6q for bipolar disorder). At the single gene level, we obtained lists of the transcripts with the most significant changes in expression and from these lists determined those with the highest degree of predictive power for classifying subjects according to diagnosis in these samples. At the gene group level, we comprehensively analyzed pairwise expression changes of more than 4,000 functional groups and cytogenetic locations, and present a novel method of displaying these data that we term "cytogenomic" mapping. Verification of selected changes in expression was performed using quantitative real-time RT-PCR. Our results provide compelling evidence for the utility of analyzing PBL RNA for changes in expression in neuropsychiatric disorders.

Synchronous Occurrence of a Follicular, Papillary and Medullary Thyroid Carcinoma in a Recurrent Goiter

The simultaneous occurrence of different types of thyroid carcinoma in a single patient is an unusual event. We report the case of a 52-year-old man with the history of two previous thyroid operations for benign goiters, who developed a recurrent goiter. The patient was referred to our department for thyroidectomy. In the pathohistological examination the specimen showed a 5 cm follicular carcinoma and a 0.3 cm papillary microcarcinoma in the right lobe as well as a 1.5 cm medullary carcinoma in the left lobe. All tumors were clearly separated from each other, representing the pure entity of each type. Postoperatively, RET germline mutation was ruled out by sequence analysis of peripheral blood leucocytes. Postoperative I-131-radioiodine scan showed multiple lung and liver metastases, while calcitonin was negative. There is no known common cause of these three different tumor types and they developed most independently from each other. The personal history of our patient was interesting in two aspects: (1) he suffered a period of severe staphylococcal sepsis with temporal immunosuppression and (2) he worked for long years as a coremaker in a foundry. This work represented possible long term exposure to inhalative carcinogenous toxins like hydrazine, which caused thyroid parafollicular cell adenomas in an animal model.

Over-Expression of AIF-1 in Liver Allografts and Peripheral Blood Correlates with Acute Rejection after Transplantation in Rats

Early and accurate detection of acute cellular rejection (ACR) is important in the management of liver allograft recipients. We hypothesized that expression of allograft inflammatory factor (AIF)-1 would be associated with liver allograft rejection as previous studies have shown that a relationship exists between kidney and heart transplantation. Indeed using rat orthotopic transplant models we found that the expression of allograft inflammatory factor-1 (AIF-1) can be detected in both allograft and peripheral blood leukocytes with peak levels detected 7 days following liver transplantation. Interestingly, AIF-1 expression increased 2-fold in acutely rejecting liver allografts compared to chronically accepted livers on days 5, 7 and 10 after transplantation. AIF-1 expression in peripheral blood leukocytes was also significantly greater in the rejection model than in the acceptance model. Flow cytometric analysis of peripheral blood leukocytes demonstrated that AIF-1 was expressed in ED2-positive cells, a marker for Kupffer cells. In vitro studies showed that AIF-1 expression in Kupffer cells was up-regulated by coculture with Th1 cytokines. However, neither LPS nor Escherichia coli (E. coli) administration had an affect on AIF-1 expression. These data indicate that high levels of AIF-1 expression reflect aggressive liver allograft rejection and suggest a role for monitoring AIF-1 in peripheral blood leukocytes as a monitor for increased immunosuppression.

Effects of cis-nonachlor, trans-nonachlor and chlordane on the immune system of Sprague–Dawley rats following a 28-day oral (gavage) treatment

The immunotoxicity of cis- and trans-nonachlor and chlordane were investigated in adult male and female Sprague–Dawley rats following a 28-day oral (gavage) treatment. Rats were randomly assigned to six experimental groups: cis-nonachlor, females; trans-nonachlor, females; technical chlordane females; cis-nonachlor, males; trans-nonachlor, males; technical chlordane, males. The immunologic endpoints included: quantification of the total serum immunoglobulin (Ig) levels and subclasses and flow cytometric analysis of peripheral blood leukocytes and T-lymphocyte subsets, evaluation of the lymphoproliferative activity of splenocytes in response to concanavalin A (Con A) and Salmonella typhimurium (STM) mitogens, and natural killer (NK) cell activity of splenocytes. Satellite experiments to examine the delayed-type hypersensitivity (DTH) response to oxazolone, and resistance to Listeria monocytogenes were set up for female rats treated with cis- or trans-nonachlor. Statistically significant ( P<0.05) effects included: increased serum immunoglobulin M (IgM) levels in the chlordane-treated females at the 25 mg/kg dose (pairwise comparison); increased serum IgG 1 and IgG 2c in the cis-nonachlor-treated males at the 2.5 and 25 mg/kg doses and increased serum IgG 2a levels at all doses; increased serum IgG 2b at the 25 mg/kg dose and decreased (dose-related) serum IgM levels in the cis-nonachlor-treated male rats; increased (linear trend) IgG 1 and IgG 2a in the cis-nonachlor-treated females with effects on IgG 2a significant at the 25 mg/kg dose compared with control; increased serum IgG 2a in the trans-nonachlor-treated male and female rats at the 2.5 mg/kg dose; increased absolute numbers (linear trend) of peripheral white blood cells, B lymphocytes, natural killer (NK) cells, T-suppressor/cytotoxic lymphocytes, and the double positive (T-helper/inducer, T-suppressor/cytotoxic) cells in the trans-nonachlor-treated females; increased (non-linear trend) lymphoproliferative activity in the Con A-stimulated splenocytes and decreased (linear trend) activity in the S. typhimurium mitogen-stimulated splenocytes of the cis-nonachlor-treated females; reduced resistance to L. monocytogenes in the cis-nonachlor (day 3, P=0.034)- and trans-nonachlor (day 2, P=0.0001)-treated females, and reduced (linear trend) NK cell activity in the cis-nonachlor-treated males. The present data indicated that the chlordane compounds tested in this study had significant effects on a number of immunologic endpoints. In comparison to technical chlordane, cis- and trans-nonachlors were more immunotoxic. Therefore, an evaluation of the risk these chlorinated compounds may pose to human health should consider the potential effects different chlordane compounds may have on the immune system.

Changes in cytokine expression in peripheral leukocytes, detected by microarray analysis, in the secretory vs proliferative phase of the menstrual cycle

Objective: Many autoimmune disorders have recognized clinical changes in pregnancy and the menstrual cycle. However the mechanisms by which hormones, menstrual cycle changes and pregnancy affect the immune system in unknown. The complexity of the immune system and abundance of immune markers necessitate global analysis. Herein, we describe high density oligonucleotide microarray profiling of peripheral blood leukocytes from the proliferative and secretory phases of the menstrual cycle. Design: Microarray analysis of peripheral blood leukocytes from the proliferative and secretory phases of the menstrual cycle. Materials/Methods: Peripheral blood was obtained from healthy female volunteers (n=5) after informed consent. Subjects were 25–34 yo with menstrual cycles of 28–30 days. Paired samples were collected from subjects in nonconception cycles in the late proliferative (cycle day 10–12) and midsecretory phase (day 20–24). Total RNA was extracted using the Paxgene Blood RNA Validation Kit. cDNA was synthesized from total RNA then biotinylated cRNAs were generated by in vitro transcription. Fragmented cRNAs were hybridized on Affymetrix HU95A microarrays, containing 12,686 human genes and ESTs. The data were analyzed with GeneChip Analysis Suite v5.0, GeneSpring v4.2& MS Excel. Results from each phase were normalized and gene expression was compared. Non-parametric testing was applied to genes showing 1.5 fold changes between the proliferative and secretory phases with a p <0.05 for statistical significance. Results: 10 cc of blood yielded 10–15 g of leukocyte RNA. 17 g of high purity, labeled cRNA was used for hybridization. 314 genes were found to be significantly up-regulated in the secretory phase compared to proliferative, including NKG2F, complement receptor C1r, fibroblast growth factor, monocyte chemotactic protein-2, C-C chemokine receptor-4, platelet derived growth factor receptor and natural killer cell stimulatory factor (IL 12). 362 genes were significantly down-regulated in the secretory phase, such as allograft inflammatory factor-1(AIF-1), pre-B cell enhancing factor (PBEF), GM-CSF receptor beta chain, leukocyte immunoglobulin-like receptor-3(LIR-3), interferon regulatory factor 1, lymphocyte activation antigen CD30, and pT49. Conclusions: We have shown significant changes in the expression of cytokines and their receptors in the menstrual cycle. While validation of these findings and further study are needed to determine their clinical significance, immune cells and their effector cytokines are actively involved in the process of implantation and maternal tolerance to a developing fetus. Peripheral changes in immune cells have been previously described in patients with recurrent spontaneous abortion and unexplained infertility. This study lays foundation for further deliniation of steroid hormone control of immune function and dysfunction in women. Supported by: NIH Training grant HD07493 and Women’s Health at Stanford.