PRR11 immunoreactivity is a weak prognostic factor in non-mucinous invasive adenocarcinoma of the lung.

INTRODUCTION: Proline-rich protein 11 (PRR11) functions in the progression of cell cycle, and silencing the PRR11 gene in lung cancer cells results in the inhibition of cellular proliferation, cell cycle progression, cell migration, invasion and colony formation. PRR11 may therefore be a therapeutic target in lung cancer. MATERIALS AND METHODS: Microarrays of surgical specimens of non-mucinous invasive adenocarcinoma of the lung, from 346 subjects that were not given preoperative therapy, were autoimmunostained with PRR11 and, except for trace and pseudo-positivity, assessed as "positive" at any proportion and intensity. RESULTS: PRR11 immunoreactivity demonstrated a tendency to associate with an aggressive phenotype (tumor size, vascular invasion, and adjuvant therapy) and some effect on overall survival (Hazard ratio 1.51). CONCLUSIONS: PRR11 may be a weak prognostic indicator of overall survival of patients with non-mucinous invasive adenocarcinoma of the lung.

Third heart sound in hospitalised patients with acute heart failure: insights from the ATTEND study.

BACKGROUND: Several previous studies have suggested that detection of a third heart sound (S3) in patients with chronic congestive heart failure is associated with adverse long-term outcomes. However, the short-term prognostic value of identifying an S3 on admission in patients with acute heart failure (AHF) is not well established. We therefore analysed the in-hospital prognostic value of detecting an S3 on admission in hospitalised patients with AHF. METHODS: The Acute Decompensated Heart Failure Syndromes (ATTEND) study investigators enrolled 4107 patients hospitalised with AHF. Investigators evaluated the presence or absence of an S3 during routine physical examination. RESULTS: On admission to hospital, 1673 patients (41%) had an S3. Patients with an S3 had a higher heart rate, higher serum level of B-type natriuretic peptide and higher creatinine levels than patients without an S3. However, there were no significant differences of systolic blood pressure, serum sodium, haemoglobin, C-reactive protein and total bilirubin between the two groups. Multivariate analysis adjusted for various markers of disease severity revealed that only the presence of an S3 was independently associated with an increase of in-hospital all cause death [adjusted odds ratio (OR), 1.69; 95% confidence interval (CI), 1.19-2.41; p = 0.003] and cardiac death (adjusted OR, 1.66; 95% CI, 1.08-2.54; p = 0.020) among the congestive physical findings related to heart failure (S3, rales, jugular venous distension and peripheral oedema). CONCLUSIONS: Detecting an S3 on admission was independently associated with adverse in-hospital outcomes in patients with AHF. Our findings suggest that careful bedside assessment is clinically meaningful.

Transcriptional CCND1 expression as a predictor of poor response to neoadjuvant chemotherapy with trastuzumab in HER2-positive/ER-positive breast cancer.

Several trials have confirmed that the pathological complete response (pCR) rates after neoadjuvant chemotherapy (NAC) are significantly lower in HER2-positive/ER-positive patients than in HER2-positive/ER-negative patients. To understand this phenomenon, we investigated the association between NAC resistance and CCND1, which is frequently overexpressed in ER-positive tumors. Pretreatment formalin-fixed tumor tissues were collected from 75 HER2-positive patients receiving NAC comprised anthracyclines, taxanes, and trastuzumab. Seventeen gene transcripts along with PIK3CA mutations were detected using MassARRAY (Sequenom, San Diego, CA). The gene expression levels were dichotomized according to the median values. The immunohistochemical expression of ER, PTEN, BCL-2, and cyclin D1 was scored. The relationship between the variables was assessed using the Spearman correlation. A logistic regression analysis was performed to detect predictors of pCR, which was defined as no invasive tumor in the breast or axilla. Forty-seven percent of the cases were ER-positive and 52 % (40/63 % in ER-positive/ER-negative) achieved a pCR. Among the ER-positive patients, the CCND1 gene expression level was 2.1 times higher than that in ER-negative patients and was significantly correlated with the expression of cyclin D1 protein. In a univariate analysis, a pCR was associated with high mRNA levels of ESR1, PGR, LMTK3, HER2, IGF1R, INPP4B, PDL-1, BCL-2, and CCND1 (P ≤ 0.05). In contrast, none of these genes were significantly correlated with a pCR among the ER-negative tumors and only EGFR was significantly correlated with a pCR. PIK3CA mutations or PTEN loss were not associated with a pCR in either group. After excluding ESR1 (r = 0.58), PGR (r = 0.64), and IGF1R (r = 0.59), the expressions of which were correlated with CCND1, a multivariate analysis revealed that CCND1 [P = 0.043; OR, 0.16] and HER2 [P = 0.012; OR, 11.2] retained its predictive value for pCR among ER-positive patients, but not among ER-negative patients. A High Level of CCND1 gene expression is a poor predictor of a pCR and provides a rationale for evaluating CDK4/6 inhibitors in HER2-positive/ER-positive breast cancer patients.

Noninvasive delivery of siRNA into the epidermis by iontophoresis using an atopic dermatitis-like model rat.

Topical application of siRNA to the skin should be an effective treatment for serious skin disorders, such as atopic dermatitis. However, it is difficult to introduce hydrophilic macromolecules, including siRNA, into the skin by conventional methods. For efficient delivery of siRNA, we examined an iontophoretic technique, since it is suitable for the delivery of charged molecules. Naked siRNA effectively accumulated in the epidermis (and not in the dermis) after iontophoretic delivery. In contrast, siRNA did not penetrate tape-stripped skin by passive diffusion. In a rat model of atopic dermatitis, skin was sensitized with ovalbumin to stimulate IL-10 mRNA expression as observed in skin lesions. Iontophoretic delivery of anti-IL-10 siRNA significantly reduced (73%) the level of IL-10 mRNA. In conclusion, we successfully delivered naked siRNA into the epidermis and concomitantly suppressed the expression of an endogenous immuno-regulatory cytokine.

Chronic middle cerebral artery occlusion: a hemodynamic and metabolic study with positron-emission tomography.

BACKGROUND AND PURPOSE: Chronic middle cerebral artery (MCA) occlusion is more common than generally thought. It is important to assess the cerebral hemodynamic status in patients with this chronic condition. We investigated the cerebral hemodynamic and metabolic disturbances in these patients in relation to the development of the collateral vasculature. MATERIALS AND METHODS: We studied 13 patients with chronic unilateral MCA occlusion who had a minor or no stroke by using positron-emission tomography (PET). PET was performed by the oxygen 15 ((15)O) gas steady-state inhalation method. The intracranial arteries were evaluated by digital subtraction angiography. We divided the patients into 2 subgroups according to whether they had a normal or increased oxygen extraction fraction (OEF) in the occluded MCA territory and compared the 2 groups. RESULTS: Of the 13 patients, 9 were classified into the normal OEF and 4 were classified into the increased OEF group. In the increased OEF group, the mean OEF values were also increased in the territories of the ipsilateral anterior cerebral artery, ipsilateral posterior cerebral artery, and contralateral MCA. The patients in the increased OEF group had more than 1 steno-occlusive lesion in the major intracranial arteries (P = .008). Three of the 4 patients in the increased OEF group also had vascular lesions in the collateral pathways to the MCA territory. CONCLUSION: Most patients with chronic MCA occlusion did not show severe hemodynamic impairment. Those with increased OEF tended to have other areas of severe hemodynamic impairment and other vascular lesions, especially in the collateral pathways.

"Schistotaxis" in cerebral blood flow on 99mTc-HMPAO single-photon emission computed tomography during a seizure following a stroke.

We describe a patient with seizures following a stroke in whom on ictal 99mTc-HMPAO single-photon emission computed tomography demonstrated cerebral blood flow "schistotaxis", i.e., focal hyperaemia corresponding to an epileptogenic focus together with an extensive hypoperfused area in the same hemisphere. This phenomenon may have been caused by haemodynamic alternation and a remote transneural effect during the seizures.

Expression of NAD(+)-dependent isocitrate dehydrogenase in brown adipose tissue.

cDNA clones significantly expressed in brown adipose tissue (BAT) but not in white adipose tissue (WAT) of rats were isolated by use of a PCR-select cDNA subtraction kit. Of the isolated clones, structural features of two of them, 2-58 and 2-67, were studied in detail. The results indicated that these clones were cDNAs encoding alpha- and beta-subunits of rat NAD(+)-dependent isocitrate dehydrogenase (NAD(+)-ICDH). Previous biochemical study suggested the importance of NAD(+)-ICDH in metabolism in BAT; however, transcript levels of individual subunits of this enzyme in BAT had never been analyzed. In the present study, using these newly isolated cDNAs, we clearly demonstrate that the expression of three subunits of NAD(+)-ICDH was the most remarkable in BAT among the various tissues analyzed.

Successful Cytoreduction with CAG (cytarabine, Aclarubicin and G-CSF) Therapy in Refractory Acute Myelogenous Leukemia before Allogeneic Stem Cell Transplantation.

Refractory acute myelogenous leukemia (AML) has a poor prognosis, and a long-term survival cannot be expected in most patients even if allogeneic bone marrow transplantation (allo-BMT) or allogeneic peripheral blood stem cell transplantation (allo-PBSCT) is performed. An abundance of residual leukemic cells and poor performance status of patients before allo-BMT are often associated with a high relapse rate and high transplant-related mortality. Thus, to improve the prognosis of patients with refractory AML undergoing allo-BMT, it is necessary to reduce the leukemic cell volume as low as possible without severe complications. In this report, we used CAG (cytarabine, aclarubicin and granulocyte colony-stimulating factor (G-CSF)) therapy for cytoreduction before allo-BMT or allo-PBSCT in five patients with refractory AML. One of them achieved complete remission (CR) by CAG therapy alone and others achieved major tumor reduction prior to BMT and PBSCT. All patients achieved CR after allo-BMT and allo-PBSCT without severe complications. Three of them have remained CR for 9, 21 and 30 months, respectively. Although the results of this feasibility study are preliminary, the pre-transplant CAG therapy for refractory AML deserves further evaluation.

[First documentation of transfusion-associated babesiosis in Japan].

A 40-year-old man received blood transfusion in December 1998 because of gastric bleeding from a peptic ulcer. One month later, he developed febrile hemolytic anemia. Administration of high doses of glucocorticoid significantly reduced the hemolysis, but did not cure the disease. To investigate the cause of the hemolysis, the patient was transferred to our hospital in May 1999. Giemsa-stained peripheral blood smears showed Babesia parasites in the red blood cells (RBC), and PCR analysis confirmed the presence of Babesia microti DNA. The parasitemia disappeared hematologically after 2 weeks of quinine and clindamycin therapy. However, parasite DNA was still detectable in the RBC. Although treatment with oral atovaquone was given for 2 weeks, parasitemia and febrile hemolysis recurred within a month after the last treatment. Fortunately, complete remission was obtained after a second 12-week course of therapy with quinine and clindamycin. PCR analysis revealed asymptomatic Babesia infection in one of eight samples from the original blood donor. The initial steroid therapy given to the patient without an accurate diagnosis seemed to have delayed augmentation of the specific antibodies (IgG) against Babesia microti, thus prolonging the parasitemia after the initial acute stage of babesiosis.

[Progression of refractory thrombocytopenia to chronic myelomonocytic leukemia accompanied by various inflammatory reactions].

A 51-year-old man was admitted for treatment of severe thrombocytopenia in May 1997. A diagnosis of MDS RA (refractory thrombocytopenia; RTC) was made by bone marrow examination, which revealed mild marrow hypoplasia and a reduced number of megakaryocytes accompanied by micromegakaryocytes and hypolobular megakaryocytes. Chromosome analysis demonstrated 46, XY, t(5;7) (q31;q22) in all 20 cells examined. The patient received only supportive therapy including platelet transfusion, until leukocytosis and monocytosis gradually developed in November 1998. In view of a marked increase in the number of monocytes (more than 3,000/microliter), a diagnosis of CMML was made in December 1998. As the leukocytosis progressed, various inflammatory symptoms such as facial erythema and endophthalmitis developed. Administration of interferon alpha (IFN alpha) unexpectedly worsened the leukocytosis and monocytosis, suggesting abnormal responses of these cells to IFN alpha. Detailed molecular analysis of these cells might reveal a novel mechanism of leukemogenesis associated with 5q31.

Kinetics of serum cytokines after allogeneic bone marrow transplantation: interleukin-5 as a potential marker of acute graft-versus-host disease.

Cytokine levels in sera from 14 patients undergoing allogeneic bone marrow transplantation (alloBMT) or donor lymphocyte infusion (DLI) were sequentially measured to evaluate the roles of cytokines in clinical graft-versus-host disease (GVHD). In clinical courses, interleukin (IL)-1 alpha, IL-1 beta, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12, tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma were measured by enzyme-linked immunosorbent assay. Among the evaluable cases, 6 patients developed acute GVHD. Serum IL-5 levels increased to more than 100 pg/mL in 5 of the 6 patients before, or simultaneously with, the clinical manifestation of acute GVHD. Elevation of IL-5 was transient in 3 patients. In the other 2 patients who showed regimen-related toxicity and/or thrombotic microangiopathy as well as acute GVHD, remarkable and sustained elevation of the serum IL-5 level was observed. In 7 patients without acute GVHD, IL-5 levels remained below 100 pg/mL. An association of acute GVHD was less prominent with TNF-alpha than with IL-5 in our study. Elevation of IL-6 was associated with infections. In 2 patients with severe extensive chronic GVHD, serum IL-10 was elevated in parallel with exacerbation of clinical symptoms. Our findings suggest that an elevated serum IL-5 level may be a marker of acute GVHD.

Novel expression of equivocal messages containing both regions of choline/ethanolamine kinase and muscle type carnitine palmitoyltransferase I.

For characterization of the detailed gene structure of human muscle type carnitine palmitoyltransferase I (M-CPTI), we analyzed the 5'-upstream region of the M-CPTI transcripts. As a result, we found a cDNA clone containing a nucleotide sequence unexpected from the reported M-CPTI gene structure in the upstream region of its 5' end. Comparison of this nucleotide sequence with that of genomic DNA showed that this sequence was derived from the 3'-untranslated region of the gene encoding choline/ethanolamine kinase-beta (CK/EK-beta) located upstream of the M-CPTI gene. Southern blot analysis showed that there was no other region homologous to the CK/EK-beta gene in the whole human genome. Thus, the overlapping transcript was concluded to be produced from the functional genes of CK/EK-beta and M-CPTI. Furthermore, cDNAs containing both exons of these genes were detected by the polymerase chain reaction using the cDNA of human heart M-CPTI obtained by specific reverse transcription from its 3'-untranslated region as a template. From these results, the production and organization of these overlapping transcripts are discussed.

[Efficacy of continuous intravenous drip infusion of disopyramide in hypertrophic obstructive cardiomyopathy during cardiogenic shock: a case report].

A 54-year-old woman was admitted to our hospital complaining of dyspnea due to hypertrophic obstructive cardiomyopathy. On admission, she was treated with 4 antiarrhythmic drugs and 2 beta-blockers. After 4 of these 6 drugs were withdrawn, the left ventricular outflow pressure gradient markedly increased and then she fell into cardiogenic shock. Therefore, disopyramide(600 mg/day) was administered by continuous intravenous drip infusion to reduce the left ventricular outflow pressure gradient. After intravenous administration of disopyramide, the left ventricular outflow pressure gradient markedly decreased from 100 to 16 mmHg and the cardiogenic shock could be improved. Continuous intravenous drip infusion of disopyramide is effective for the treatment of cardiogenic shock due to severe left ventricular outflow obstruction in patients with hypertrophic obstructive cardiomyopathy.

Soluble fas ligand in natural killer cell lymphoma.

We measured serum soluble Fas ligand (sFasL) in a patient with natural killer cell lymphoma, and investigated relationship between sFasL and liver dysfunction. An elevated level of sFasL was decreased after local radiation therapy, and liver function improved. When lymphoma relapsed, liver dysfunction reappeared and the level of sFasL increased parallelly. Lymphoma cells expressed mRNA of FasL. This suggested that this liver dysfunction was induced by some remote effectors, and sFasL was one of candidates of these effectors.

Levels of soluble FasL and FasL gene expression during the development of graft-versus-host disease in DLT-treated patients.

Three patients with different clinical symptoms of graft-versus-host disease (GVHD) who had received donor lymphocyte transfusion (DLT) for the treatment of relapsed leukaemia after an allogeneic bone marrow transplantation (BMT) from HLA-matched sibling donors were analysed for the presence of soluble FasL (sFasL) in the sera and for the expression of the Fas ligand (FasL) gene in the peripheral blood mononuclear cells (PBMNC). Two patients who demonstrated liver damage with increased levels of serum bilirubin showed significantly increased levels of serum sFasL. The increase in the sFasL level was observed prior to the increase in the bilirubin during the clinical courses of both patients. The high dose of methyl predonisolone administered to one of these patients greatly reduced the levels of sFasL in the serum. The bilirubin levels were also reduced thereafter. The third patient (without liver damage) did not show any increase in the serum sFasL level. The expression of the FasL gene in the PBMNC of these three patients was examined. All three patients showed increased levels of the FasL gene expression during their clinical courses. However, only one patient showed a parallel alteration of FasL gene expression with sFasL in the serum. These cases provide evidence that the Fas/FasL system is closely associated with human GVHD, especially in the development of liver GVHD.

Coagulation abnormalities and thrombotic microangiopathy following bone marrow transplantation from HLA-matched unrelated donors in patients with hematological malignancies.

Thrombotic microangiopathy is a well-known heterogeneous disorder that occurs as a complication of allogenic bone marrow transplantation (BMT). We evaluated 12 consecutive patients receiving HLA-matched unrelated BMT and 12 consecutive recipients of HLA-identical related BMT for the development of bone marrow transplantation-associated thrombotic microangiopathy (BMT-TM) on days 30 and 60 following BMT. A diagnosis was made in four of 12 (33.3%) unrelated compared to none of 12 (0%) HLA-identical cases. Levels of serum lactic dehydrogenase (LDH), fibrin degraded products (FDP) and de novo thrombocytopenia were elevated in eight of 12 patients (66.7%) receiving unrelated donor BMT, and none of the patients receiving related donor BMT. Our findings suggest clinical or subclinical microangiopathic changes may occur frequently in unrelated donor BMT. FDP elevation is possibly an important marker of microangiopathic changes as early complications of BMT.

Contribution of phosphodiesterase isozymes to the regulation of the L-type calcium current in human cardiac myocytes.

1. To determine the contribution of the various phosphodiesterase (PDE) isozymes to the regulation of the L-type calcium current (ICa(L)) in the human myocardium, we investigated the effect of selective and non-selective PDE inhibitors on ICa(L) in single human atrial cells by use of the whole-cell patch-clamp method. We repeated some experiments in rabbit atrial myocytes, to make a species comparison. 2. In human atrial cells, 100 microM pimobendan increased ICa(L) (evoked by depolarization to +10 mV from a holding potential of -40 mV) by 250.4 +/- 45.0% (n = 15), with the concentration for half-maximal stimulation (EC50) being 1.13 microM. ICa(L) was increased by 100 microM UD-CG 212 by 174.5 +/- 30.2% (n = 10) with an EC50 value of 1.78 microM in human atrial cells. These two agents inhibit PDE III selectively. 3. A selective PDE IV inhibitor, rolipram (1-100 microM), did not itself affect ICa(L) in human atrial cells. However, 100 microM rolipram significantly enhanced the effect of 100 microM UD-CG 212 on ICa(L) (increase with UD-CG 212 alone, 167.9 +/- 33.9, n = 5; increase with the two agents together, 270.0 +/- 52.2%; n = 5, P < 0.05). Rolipram also enhanced isoprenaline (5 nM)-stimulated ICa(L) by 52.9 +/- 9.3% (n = 5) in human atrial cells. 4. In rabbit atrial cells, ICa(L) at +10 mV was increased by 22.1 +/- 9.0% by UD-CG 212 (n = 10) and by 67.4 +/- 12.0% (n = 10) by pimobendan (each at 100 microM). These values were significantly lower than those obtained in human atrial cells (P < 0.0001). Rolipram (1-100 microM) did not itself affect ICa(L) in rabbit atrial cells. However, ICa(L) was increased by 215.7 +/- 65.2% (n = 10) by the combination of 100 microM UD-CG 212 and 100 microM rolipram. This value was almost 10 times larger than that obtained for the effect of 100 microM UD-CG 212 alone. 5. These results imply a species difference: in the human atrium, the PDE III isoform seems dominant, whereas PDE IV may be more important in the rabbit atrium for regulating ICa(L). However, PDE IV might contribute significantly to the regulation of intracellular cyclic AMP in human myocardium when PDE III is already inhibited or when the myocardium is under beta-adrenoceptor-mediated stimulation.