From the ryanodine receptor to cardiac arrhythmias.

Cardiac contraction is activated by an increase of intracellular calcium concentration ([Ca(2+)](i)), most of which comes from the sarcoplasmic reticulum (SR) where it is released, via the ryanodine receptor (RyR), in response to Ca(2+) entering the cell on the L-type Ca(2+) current. This phenomenon is termed Ca(2+)-induced Ca(2+) release (CICR). However, under certain circumstances, the SR can become overloaded with Ca(2+) and once a threshold SR Ca(2+) content is reached Ca(2+) is released spontaneously. Such spontaneous Ca(2+) release from the SR propagates as a Ca(2+) wave by CICR. Some of the Ca(2+) released during a wave is removed from the cell on the electrogenic Na - Ca exchanger resulting in depolarization. This is the cellular mechanism producing delayed afterdepolarizations and is common to those arrhythmias produced by digitalis toxicity and right ventricular outflow tract tachycardia. More recently it has been suggested that arrhythmogenic Ca(2+) waves can also occur if the properties of the RyR are altered, resulting in increase of RyR open probability, for example by phosphorylation. However, in this review experimental evidence will be presented to support the view that such arrhythmias still require a threshold SR Ca(2+) content to be exceeded and that this threshold is decreased by increasing RyR open probability.

What role does modulation of the ryanodine receptor play in cardiac inotropy and arrhythmogenesis?

In this article we review the role of the Ryanodine Receptor (RyR) in cardiac inotropy and arrhythmogenesis. Most of the calcium that activates cardiac contraction comes from the sarcoplasmic reticulum (SR) from where it is released through the RyR. The amplitude of the systolic Ca transient depends steeply on the SR Ca content and it is therefore important that SR content be regulated. This regulation occurs via changes of SR Ca content affecting systolic Ca and thence sarcolemmal Ca fluxes. In the steady state, the cardiac myocyte must be in Ca flux balance on each beat and this has implications for understanding even simple inotropic manoeuvres. The main part of the review considers the effects of modulating the RyR on systolic Ca. Potentiation of RyR opening produces an increase of the amplitude of the Ca transient but this effect disappears within a few beats because the increased sarcolemmal efflux of Ca decreases SR Ca content. We conclude that it is therefore unlikely that potentiation of the RyR by phosphorylation plays a dominant role in the actions of positive inotropic agents such as beta-adrenergic stimulation. Some cardiac arrhythmias result from release of Ca from the SR in the form of waves. This is best known to occur when the SR is overloaded with calcium. Mutations in the RyR also produce cardiac arrhythmias attributed to Ca waves due to leaky RyRs and a similar leak has been suggested to contribute to arrhythmias in heart failure. We show that, due to compensatory changes of SR Ca content, simply making the RyR leaky does not produce Ca waves in the steady state and that SR Ca content is critical in determining whether Ca waves occur.

Granulocyte-colony stimulating factor induced intranuclear endonuclease in murine leukemia cell line.

We have reported that murine leukemia cell line (C2M-A5) induced apoptosis by G-CSF. To clarify the mechanism, mRNA expression of apoptosis-related genes was studied. It revealed transient over-expression of c-myc, H-ras and p53 and down-expression of bcl-2. These changes were known as triggers of endonuclease induction. After 96 h culture with G-CSF, apoptosis was occurred simultaneously with endonuclease (37 kd) activation. This endonuclease induced the digestion of double-strand DNA and might be associated with caspase3. Although G-CSF accelerates cell growth and prevents apoptosis in general, it is a contradictory effect. We concluded that G-CSF induced endogenous endonuclease activity in C2M-A5.

Expression of functional granulocyte colony-stimulating factor receptors on human B-lymphocytic leukemia cells.

We analyzed the expression of cell surface antigens and granulocyte colony-stimulating factor (G-CSF) receptors using flow cytometry, the expression of G-CSF mRNA receptor, using reverse transcription (RT)-PCR, and tested the effect of G-CSF on leukemia colony formation. A total of 14 lymphocytic leukemia patients were examined, seven with acute lymphocytic leukemia (ALL), two with adult T-cell leukemia (ATL), two with B-chronic lymphocytic leukemia (CLL), two with chronic myelocytic leukemia in lymphoid blastic crisis (CML-LBC), and one with plasma cell leukemia (PCL). The presence of G-CSF receptors was demonstrated in 4/14 (29%) patients, two with ALL, one with CLL, and one with CML-LBC, and was associated with stimulation of leukemia clonogenic cell growth by G-CSF. In addition, all four positive leukemia cell types expressed typical B-cell antigens. Our results indicated that G-CSF receptors are expressed on some portion of B-lymphoid leukemia and that their receptors are functional as growth stimulators.

[Salvage therapy in relapsed or refractory malignant lymphoma].

High dose therapy(HDT) followed by autologous stem cell transplantation(ASCT) is a therapeutic options in chemotherapy-sensitive aggressive non-Hodgkin's lymphoma(NHL) and Hodgkin's disease at relapse. Of the patients with NHL, primary refractory disease should also be treated with such therapy. In patients with indolent lymphoma at relapse, disease free survival after treatment with purged ASCT has been shown to reach a plateau, although the therapy is a matter for debate at present. Anti-CD-20 monoclonal antibody in combination with standard- or high-dose chemotherapy is also quite effective for indolent NHL at relapse. In aggressive NHL with high- or high-intermediate international prognostic index, Burkitt's lymphoma, and mantle cell lymphoma. ASCT as front-line therapy might improve the clinical outcome.

[Atypical chronic myeloid leukemia presenting with trilineage dysplasia and IgG (lambda) type monoclonal gammopathy].

A 78-year-old man was diagnosed as leukocytosis in February 1994. Physical examination revealed marked hepatosplenomegaly. A peripheral blood examination disclosed 95,090/microliter leukocytes without hiatus leukemicus, 6.5 g/dl Hb, and 15.0 x 10(4)/microliter platelets. The neutrophil alkaline phosphatase score was 27, and serum VB12 was above 1,600pg/ml. IgG was identified as monoclonal immunoglobulin of type lambda. Bone marrow specimens demonstrated marked granulocytic hyperplasia. Neither the Philadelphia chromosome (Ph1) nor BCR gene rearrangement was detected; hence, the diagnosis of Ph1 (-) chronic myeloid leukemia (CML) was made. The patient was treated with hydroxyurea and low-dose VP-16 with no improvement, and died of pneumonia and sepsis in June 1995. This case was considered to be consistent with atypical CML (aCML) according to the FAB classification because monocytosis was not observed. It seems likely and interesting that the coexistent monoclonal gammopathy and aCML might have arisen from common abnormal hematopoietic stem cells.

Efficacy of exogenous fibronectin in wound healing in malnourished rats.

BACKGROUND/PURPOSE: Fibronectin (FN) plays an important role in fibrin matrix formation during the wound healing process. The authors investigated whether exogenous FN increases the bursting strength (BS) of surgical wounds in malnourished rats. METHODS: Ninety rats were grouped according to three nutritional conditions (n = 30 in each group). All animals underwent a transverse celiotomy after 3 weeks of feeding, and FN (2 mg/body/day) was given postoperatively to 15 rats in each of the three groups. Wound BS was measured on postoperative days (PODs) 3, 5, and 7 after removal of the sutures. RESULTS: BS in normally nourished rats (group N; 142.6+/-23.4 mmHg) was significantly higher than that in protein malnourished rats (group PM, 110.2+/-11.2 mm Hg) and protein/ calorie (Cal)-malnourished rats (group PCM, 76.5+/-10.7 mm Hg) on POD 7 (P<.01). However, BS values for groups PM + FN (147.0+/-21.1 mmHg) and PCM + FN (115.1+/-28.9 mm Hg) were intensified significantly in comparison with groups PM and PCM (P<.01). Plasma FN levels in rats of the three FN nontreated groups were similarly decreased on POD 3 or 5, but returned to the preoperative level on POD 7, whereas those for the other three FN-treated groups increased after POD 3. CONCLUSION: Intravenous administration of FN might strengthen the weakened wounds of malnourished animals.

[Plasma cell leukemia associated with monocytosis].

A 51-year-old man was admitted to our hospital in December 1993, because of fatigue. Peripheral blood tests showed a WBC of 49,400/microliter with 36% plasma cells and 35% monocytes, Hb 14.5 g/dl, and Plt 137,000/microliter. Bone marrow aspirate revealed hypercellularity with 48.7% plasma cells and 22.4% monocytes. Plasma cells in blood were positive for CD38 and PCA-1. Serum calcium, IgA and M-CSF levels were elevated to 14.1 mg/dl, 2,337 mg/dl and 2.7 ng/ml, respectively. Immunoelectrophoresis of serum and urine revealed IgA lambda type M protein and lambda type Bence Jones protein, respectively. Rearrangements of immunoglobulin heavy chain and light chain were demonstrated by Southern blotting analysis. Plasma cell leukemia (IgA lambda type) was diagnosed. He was treated with combination chemotherapy and IFN-alpha and achieved complete remission. However, he suffered a meningeal relapse in February 1995, and died in April 1996. It seems likely that the enhanced production of M-CSF by myeloma cells and/ or activated B cells stimulated monocyte production.

Granulocyte-colony stimulating factor induced apoptosis in radiation-induced murine leukemia cell line.

Cytokines regulate proliferation and differentiation of hematopoietic progenitor cells. Recently it has been clarified that physiological cell death, apoptosis plays important role of hematopoiesis. So we evaluated the effects of granulocyte-colony stimulating factor (G-CSF) on leukemic cells, especially focused on apoptosis. Intravenous inoculation of radiation-induced murine leukemia cell line, C2M-A5 into the parent C3H mice resulted in the development of myeloid leukemia. However, the leukemic death of the mice was completely suppressed by the daily subcutaneous injection of recombinant human (rh)G-CSF from the next day (Bessho M. et al., Leuk Res 1989:13:1001-1007). In the in vitro study using C2M-A5 cells, we found that apoptosis appears on the cells at 48 hours after addition of G-CSF in culture. The cells in this stage lost the leukemogenicity to C3H mice (Bessho M. et al., Leukemia 8:1185-1190:1994). To clarify the mechanism of the induction of apoptosis by G-CSF we studied cell cycle and molecular changes in C2M-A5 cells cultured in medium with or without rhG-CSF by means of using the flowcytometry and Northern and Western blot analyses. After addition of rh G-CSF to culture, C2M-A5 cells removed to S phase, next arrested at G0/G1 phase on and after 24 hours, and 48 hours later, apoptosis was observed. Overexpression of mRNAs for c-myc (3-24 hours later) and for p53 (6-24 hours later), were observed in the cell cultured in rhG-CSF administered medium with a concomitant down-expression of bcl-2 mRNA (from 6 hours later). Tyrosine-phosphorylated protein (17 kd) appeared at 48 hours after administration of rhG-CSF to cell culture. This protein was suggested for specific apoptosis induction by rhG-CSF. These results are summarized as follows. (1) rhG-CSF induced apoptosis to C2M-A5 and deprived its leukemogenicity to mice. (2) Induction of apoptosis was associated with cell cycle and correlated to the changes of the expression rates of c-myc,p53, and bcl-2. (3) Tyrosine kinase may play an important role in apoptosis induction to C2M-A5 by rhG-CSF.

Immunologic reaction and genetic factors in biliary atresia.

The characteristic histopathological features seen in the livers of patients with biliary atresia (BA) are very similar to those of primary biliary cirrhosis, which is an autoimmune disease. To clarify whether BA liver possess an immunological response similar to that in primary biliary cirrhosis, we studied HLA-DR expression in liver tissue of BA patients, using a HLA-DR staining method, and determined the frequency of HLA types in BA patients and their families. HLA-DR was expressed by the bile duct epithelium in 11 of 16 liver specimens obtained from 13 BA patients. By contrast, HLA-DR was not expressed in liver specimens from 6 patients with congenital biliary dilatation. Among the HLA types seen in BA patients and their families, HLA-A33, -B44 and -DR6 were frequently expressed in blood. These results suggest that certain immunological factors and disease-susceptible genes might be involved in the etiology of BA.

Acute gastric outlet obstruction following the administration of prostaglandin: an additional case.

We present a case of neonatal acute gastric outlet obstruction related to prostaglandin-induced gastric foveolar hyperplasia, which developed following infusion of prostaglandin E1 (PGE1) for treatment of hypoplastic left heart syndrome. Abdominal distension occurred after administration of PGE1 in a cumulative dose of 2914 microg/kg. Ultrasonography performed after a cumulative dose of 5074 microg/kg had been administered disclosed a lobulated thickening of the gastric mucosa with a brush-like appearance composed of alternately echogenic and hypoechoic, vertically oriented lines. These ultrasonographic findings corresponded to the histological abnormalities of gastric foveolar hyperplasia with impacted interfoveolar mucin products and dilated mucosal glands. The development of gastric outlet obstruction in our patient, a relatively rare manifestation of prostaglandin-induced foveolar hyperplasia, might have been related to the unusually high cumulative dose of PGE1.

Characterization of platelet activity in neuroblastoma.

A study was conducted to characterize the platelet aggregation induced by neuroblastoma tissue to investigate the mechanism of hypercoagulability in patients with neuroblastoma. The patients whose tumor tissues were examined had been shown clinically to have enhanced platelet activity. Platelet aggregation induced by neuroblastoma tissue extract was compared with that of other pediatric tumors. The effects of pretreatment with an antithrombin agent and prostacyclin (PGI2) on the platelet aggregation induced by tumor tissue extracts were also evaluated. Tissue extracts of 12 of 15 neuroblastomas, 3 of 3 Wilms' tumors, and 1 pheochromocytoma were demonstrated to have an activity that potentiated platelet aggregation in vitro. The platelet aggregation induced by tissue extracts of neuroblastomas and other tumor tissues was suppressed almost completely by pretreatment with a PGI2 analogue. The aggregation induced by neuroblastomas and the pheochromocytoma was also suppressed by pretreatment with an antithrombin agent, argatroban, whereas the aggregation induced by Wilms' tumors was not suppressed by this agent. These results suggest that (1) malignant tumors in children also have some chemical substances that sensitize platelet activity, such as those in adult cancers, and (2) thrombin is one of the mediators stimulating platelet aggregation in cases of neuroblastoma, although it is unlikely to be a contributing factor in other pediatric malignancies such as Wilms' tumor.