Activation of RHOA-VAV1 signaling in angioimmunoblastic T-cell lymphoma.

Somatic G17V RHOA mutations were found in 50-70% of angioimmunoblastic T-cell lymphoma (AITL). The mutant RHOA lacks GTP binding capacity, suggesting defects in the classical RHOA signaling. Here, we discovered the novel function of the G17V RHOA: VAV1 was identified as a G17V RHOA-specific binding partner via high-throughput screening. We found that binding of G17V RHOA to VAV1 augmented its adaptor function through phosphorylation of 174Tyr, resulting in acceleration of T-cell receptor (TCR) signaling. Enrichment of cytokine and chemokine-related pathways was also evident by the expression of G17V RHOA. We further identified VAV1 mutations and a new translocation, VAV1-STAP2, in seven of the 85 RHOA mutation-negative samples (8.2%), whereas none of the 41 RHOA mutation-positive samples exhibited VAV1 mutations. Augmentation of 174Tyr phosphorylation was also demonstrated in VAV1-STAP2. Dasatinib, a multikinase inhibitor, efficiently blocked the accelerated VAV1 phosphorylation and the associating TCR signaling by both G17V RHOA and VAV1-STAP2 expression. Phospho-VAV1 staining was demonstrated in the clinical specimens harboring G17V RHOA and VAV1 mutations at a higher frequency than those without. Our findings indicate that the G17V RHOA-VAV1 axis may provide a new therapeutic target in AITL.

Identification of cell-type-specific mutations in nodal T-cell lymphomas.

Recent genetic analysis has identified frequent mutations in ten-eleven translocation 2 (TET2), DNA methyltransferase 3A (DNMT3A), isocitrate dehydrogenase 2 (IDH2) and ras homolog family member A (RHOA) in nodal T-cell lymphomas, including angioimmunoblastic T-cell lymphoma and peripheral T-cell lymphoma, not otherwise specified. We examined the distribution of mutations in these subtypes of mature T-/natural killer cell neoplasms to determine their clonal architecture. Targeted sequencing was performed for 71 genes in tumor-derived DNA of 87 cases. The mutations were then analyzed in a programmed death-1 (PD1)-positive population enriched with tumor cells and CD20-positive B cells purified by laser microdissection from 19 cases. TET2 and DNMT3A mutations were identified in both the PD1+ cells and the CD20+ cells in 15/16 and 4/7 cases, respectively. All the RHOA and IDH2 mutations were confined to the PD1+ cells, indicating that some, including RHOA and IDH2 mutations, being specific events in tumor cells. Notably, we found that all NOTCH1 mutations were detected only in the CD20+ cells. In conclusion, we identified both B- as well as T-cell-specific mutations, and mutations common to both T and B cells. These findings indicate the expansion of a clone after multistep and multilineal acquisition of gene mutations.

A phase I/II trial of intrabone marrow cord blood transplantation and comparison of the hematological recovery with the Japanese nationwide database.

Intrabone marrow cord blood transplantation (IB-CBT) was proposed as a promising treatment modality to improve hematological recovery. However, clinical advantages of IB-CBT over conventional IV CBT have been unclear. We conducted a prospective single-center trial of IB-CBT to evaluate its safety and superiority in terms of hematological recovery. Fifteen adults with hematological malignancies were enrolled. A thawed and unwashed single cord blood unit was injected into the bilateral superior-posterior iliac crests under local anesthesia. Engraftments of neutrophils and platelets were achieved in 13 cases, with medians of 17 and 45 days, respectively. For the control, we extracted data from the Japanese nationwide database and compared the hematological recovery of contemporaneously transplanted 1135 CBT cases. Multivariate analysis revealed that IB-CBT enhanced platelet recovery (hazard ratio, 2.13; P=0.007), but neutrophil recovery did not differ significantly (hazard ratio, 1.70; P=0.19). Better donor chimerism was seen in the bone marrow of the ilium than of the sternum on day 14, suggesting that the local hematopoiesis at the injected site was established earlier than that at the remote bone marrow site. Collectively, IB-CBT was well tolerated and may enhance local engraftment, which promotes prompter platelet recovery than does IV-CBT.

Hes1 suppresses acute myeloid leukemia development through FLT3 repression.

In leukemogenesis, Notch signaling can be up and downregulated in a context-dependent manner. The transcription factor hairy and enhancer of split-1 (Hes1) is well-characterized as a downstream target of Notch signaling. Hes1 encodes a basic helix-loop-helix-type protein, and represses target gene expression. Here, we report that deletion of the Hes1 gene in mice promotes acute myeloid leukemia (AML) development induced by the MLL-AF9 fusion protein. We then found that Hes1 directly bound to the promoter region of the FMS-like tyrosine kinase 3 (FLT3) gene and downregulated the promoter activity. FLT3 was consequently upregulated in MLL-AF9-expressing immortalized and leukemia cells with a Hes1- or RBPJ-null background. MLL-AF9-expressing Hes1-null AML cells showed enhanced proliferation and ERK phosphorylation following FLT3 ligand stimulation. FLT3 inhibition efficiently abrogated proliferation of MLL-AF9-induced Hes1-null AML cells. Furthermore, an agonistic anti-Notch2 antibody induced apoptosis of MLL-AF9-induced AML cells in a Hes1-wild type but not a Hes1-null background. We also accessed two independent databases containing messenger RNA (mRNA) expression profiles and found that the expression level of FLT3 mRNA was negatively correlated with those of HES1 in patient AML samples. These observations demonstrate that Hes1 mediates tumor suppressive roles of Notch signaling in AML development, probably by downregulating FLT3 expression.

Reduced TET2 function leads to T-cell lymphoma with follicular helper T-cell-like features in mice.

TET2 (Ten Eleven Translocation 2) is a dioxygenase that converts methylcytosine (mC) to hydroxymethylcytosine (hmC). TET2 loss-of-function mutations are highly frequent in subtypes of T-cell lymphoma that harbor follicular helper T (Tfh)-cell-like features, such as angioimmunoblastic T-cell lymphoma (30-83%) or peripheral T-cell lymphoma, not otherwise specified (10-49%), as well as myeloid malignancies. Here, we show that middle-aged Tet2 knockdown (Tet2(gt/gt)) mice exhibit Tfh-like cell overproduction in the spleen compared with control mice. The Tet2 knockdown mice eventually develop T-cell lymphoma with Tfh-like features after a long latency (median 67 weeks). Transcriptome analysis revealed that these lymphoma cells had Tfh-like gene expression patterns when compared with splenic CD4-positive cells of wild-type mice. The lymphoma cells showed lower hmC densities around the transcription start site (TSS) and higher mC densities at the regions of the TSS, gene body and CpG islands. These epigenetic changes, seen in Tet2 insufficiency-triggered lymphoma, possibly contributed to predated outgrowth of Tfh-like cells and subsequent lymphomagenesis. The mouse model described here suggests that TET2 mutations play a major role in the development of T-cell lymphoma with Tfh-like features in humans.

Neutralization of the γ-secretase activity by monoclonal antibody against extracellular domain of nicastrin.

Several lines of evidence suggest that aberrant Notch signaling contributes to the development of several types of cancer. Activation of Notch receptor is executed through intramembrane proteolysis by γ-secretase, which is a multimeric membrane-embedded protease comprised of presenilin, nicastrin (NCT), anterior pharynx defective 1 and PEN-2. In this study, we report the neutralization of the γ-secretase activity by a novel monoclonal antibody A5226A against the extracellular domain of NCT, generated by using a recombinant budded baculovirus as an immunogen. This antibody recognized fully glycosylated mature NCT in the active γ-secretase complex on the cell surface, and inhibited the γ-secretase activity by competing with the substrate binding in vitro. Moreover, A5226A abolished the γ-secretase activity-dependent growth of cancer cells in a xenograft model. Our data provide compelling evidence that NCT is a molecular target for the mechanism-based inhibition of γ-secretase, and that targeting NCT might be a novel therapeutic strategy against cancer caused by aberrant γ-secretase activity and Notch signaling.

Chilling-injury and disturbance of ion homeostasis in the coxal muscle of the tropical cockroach (Nauphoeta cinerea).

Adults of warm- and cold-acclimated tropical cockroaches, Nauphoeta cinerea were exposed to low temperatures of 0 or 5 degrees C for various time intervals (hours to days). Development of chilling-injury (defects in crawling and uncoordinated movements) and mortality during the exposure were assessed and correlated with the changes in concentrations of metal ions (Na(+), K(+) and Mg(2+)) in the haemolymph and coxal muscle tissue. Warm-acclimated insects entered chill-coma at both low temperatures. In their haemolymph, the [Na(+)] and [Mg(2+)] linearly decreased and [K(+)] increased with the increasing time of exposure. The rate of concentration changes was higher at 0 than at 5 degrees C. The concentration changes resulted in gradually dissipating equilibrium potentials across the muscle cell membranes. For instance, E(K) decreased from -49.8 to -20.7 mV during 7 days at 5 degrees C. Such a disturbance of ion homeostasis was paralleled by the gradual development of chilling-injury and mortality. Most of the cockroaches showed chilling-injury when the molar ratio of [Na(+)]/[K(+)] in their haemolymph decreased from an initial of 4.4 to 2.1-2.5. In contrast, the cold-acclimated cockroaches did not enter chill-coma. They maintained constant concentrations of ions in their haemolymph, constant equilibrium potentials across muscle cell membranes and the development of chilling-injury was significantly suppressed at 5 degrees C for 7 days.

High-grade cytomegalovirus antigenemia after hematopoietic stem cell transplantation.

Clinical impact of high-grade (HG) cytomegalovirus (CMV) antigenemia after hematopoietic stem cell transplantation has not been clarified. Therefore, in order to investigate the risk factors and outcome for HG-CMV antigenemia, we retrospectively analyzed the records of 154 Japanese adult patients who underwent allogeneic hematopoietic stem cell transplantation for the first time from 1995 to 2002 at the University of Tokyo Hospital. Among 107 patients who developed positive CMV antigenemia at any level, 74 received risk-adapted preemptive therapy with ganciclovir (GCV), and 17 of these developed HG-antigenemia defined as > or = 50 positive cells per two slides. The use of systemic corticosteroids at > or = 0.5 mg/kg/day at the initiation of GCV was identified as an independent significant risk factor for HG-antigenemia. Seven of the 17 HG-antigenemia patients developed CMV disease, with a cumulative incidence of 49.5%, which was significantly higher than that in the low-grade antigenemia patients (4%, P<0.001). However, overall survival was almost equivalent in the two groups. In conclusion, the development of HG-antigenemia appeared to depend on the profound immune suppression of the recipient. Although CMV disease frequently developed in HG-antigenemia patients, antiviral therapy could prevent a fatal outcome.

Cardiac complications after haploidentical HLA-mismatched hematopoietic stem cell transplantation using in vivo alemtuzumab.

Alemtuzumab is a humanized monoclonal antibody directed against human CD52 with a strong lympholytic effect. We have performed unmanipulated hematopoietic stem cell transplantation (HSCT) from 2- or 3-locus-mismatched family donors in 14 patients using in vivo alemtuzumab. All achieved complete donor cell engraftment and grade III-IV acute graft-versus-host disease was observed in only one patient. However, eight of the 14 patients developed grade II-IV cardiac complications according to Bearman's criteria. Next, we retrospectively analyzed the records of 142 adult patients who underwent allogeneic HSCT from 1995 to 2004 to evaluate whether the use of alemtuzumab was an independent risk factor for cardiac complications. Among several factors that increased the incidence of grade II-IV cardiac complications with at least borderline significance, a multivariate analysis identified the cumulative dose of anthracyclines (P=0.0016) and the use of alemtuzumab (P=0.0001) as independent significant risk factors. All of the cardiac complications in the alemtuzumab group were successfully treated with diuretics and/or catecholamines. Patient selection and close monitoring of cardiac function may be important in HLA-mismatched HSCT using in vivo alemtuzumab.

Predictors for severe cardiac complications after hematopoietic stem cell transplantation.

The value of pre-transplant factors for predicting the development of cardiac complications after transplantation has been inconsistent among studies. We analyzed the impact of pre-transplant factors on the incidence of severe cardiac complications in 164 hematopoietic stem cell transplant recipients. We identified eight patients (4.8%) who experienced grade III or IV cardiac complications according to the Bearman criteria. Seven died of cardiac causes a median of 3 days after the onset of cardiac complications. On univariate analysis, both the cumulative dose of anthracyclines and the use of anthracyclines within 60 days before transplantation affected the incidence of severe cardiac complications (P=0.0091 and 0.011). The dissociation of heart rate and body temperature, which reflects "relative tachycardia", was also associated with a higher incidence of cardiac complications (P=0.024). None of the variables obtained by electrocardiography or echocardiography were useful for predicting cardiac complications after transplantation, although the statistical power might not be sufficient to detect the usefulness of ejection fraction. On a multivariate analysis, the cumulative dose of anthracyclines was the only independent significant risk factor for severe cardiac complications. We conclude that the cumulative dose of anthracyclines is the most potent predictor of cardiac complications and the administration of anthracyclines should be avoided within two months before transplantation.

Increased incidence of acute graft-versus-host disease with the continuous infusion of cyclosporine A compared to twice-daily infusion.

We retrospectively compared the incidence of acute graft-versus-host disease (GVHD) before and after September 1999, when we changed the mode of cyclosporine A (CsA) administration from twice-daily infusions (TD) (n=58) to continuous infusion (CIF) (n=71). The incidence of grade II-IV acute GVHD in the CIF group (56%) was significantly higher than that in the TD group (27%, P=0.00022). Multivariate analysis identified only two independent significant risk factors for the development of grade II-IV acute GVHD; CIF of CsA (relative risk 2.59, 95% CI 1.46-4.60, P=0.0011) and the presence of HLA mismatch (2.01, 95% CI 1.15-3.53, P=0.014). The incidence of relapse was significantly lower in the CIF group when adjusted for disease status before transplantation (0.41, 95% CI 0.18-0.95, P=0.038), which resulted in better disease-free survival in high-risk patients (43 vs 16% at 2 years, P=0.039), but not in standard-risk patients (72 vs 80%, P=0.45). CIF of CsA with a target level of 250-400 ng/ml may not be appropriate for GVHD prophylaxis in standard-risk patients.

Male predominance among Japanese adult patients with late-onset hemorrhagic cystitis after hematopoietic stem cell transplantation.

Late-onset hemorrhagic cystitis (LHC) after hematopoietic stem cell transplantation (HSCT) is mainly caused by viral infections. We retrospectively analyzed the records of 141 Japanese adult patients who underwent a first allogeneic HSCT from 1995 to 2002. In all, 19 patients developed LHC a median of 51 days after HSCT. Adenovirus (AdV) was detected in the urine of 10 LHC patients, of whom eight had AdV type 11. Five of the six available serum samples from these patients were also positive for AdV type 11, but the detection of AdV in serum was not associated with a worse outcome. Male sex and the development of grade II-IV acute graft-versus-host disease were identified as independent significant risk factors for LHC. Male predominance was detected in LHC after HSCT, as has been previously shown in children with AdV-induced acute HC. The detection of AdV DNA in serum did not predict a poor outcome.

Relation with preoperative fructosamine and autonomic nerve function and blood pressure during anesthesia in diabetics: a retrospective study.

Many diabetics may have a high risk involving the cardiovascular system. In an attempt to predict the intraoperative risks of diabetics during anesthesia, we evaluated retrospectively the relationship among the biochemical assay or autonomic nerve function obtained as parts of the preoperative examination, and the blood pressure changes relating to the stimulation of intubation and extubation for anesthesia. In 40 diabetic surgical patients examined the biochemical assay (HbA1c, fructosamine level and blood glucose level) beforehand, the autonomic nerve function was quantified preoperatively by analysis of ECG R-R variability recorded in supine and subsequent standing position using an HRV analyzer, and some parameters of autonomic nerve function especially responsive sympathetic nerve activities were obtained. We assessed the correlation with systolic blood pressure changes in these cases at intubation for general anesthesia comparing to similar conditioned 40 non-diabetics. A diabetics with low vagal activity became larger systolic blood pressure afterdrop at tracheal intubation for anesthesia (r=0.513, p<0.001). Otherwise the blood pressure afterdrop at extubation became larger in a non-diabetics with high sympathetic activity (r=0.502, p<0.001). The preoperative fructosamine concentration in diabetics correlated positively with the responsive sympathetic nerve irritability index; "mRR(sup)-RRmin(std)" (r=0.432, p<0.05) and the responsive sympathetic nerve excitability index; "mRR(sup-std)" (r=0.448, p<0.05). However HbA1c had no correlation with these parameters of autonomic nerve function and blood pressure rise at tracheal intubation. Because of above correlation with blood pressure rise at intubation for anesthesia induction, the preoperative fructosamine examination and the responsive sympathetic nerve function test must be useful preoperative examination for detection of the unexpected heart events of diabetic patients during operation.

Excitation of oxytocin cells in the hypothalamic supraoptic nucleus by electrical stimulation of the dorsal penile nerve and tactile stimulation of the penis in the rat.

In urethane-anaesthetized male rats, electrical stimulation of the dorsal penile nerve (DPN) excited 29 of 48 (60%) oxytocin cells in the contralateral supraoptic nucleus, whereas only 5 of 28 (18%) vasopressin cells were excited by the stimulation. The stimulus applied to the ipsilateral DPN to the recorded neurone also excited a similar proportion of oxytocin cells (25 of 43; 58%). Tactile stimulation of the glans penis excited 7 of 12 (58%) oxytocin cells, whereas the same stimulation excited only 3 of 16 (19%) vasopressin cells. The results suggest that sensory information arising from the penis preferentially excites oxytocin cells in the supraoptic nucleus.

[Intraoperative CT imaging system using a mobile CT scanner gantry mounted on floor-embedded rails for neurosurgery].

Many neurosurgeons prefer to use intraoperative computed tomographic (CT) scanning, when possible, to check whether there is residual lesion or unexpected bleeding. We report a practical intraoperative CT imaging system using a high-speed CT scanner installed in the operating room along with a digitally controlled neurosurgical operating table. We designed a rail-track system to mobilize the CT gantry. The gantry is fixed onto a motorized carrier that can be moved smoothly on a rail-track embedded in the floor and with a maximum reach of 2.85 m from the room's wall to the operating table. The longitudinal motion of the operating table is easily adjusted by a foot switch from manual control to automatic control directly from the CT scanner's computer like an ordinary CT scanner bed in increments of 2, 5 or 10 mm during CT scanning. Either a carbon-made radiolucent head frame or carbon-made head plate is used as a headrest. Using this CT scanner system, pre- and intraoperative CT scannings were performed on 46 patients with brain tumors, cervical lesions or other intracranial lesions. We could operate on the patient with enough working space between the mobile CT gantry and the operating table for microneurosurgery. We could obtain intraoperative CT imaging of a patient on the operating table while the surgical wound remained open, the surgical drapes kept in place, and the surgical position unchanged, saving time in intraoperative CT scanning and preparation for further surgery when needed. This intraoperative CT imaging system installed in the operating room should be useful for neurosurgery.

Effects of growth hormone-releasing hormone (GRF) analogs, bovine and rat GRF on growth hormone secretion in cattle in vivo.

Effects of bovine and human growth hormone-releasing hormone (GRF) analogs (bGRF(1-29)-NH2: bGRF-29, [D-Ala2, Ala15]-bGRF-29, [D-Ala2]-hGRF(1-29)-NH2: [D-Ala2]-hGRF-29), bovine GRF (bGRF(1-44)-NH2: bGRF-44), as well as rat GRF (rGRF) on GH release were studied in female calves. Intravenous (i.v.) bolus injections of 0.25 microg/kg BW of bGRF-29, [D-Ala2, Ala15]-bGRF-29, and [D-Ala2]-hGRF-29 stimulated GH release. Plasma GH levels began to rise 10 min after the injection of each peptide, and significant increases in GH concentrations were obtained at 60, 180 and 150 min after the injection of bGRF-29, [D-Ala2, Ala15]-bGRF-29 and [D-Ala2]-hGRF-29, respectively. The concentrations of GH 80 min after the injection of [D-Ala2, Ala15]-bGRF-29 were significantly higher than those after the injection of [D-Ala2]-hGRF-29 (except at 80 and 90 min) or bGRF-29. The i.v. bolus injections of 0.25 microg/kg BW of bGRF-44 and rGRF stimulated GH release, and the GH-releasing potency of rGRF was approximately equal to that of bGRF-44. The plasma GH responses to the repeated i.v. injection of bGRF-29 or [D-Ala2, Ala15]-bGRF-29 at 2-h intervals were examined. bGRF-29 acutely increased plasma GH levels after each injection, and the high GH levels decreased to the basal values within 2 h. In contrast, high GH levels induced by [D-Ala2, Ala15]-bGRF-29 were gradually decreased but not lowered to basal values throughout the experiment. These results show that [D-Ala2, Ala15]-bGRF-29 has longer-lasting and greater GH-releasing activity than the other GRF analogs in female calves, and the GH-releasing potency of rat GRF is approximately equal to that of bovine GRF in cattle in vivo.

Afferents originating from the dorsal penile nerve excite oxytocin cells in the hypothalamic paraventricular nucleus of the rat.

Electrical stimulation of the dorsal penile nerve (DPN) produced orthodromic excitation in about half of oxytocin cells in the paraventricular nucleus (PVN). In contrast, less than 10% of vasopressin cells were excited. Tactile stimulation of the glans penis by a paintbrush produced excitation in 40% of oxytocin cells. Castration did not prevent activation of oxytocin cells. These results suggest that somatosensory information from the penis is transmitted to the PVN through the DPN and that such afferent input preferentially innervates oxytocin cells.

Retinal complications with elevated circulating plasma C5a associated with interferon-alpha therapy for chronic active hepatitis C.

Retinal hemorrhage is a complication of interferon therapy of unknown pathogenesis. We report two chronic active hepatitis C patients who developed retinal hemorrhage and/or cotton wool patches during interferon-alpha therapy 4 and 12 wk after beginning treatment. At the time of the hemorrhage, plasma-activated complement 5, a known potent intravascular aggregator of granulocytes, increased to 54 ng/ml in one patient and to 29 ng/ml in the other patient. When the hemorrhage resolved, it decreased to under 5 ng/ml. Our cases suggest that complement activation occurs in patients treated with interferon-alpha and that activation of complement 5 can lead to retinal capillary infarction and retinal hemorrhage. High levels of activated complement 5 may predict retinal artery infarction or perhaps microvascular emboli in the other organs.

Brain energy metabolism and blood flow during sevoflurane and halothane anesthesia: effects of hypocapnia and blood pressure fluctuations.

The effects of halothane and sevoflurane on cat brain energy metabolism and regional cerebral blood flow (rCBF) were evaluated during normo- and hypocapnia. Brain energy status was evaluated with phosphorous nuclear magnetic resonance spectroscopy (31P-MRS) and rCBF was measured by the hydrogen clearance method. A high concentration of halothane (3 MAC) impaired brain energy metabolism, while even a higher concentration of sevoflurane (4 MAC) had no untoward effect on brain energy metabolism. At 3 MAC of halothane, there were measurable decreases in brain phosphocreatine (69% of the control) and increases in brain inorganic phosphate (about 250% of control Pi), even though CBF was about 70% of the control value. During hypocapnia, the phosphocreatine levels began to decrease at a Paco2 of 2.7 kPa with 2 MAC of sevoflurane (90% of the control), and at a Paco2 of 4.0 kPa with 2 MAC of halothane (92% of the control). rCBF had decreased to less than 50% of the control value when Paco2 was < or = 2.7 kPa with 2 MAC of sevoflurane and < or = 4.0 kPa with 2 MAC of halothane. Abnormal brain energy metabolism was only observed when rCBF was decreased to less than half of the control (non-anesthetized and normocapnic) value. Following administration of a vasopressor, metaraminol, the abnormal brain energy metabolism induced by 2 MAC of halothane at a Paco2 of 1.33 kPa was normalized in parallel with the improved rCBF values. We conclude that hyperventilation and fluctuating blood pressure contribute to the occurrence of abnormal brain energy metabolism during halothane and sevoflurane anesthesia. This is more pronounced with halothane than with sevoflurane. The hypocapnia-induced abnormality during exposure to 2 MAC of either agent was due to decreased CBF associated with low perfusion pressure, indicating that there was no direct effect of these anesthetics on cerebral energy metabolism.