Periodic acoustic pulse generation by mode locked thermoacoustic oscillator.

This study documents periodic acoustic pulse generation by heat in a thermoacoustic oscillator system constructed from a gas-filled acoustic resonance tube having a porous medium called a stack. When the system's dissonancy was enhanced by a local cross-sectional area change in the resonance tube, the periodic pulsed state was turned to a quasiperiodic state. This observation suggests that the acoustic pulse was created through the mode-locking of internal oscillation modes. Propagation of pulsed acoustic intensity in the resonance tube was evidenced by simultaneous measurements of acoustic pressure and axial acoustic particle velocity of the gas.

Synchronization of thermoacoustic quasiperiodic oscillation by periodic external force.

Quasiperiodic oscillations can occur in nonequilibrium systems where two or more frequency components are generated simultaneously. Many studies have explored the synchronization of periodic and chaotic oscillations; however, the synchronization of quasiperiodic oscillations has not received much attention. This study experimentally documents forced synchronization of the quasiperiodic state and the internally locked state of a thermoacoustic oscillator system. This system consists of a gas-filled resonance tube with a nonuniform cross-sectional area. The thermoacoustic oscillator was designed and built in such a way that nonlinear interactions between the fundamental acoustic oscillation mode and the third mode of the gas column are controlled by a temperature difference that is locally created in the resonance tube. Bifurcation diagrams were mapped out by changing the forcing strength and frequency. Separated Arnold tongues were found and both modes were entrained to the external force through complete synchronization. A saddle-node bifurcation was observed in the route from partial to complete synchronization when the forcing strength was relatively weak. However, a Hopf (torus-death) bifurcation was observed when the forcing was relatively strong. In the internally locked state, the bifurcation occurred after the internal locking was broken down by the external force.

An MDS-specific frailty index based on cumulative deficits adds independent prognostic information to clinical prognostic scoring.

The frailty index (FI) is based on the principle that the more deficits an individual has, the greater their risk of adverse outcomes. It is expressed as a ratio of the number of deficits present to the total number of deficits considered. We developed an MDS-specific FI using a prospective MDS registry and assessed its ability to add prognostic power to conventional prognostic scores in MDS. The 42 deficits included in this FI included measurements of physical performance, comorbidities, laboratory values, instrumental activities of daily living, quality of life and performance status. Of 644 patients, 440 were eligible for FI calculation. The median FI score was 0.25 (range 0.05-0.67), correlated with age and IPSS/IPSS-R risk scores and discriminated overall survival. With a follow-up of 20 months, survival was 27 months (95% CI 24-30.4). By multivariate analysis, age >70, FI, transfusion dependence, and IPSS were significant covariates associated with OS. The incremental discrimination improvement of the frailty index was 37%. We derived a prognostic score with five risk groups and distinct survivals ranging from 7.4 months to not yet reached. If externally validated, the MDS-FI could be used as a tool to refine the risk stratification of current clinical prognostication models.

Advances in the treatment of relapsed/refractory chronic lymphocytic leukemia.

Treatment of chronic lymphocytic leukemia (CLL) has advanced with the introduction of chemoimmunotherapy (CIT) agents that have improved the outcomes of frontline therapy. However, most treated patients will relapse and require subsequent therapy. This review focuses on recent advances in the treatment of relapsed or refractory CLL. Until recently, treatment options for relapsed CLL were of limited efficacy. Retreatment with fludarabine, cyclophosphamide, and rituximab (FCR) was recommended for patients with a durable response to first-line FCR, although acquired genetic aberrations, impaired marrow reserve, and comorbidities often made this suboptimal therapy for many patients. New options include two agents targeting B cell receptor (BCR) signaling pathways (ibrutinib and idelalisib) and a B cell lymphoma-2 (BCL-2) inhibitor (venetoclax). Allogeneic hematopoietic stem cell transplantation (HSCT) remains a potentially curative option for younger patients with a suitable donor.

The impact of UGT1A8, UGT1A9, and UGT2B7 genetic polymorphisms on the pharmacokinetic profile of mycophenolic acid after a single oral dose in healthy volunteers.

We studied whether polymorphisms in the UGT1A8, UGT1A9, and UGT2B7 genes, the enzymes producing the phenolic (MPAG) and acyl (AcMPAG) glucuronides of mycophenolic acid (MPA), could contribute to the interindividual variation observed in mycophenolate mofetil (MMF) pharmacokinetics (PKs). This study enrolled 17 healthy volunteers with no polymorphisms (controls) and 17 carriers of UGT1A9 -275/-2152 selected among 305 individuals genetically screened for UDP-glucuronosyltransferase (UGT) polymorphisms. Additional investigative groups included carriers of UGT1A8*2 (A173G) (n=9), UGT1A8*3 (C277Y) (n=4), and UGT1A9*3 (M33T) (n=5). Genetic analysis also included UGT2B7 to detect UGT2B7*2 (His268Tyr) and the promoter haplotype -1248A>G, -1241T>C, -1054T>C, -842G>A, -268A>G, -102T>C. Kinetics were measured in plasma and urine after a single 1.5 g oral dose of MMF, by high-performance liquid chromatography coupled with tandem mass spectrometry, over 12 h after drug intake. Compared to controls, MPA exposure was significantly lower for UGT1A9 -275/-2152 carriers, with no significant changes in MPAG. The estimates of enterohepatic (re)cycling (area under the concentration-time curve (AUC6-12 h/AUC0-12 h)) were significantly lower for MPA, MPAG, and AcMPAG in UGT1A9 -275/-2152 subjects. Compared with controls, UGT1A9*3 carriers had higher MPA and AcMPAG exposure, whereas homozygosity for the UGT1A8*2 allele and heterozygosity for UGT1A8*3 allele had no impact on MPA PKs. Compared with UGT2B7*1/*1 individuals (n=10), UGT2B7*2/*2 subjects (n=17) presented significantly higher free MPA C(max) values and elevated free and total MPA. Results indicate that after a single oral dose of MMF in healthy volunteers, specific UGT genotypes significantly alter MPA PKs and this clearly warrants additional studies with complete and detailed genetic profiling of UGT1A8, UGT1A9, and UGT2B7 genes.

Lack of emergence of cytomegalovirus UL97 mutations conferring ganciclovir (GCV) resistance following preemptive GCV therapy in allogeneic stem cell transplant recipients.

Fifty allogeneic stem cell transplant recipients were enrolled in a prospective cytomegalovirus pp65 antigenemia-guided preemptive therapy trial. Among these, 10 of 34 patients who received ganciclovir exhibited sustained and/or recurrent antigenemia despite treatment. Thirteen leukocyte preparations from these 10 subjects were screened for the presence of the most frequent cytomegalovirus UL97 mutations conferring ganciclovir resistance. None of these mutations were detected after mean and median ganciclovir exposures of 31.6 and 28.0 days, respectively.

Evidence of a founder effect for the protein C gene 3363 inserted C mutation in thrombophilic pedigrees of French origin.

We have previously reported that the 3363 inserted (Ins) C mutation in exon 6 of the protein C gene was present in four unrelated French patients and in four French Canadian families with type I protein C deficiency as well as in a large Vermont protein C deficient kindred of French Canadian origin. The present study was designed to investigate the likelihood of the existence of a founder effect for this mutation in protein C deficient individuals of French origin living in France, Quebec and Vermont. In order to demonstrate a possible founder effect for the 3363 InsC mutation, we have previously constructed a high-resolution genetic map to locate several highly polymorphic markers close to the protein C locus. Thereafter, the markers D2S347, D2S2339, D2S383, D2S2271 and D2S2215 were genotyped in 117 heterozygotes from France (n = 7), Quebec (n = 36) or Vermont (n = 74). The allelic frequency distribution of these five markers was also determined in fifty control French Canadian subjects and thirty-two unaffected members of the Vermont kindred with normal protein C levels and compared with their frequency in our cohort of heterozygotes. Our data suggest that patients from Quebec and Vermont carry a common haplotype at the protein C locus. Moreover, in order to study the evolutionary history of the 3363 InsC mutation, we traced back the ascending genealogy of one proband in each of the families with this mutation. These results showed that the 3363 InsC mutation was most probably introduced in North America by a couple of French settlers who established themselves in 1669 on Isle d'Orleans located near Quebec City. All heterozygotes for the 3363 InsC mutation living in North America are related to these founders within 10 generations. Thus, these families afford a unique opportunity to evaluate the role of the protein C system in thrombophilia due to the high degree of linkage disequilibrium at the protein C gene, which in essence holds that variable more constant than in a more heterogeneous population.

Persistent polyclonal B-cell lymphocytosis: further evidence for a genetic disorder associated with B-cell abnormalities.

Persistent polyclonal B-cell lymphocytosis (PPBL) is an intriguing disorder diagnosed predominantly in women, usually cigarette smokers, characterized by an increase in the number of polyclonal B lymphocytes. Abnormality of the B-cell population is also evidenced by the presence of multiple bcl-2/Ig gene rearrangements and the finding of an additional long arm chromosome 3q+ (i3)(q10) within a significant proportion of B cells. The physiopathology of PPBL is unknown but its association with the HLA DR7 phenotype suggests a possible genetic disorder. To further determine whether PPBL has a genetic predisposition, we have undertaken an extensive study in a large family of a patient diagnosed with PPBL. Three individuals among the first-degree relatives presented all the criteria for a diagnosis of PPBL. A slight increase in serum IgM without evidence of B-cell proliferation was shown in two additional siblings. Multiple bcl-2/Ig gene rearrangements, a typical feature of PPBL, were identified in 8/10 individuals among first-degree relatives. A statistically significant association was found between the presence of these rearrangements and of a paternal HLA haplotype. We conclude that PPBL has a familial occurrence suggesting an underlying genetic defect. The development of the complete syndrome probably relies on unidentified additional co-factors.

Lack of CD40-dependent B-cell proliferation in B lymphocytes isolated from patients with persistent polyclonal B-cell lymphocytosis.

Persistent B-cell lymphocytosis (PPBL) is a haematological disorder diagnosed primarily in adult female smokers that is characterized by a polyclonal increase in peripheral blood B lymphocytes and a moderate elevation of serum IgM. B lymphocyte-associated cellular abnormalities, such as the occurrence of multi-lobed nuclei, increased bcl2/Ig gene rearrangements and the identification of an extra long-arm chromosome (i3)(q10) in the B-cell population, indicate that PPBL could be part of a multi-step process leading to the emergence of a malignant B lymphoproliferation. However, the resulting impact on cellular functional properties remains to be elucidated. Our goal was to address that aspect via the study of B-cell activity following stimulation through CD40, a key molecule of the tumour necrosis factor receptor superfamily involved in B lymphocyte development. In contrast to normal B cells, PPBL B lymphocytes were unable to respond to the proliferative signal delivered in vitro by CD40, indicating a defect in the CD40 activation pathway. Polymerase chain reaction amplification and sequencing of the receptor as well as FACScan analysis of patient B lymphocytes dismissed the possibility of a defect in either CD40 structure or expression. Moreover, Western blot analysis of tyrosine phosphorylation, an early event in the CD40-signalling cascade, was similar in patients and controls, leading to the conclusion that the defect affecting B lymphocytes in PPBL patients is probably located downstream of that signalling cascade.

Quantitative analysis of cytomegalovirus (CMV) viremia using the pp65 antigenemia assay and the COBAS AMPLICOR CMV MONITOR PCR test after blood and marrow allogeneic transplantation.

The performance of a commercially available qualitative PCR test for plasma (AMPLICOR CMV Test; Roche Diagnostics) and a quantitative PCR test for plasma and leukocytes (COBAS AMPLICOR CMV MONITOR Test; Roche Diagnostics) was evaluated with samples from 50 blood or marrow allogeneic transplant recipients who received short courses of sequential ganciclovir therapy (2 weeks intravenously followed by 2 weeks orally) based on a positive cytomegalovirus (CMV) pp65 antigenemia (AG) assay. The number of persons with a positive CMV test was significantly higher for leukocyte-based assays (AG, 67.5%; PCR, 62.5%) compared to both quantitative and qualitative PCR tests of plasma (42.5 and 35%, respectively). One person developed CMV disease during the study despite a negative AG assay; in this particular case, all PCR assays were found to be positive 10 days before his death. There was a trend for earlier positivity after transplantation and more rapid negativity after initiation of ganciclovir for the tests performed on leukocytes. The mean number of CMV copies as assessed by PCR was significantly higher in leukocytes than in plasma (P = 0.02). Overall, excellent agreement (kappa coefficient, >0.75) was found only between the two PCR assays (qualitative and quantitative) based on plasma. These results suggest that either the pp65 AG assay or the COBAS AMPLICOR CMV MONITOR Test using leukocytes could be used to safely monitor CMV viremia in related allogeneic blood or marrow transplant recipients. Such a strategy will result in preemptive treatment for about two-thirds of the persons with a relatively low rate (<33%) of secondary viremic episodes following short courses of ganciclovir therapy.

Type I protein C deficiency in French Canadians: evidence of a founder effect and association of specific protein C gene mutations with plasma protein C levels.

Protein C (PROC) deficiency is one of the most common autosomal codominant diseases. Although more than 150 germline mutations in the PROC gene have been described around the world, the spectrum of mutations among French Canadians is unknown. We have identified one frameshift (3363 ins C) and two missense mutations (R178Q and T298M) in 7 French Canadian families with type I PROC deficiency. In order to demonstrate a possible founder effect for the 3363 ins C mutation, we have constructed a high-resolution genetic map to locate several highly polymorphic markers close to PROC locus. We have then genotyped five markers in 36 heterozygotes for the 3363 ins C mutation. Our data suggest that these patients carry a common haplotype at the PROC locus. Immunologic plasma PROC levels of heterozygotes and genetically normal relatives were also correlated with the nature of the mutation in the coding sequence and with the genotype of three polymorphisms in the PROC promoter. We found that the mean immunologic plasma PROC levels were lower in heterozygotes for the frameshift mutation 3363 ins C compared to heterozygotes for one of the two missense mutations R178Q and T298M (0.46 vs 0.61; P = 0.0004). Moreover, this difference cannot be explained by the genetic variation of the three polymorphisms in the PROC promoter which accounts for only 10.4% of the variation of immunologic PROC levels in non-deficient subjects. These results suggest that the nature of the mutation in the coding sequence of PROC gene may modulate immunologic plasma PROC levels.

Large cell lymphoma complicating persistent polyclonal B cell lymphocytosis.

Persistent polyclonal B cell lymphocytosis (PPBL) is a rare lymphoproliferative disorder of unclear natural history and its potential for B cell malignancy remains unknown. We describe the case of a 39-year-old female who presented with stage IV-B large cell lymphoma 19 years after an initial diagnosis of PPBL; her disease was rapidly fatal despite intensive chemotherapy and blood stem cell transplantation. Because we had recently identified multiple bcl-2/Ig gene rearrangements in blood mononuclear cells of patients with PPBL, we sought evidence of this oncogene in this particular patient: bcl-2/Ig gene rearrangements were found in blood mononuclear cells but not in lymphoma cells. Owing to the possible role of Epstein-Barr virus (EBV) in the pathogenesis of PPBL, we also hypothesized our patient might have an EBV-related lymphoproliferative disorder. Despite serologies consistent with past exposure to this virus, it was not found in lymphoma cells using a sensitive polymerase chain reaction technique. We conclude that non-Hodgkin's lymphoma may occur during the course of PPBL. However, longer follow-up in more patients will be needed in order to better clarity the risk of hematologic malignancy in patients with PPBL.

All patients with persistent polyclonal B cell lymphocytosis present Bcl-2/Ig gene rearrangements.

The bcl-2 gene belongs to a class of oncogenes involved in the inhibition of apoptosis. Most follicular lymphomas are associated with the t(14;18) translocation that juxtaposes the bcl-2 gene located on chromosome 18 to the immunoglobulin gene locus located on chromosome 14. Consequently, the bcl-2 gene is overly expressed and leads to an accumulation of mature clonal B cells. Prolonged survival of the B cell clone appears to be the early event in tumorigenesis, creating an increased risk of cumulative mutations. Interestingly, bcl-2/Ig gene rearrangements may be identified in nearly 50% of normal individuals but the outcome of normal individuals carrying high levels of t(14;18) is not well defined. Persistent polyclonal B cell lymphocytosis (PPBL) is a unique polyclonal lymphoproliferative disorder mostly restricted to women. We have recently demonstrated that PPBL is also associated with multiple bcl-2/Ig gene rearrangements. In this report, we have extended our analysis to additional patients and demonstrated that all patients presented multiple detectable t(14;18) translocated clones. In addition, Bcl-2 protein expression was increased. Our findings, along with the clinical features of PPBL, make this disorder an exceptional model for the study of B-cell homeostasis.

Multiple bcl-2/Ig gene rearrangements in persistent polyclonal B-cell lymphocytosis.

Persistent polyclonal B-cell lymphocytosis is a benign lymphoproliferative disorder of unknown aetiology occurring exclusively in women, characterized by typical binucleated lymphocytes, polyclonal expansion of B cells and elevated serum IgM. Owing to the role of Bcl-2 oncogene in inhibition of apoptosis, we have investigated the presence of the bcl-2/Ig gene rearrangement. Bcl-2/Ig gene rearrangement was determined by polymerase chain reaction targeting the usual breakpoint regions of the t(14;18). Bcl-2/Ig gene rearrangement was identified in all six patients and, more importantly, multiple rearrangements were present in five patients. The frequency of the bcl-2/Ig gene rearrangement is estimated to be of one translocation in 1 x 10(2) to 1 x 10(3) peripheral blood mononuclear cells. We conclude that persistent polyclonal B-cell lymphocytosis is associated with bcl-2/Ig gene rearrangement. These findings are of clinical importance because these patients may be misdiagnosed as having a leukaemic expression of non-Hodgkin's lymphoma.

Treatment of essential thrombocythemia during pregnancy with interferon-alpha.

BACKGROUND: Only a few cases of essential thrombocythemia in pregnant women have been reported, and the management of this myeloproliferative disorder during pregnancy remains uncertain. We report a successful pregnancy in a patient who had essential thrombocythemia and who was treated with interferon-alpha, and we review the literature for the outcome of similar patients. CASE: A 32-year-old woman, gravida 4, para 3, aborta 0, presented at 18 weeks' gestation with two episodes of amaurosis fugax and an elevated platelet count of 2300 x 10(9)/L. The initiation of interferon-alpha led to a progressive fall of the platelet level, with no occurrence of thrombotic or hemorrhagic manifestations. Serial ultrasound examinations revealed normal fetal and placental development. The patient was delivered of a male infant at 37 weeks. Both child and placenta were normal on examination. CONCLUSION: Our case and the current available data suggest that interferon-alpha may be the best therapeutic option for pregnant patients with essential thrombocythemia in whom myelosuppression is required.

Granulocyte recovery after sequential transfusion of mobilized blood stem cell concentrates in syngeneic recipients.

Three patients received sequential transfusions of G-CSF-mobilized peripheral blood stem cells from their identical twin in an attempt to abrogate neutropenia. Blood stem cells were harvested by leukapheresis in the healthy donor twins following mobilization with rhG-CSF at 5 micrograms/kg/day subcutaneously for at least 5 days. An average of 2.2 x 10(7) CFU-GM (range: 1.4-3.3) were collected and transfused without further manipulation. One patient, transfused with a CFU-GM dose of 3 x 10(7) on day +6 after a syngeneic marrow transplant, experienced near-complete elimination of absolute neutropenia until spontaneous engraftment occurred on day +11. In the other two patients, we unexpectedly observed a transient granulopoietic inhibition, possibly related to the high T cell content of the blood stem cell transfusions.

Evidence for restricted diversity of antigen-specific human antibodies in immunized hu-PBL-SCID mice.

Previous studies have revealed that specific human humoral immune responses could be produced in immunized SCID mice after engraftment of human lymphocytes (hu-PBL-SCID). On the other hand, the engrafted repertoire of B cell clones is known to be skewed in hu-PBL-SCID with the corresponding production of only a limited set of major human antibodies. In this work, we have analyzed the diversity of tetanus toxoid-specific human antibodies produced in immunized hu-PBL-SCID mice in comparison with the total serum antibody population using zone electrophoresis followed by blotting. The results showed that the diversity of tetanus toxoid-specific antibody population was more restricted than that of the total human antibody population, with some animal sera containing a single band of tetanus toxoid-specific antibody molecule, in clear contrast to the polyclonal response of the PBL donor. Absorption experiments showed tetanus toxoid-specific antibodies could account for a significant proportion (up to 10%) of the total human antibodies present in hu-PBL-SCID mouse sera. The inability to expand a high number of different antigen-specific B cell clones in immunized hu-PBL-SCID mice represents an important intrinsic limitation of this animal model which may be caused by defects in T cell help.

Hematopoietic engraftment from a minimal number of apheresis procedures after mobilization of peripheral blood stem cells with chemotherapy and rhG-CSF.

In a cohort of 13 patients, peripheral blood stem cells (PBSC) were harvested by apheresis after mobilization with chemotherapy and rhG-CSF. Nine patients who had excellent mobilization were transplanted with PBSC concentrates from a minimal number of apheresis procedures (mean of 1.5, range = 1-3). During collection, the number of circulating progenitors was on average 50 times higher than those observed at the steady state in the peripheral blood of healthy unstimulated individuals. The mean number of CFU-GM/kg reinfused per patient was 28.1 x 10(4) (range = 18.0-50 x 10(4)). The use of rhG-CSF, at either 1 or 5 micrograms/kg/day, resulted in a significantly greater yield of CFU-GM per mononuclear cells than that observed previously in a comparable group of patients receiving chemotherapy alone. Prompt and durable engraftment occurred after myeloablative chemotherapy. The average duration of absolute neutropenia was 9 days. Transfusion requirements were low with an average of four packed red cell units and two platelet transfusions per patient. The shortest follow-up is 5 months and the longest is 20+ months. The convenience of this new approach to support myeloablative therapy offers new possibilities for the administration of a higher dose-intensity of chemotherapeutic agents. A limited number of apheresis procedures timely harvested will improve the cost effectiveness of transplant programs.

Efficient preparation of human monoclonal antibody-secreting heterohybridomas using peripheral B lymphocytes cultured in the CD40 system.

The use of peripheral B lymphocytes in the successful preparation of human monoclonal antibodies by hybridoma technology is highly dependent on lymphocyte activation procedures. We studied the ability of peripheral human B lymphocytes cultured in vitro and activated through their CD40 antigen (CD40 system) (Banchereau et al., 1991) to form antibody-secreting heterohybridomas after fusion with murine X63Ag8.653 myeloma cells. The frequency of antibody-secreting heterohybridomas formation was greatly increased (15 times) by culture of B cells in the CD40 system. The CD40 system offers many advantages over other procedures of B lymphocyte activation representing a significant technological advance in the preparation of human monoclonal antibodies by standard hybridoma technology.