Hyperacute graft-versus-host disease and NKT cells.
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Adult↗
Biomedical subjects
K Izutsu
Publications and source records attributed to K Izutsu.
Freeze-concentration separates proteins and polymer excipients into different amorphous phases.
PURPOSE: To study the miscibility of proteins and polymer excipients in frozen solutions and freeze-dried solids as protein formulation models. METHODS: Thermal profiles of frozen solutions and freeze-dried solids containing various proteins (lysozyme, ovalbumin, BSA), nonionic polymers (Ficoll, polyvinylpyrrolidone [PVP]), and salts were analyzed by differential scanning calorimetry (DSC). The polymer miscibility was determined from the glass transition temperature of maximally freeze-concentrated solute (Tg') and the glass transition temperature of freeze-dried solid (Tg). RESULTS: Frozen Ficoll or PVP 40k solutions showed Tg' at -22 degrees C, while protein solutions did not show an apparent Tg'. All the protein and nonionic polymer combinations (5% w/w, each) were miscible in frozen solutions and presented single Tg's that rose with increases in the protein ratio. Various salts concentration-dependently lowered the single Tg's of the proteins and Ficoll combinations maintaining the mixed amorphous phase. In contrast, some salts induced the separation of the proteins and PVP combinations into protein-rich and PVP-rich phases among ice crystals. The Tg's of these polymer combinations were jump-shifted to PVP's intrinsic Tg' at certain salt concentrations. Freeze-dried solids showed varied polymer miscibilities identical to those in frozen solutions. CONCLUSIONS: Freeze-concentration separates some combinations of proteins and nonionic polymers into different amorphous phases in a frozen solution. Controlling the polymer miscibility is important in designing protein formulations.
Calorimetry, Differential Scanning↗
TT virus in bone marrow transplant recipients.
TT virus (TTV) is a newly discovered transfusion-transmissible DNA virus, which may cause posttransfusion hepatitis. The virus was detected in 12% of Japanese blood donors. The aim of the study is to investigate the prevalence and clinical influence of TTV in bone marrow transplant (BMT) recipients. Sera from 25 BMT recipients obtained 6 to 12 weeks after the transplant were examined for TTV-DNA by the seminested polymerase chain reaction. Serial samples were additionally analyzed in patients with TTV-DNA. Fifteen of 25 recipients (60%) were positive for TTV-DNA after transplant, whereas it was detected in only two of 20 BMT donors (10%). In patients positive for TTV-DNA before BMT, the amount of TTV-DNA decreased to an undetectable level during the myelosuppressed period after BMT. We also found that there was a novel group of TTV, G3, classified by the nucleotide sequences. The median peak alanine aminotransferase (ALT) levels were 135.0 IU/L and 116.5 IU/L (normal range, 4 to 36 IU/L) in TTV-positive and TTV-negative recipients, respectively. In one of the seven TTV-positive patients who developed hepatic injury (ALT > 150 IU/L), a serial change in the serum TTV titer showed a good correlation with the ALT level. We concluded that (1) the prevalence of TTV is high in BMT recipients, (2) TTV might be replicated mainly in hematopoietic cells, (3) transfusion-transmitted TTV may cause persistent infection, (4) a novel genetic group of TTV, G3, was discovered, and (5) TTV does not seem to frequently cause hepatic injury, although one patient was strongly suggested to have TTV-induced hepatitis.
Adolescent↗
Successful closure of coronary-bronchial artery fistula with vein graft-coated stent.
A coronary-bronchial fistula and aneurysmal dilatation of the proximal part of the fistula was successfully closed using an autologous vein graft-coated stent (Palmaz-Schatz stent). This is the first report that demonstrates the feasibility of the vein-coated stent for the treatment of congenital disease.
Aged↗
TT virus in frequently transfused patients.
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Adult↗
Increased serum soluble Fas ligand associated with recurrent B-cell non-Hodgkin's lymphoma after autologous peripheral blood stem cell transplantation.
Fas-ligand (FasL) is a member of the tumor necrosis factor family and transmits apoptotic cell death signal by binding to its receptor, Fas. FasL is expressed on the cell surface of activated T-cell and natural killer (NK) cell. It has been shown that the FasL can be released from the cell surface by metalloproteinase. The serum soluble FasL (sFasL) is increased in some patients with NK cell lymphoma/large granular lymphocytic leukemia. We have recently seen a patient with recurrent B-cell lymphoma accompanied with an increased serum sFasL level after autologous peripheral blood stem cell transplantation. The sFasL was markedly decreased with the tumor regression induced by the chemotherapy. We present here the first case of an elevated serum sFasL level associated with B-cell lineage malignancy and discuss the possible clinical value of sFasL.
Adult↗
[X-linked sideroblastic anemia].
A 20-year-old man presented with microcytic hypochromic anemia (hemoglobin: 9.3 g/dl, MCV: 82.0 fl, MCHC: 29.5 g/dl) and dimorphism RBCs in circulating blood (RDW: 26.8%). Ringed sideroblasts accounted for 29.5% of bone marrow erythroblasts. Iron overload was also observed. Because the patient had a clear family history of anemia, he was given a diagnosis of X-linked sideroblastic anemia. The activity of delta-aminolevulinic acid synthase (ALAS) in bone marrow erythroblasts was low. However, we did not detect mutation of the gene for ALAS. The patient has responded well to a treatment regimen consisting of oral vitamin B6, Fe-chelation therapy, and phlebotomy.
Adult↗
Increased soluble Fas-ligand in sera of bone marrow transplant recipients with acute graft-versus-host disease.
Acute graft-versus-host disease (aGVHD) is a major complication following allogeneic bone marrow transplantation (BMT). Recently, accumulating evidence indicates that the Fas/Fas ligand (FasL) system is implicated in the pathogenesis of aGVHD in murine models. We determined the serum levels of soluble FasL (sFasL) in BMT recipients using an enzyme-linked immunosorbent assay. The serum sFasL was suppressed during the period of myelosuppression following the preparative regimen and subsequently increased with hematopoietic reconstitution after BMT. In patients with aGVHD, the serum sFasL level was significantly higher than in those without aGVHD. In the mixed lymphocyte reaction assay, sFasL in the supernatants was increased with a significant correlation to the level of 3H-thymidine uptake. Our findings suggest that the Fas/FasL system is activated by allogeneic stimulation and may have close correlation to the development of aGVHD in human BMT.
Adolescent↗
Control of the pulmonary arterial resistance by the use of the oscillated assist flow.
In the clinical application of supporting circulation, the treatment of a patient with pulmonary hypertension is very important. We developed the electromagnetically driven vibrating flow pump (VFP) as a totally implantable type ventricular assist system. The artificial heart driven by electromagnetic forces creates high speed oscillation flow around 10-50 Hz. Assistance by high-speed oscillation flow has an interesting influence on the cardiovascular system. In this study, we carried out research on the influence such oscillation flow had on the pulmonary arterial vessels, and the supporting flow wave-form that controlled pulmonary vascular resistance was considered. Six healthy adult goats of both sexes were used in the experiments. We carried out inhalation anesthesia and performed intubation. The thorax was opened through left fourth rib resection. Right heart bypass was performed from the right atrium to the pulmonary artery. The flow of right heart assistance was maintained within 20-25% of total flow. Our purpose was to add flow of a specific high frequency to the right heart circulation. The hemodynamic parameters were recorded on a magnetic tape data recorder and input into a computer through an A-D converter. A result identified was that the pulmonary vascular resistance changed according to the alteration of the driving frequency of the VFP even during the same flow assistance. The resistance of the pulmonary arterial vessels became smaller when the driving of the VFP of 30 Hz was added to the right heart circulation. This was significant even when compared with continuous flow right heart assist. The characteristics of impedance appeared to have interesting alterations as well. Control of pulmonary vascular resistance by right heart assistance becomes possible if these results are applied. Accordingly, it may become one of the choices for treatment of a patient with pulmonary hypertension.
Animals↗
[Polyclonal plasmacytosis in peripheral blood and leukocytoclastic vasculitis in Castleman's disease].
A 62 year-old man had fever, splenomegaly, systemic lymphadenopathy and palpable petechiae. Laboratory studies showed leukocytosis (10,200/microliters) with plasma cells (23%) and polyclonal gammopathy. Southern blot analysis of peripheral blood mononuclear cells showed no rearrangement of immunoglobulin genes, indicating polyclonal plasmacytosis in peripheral blood. A diagnosis of Castleman's disease was made by the cervical lymph node biopsy, which showed onion-skin structures in the germinal centers and plasmacytosis in the interfollicular region. A skin biopsy of the purpura lesion disclosed leukocytoclastic vasculitis. This is an unusual case of Castleman's disease presenting plasmacytosis in peripheral blood and leukocytoclastic vasculitis.
Castleman Disease↗
Influence of thrombopoietin on platelet activation in myeloproliferative disorders.
Although thrombopoietin itself does not influence platelet aggregation, it enhances platelet activation in response to certain agonists. We evaluated the effects of thrombopoietin on platelet activation using platelet-rich plasma from 16 patients with myeloproliferative disorders (MPD group) and 16 healthy volunteers (control group). Preincubation with thrombopoietin significantly enhanced platelet aggregation stimulated by ADP, collagen, or epinephrine in the MPD group as well as the control group. However, aggregation induced by 3 micro ADP or 16 microM epinephrine showed significantly less augmentation by thrombopoietin in the MPD group than in the control group. Thrombopoietin significantly shortened the lag time between the addition of 3 microM ADP or 16 microM epinephrine and initiation of secondary aggregation and the lag time between addition of 2 microg/ml collagen and initiation of aggregation in both groups. When platelet-rich plasma was used without adjustment of the platelet count, thrombopoietin itself induced aggregation in two patients. Hypoaggregation after addition of 0.5 microg/ml collagen was observed in seven out of nine patients with normal thrombopoietin levels and only one of six patients with high levels (P = 0.04). Enhancement of 0.5 microg/ml collagen-induced aggregation by thrombopoietin was seen in five out of nine patients with normal thrombopoietin levels and none of the six patients with elevated levels (P = 0.04). These results indicate that platelet activation by certain agonists is enhanced by thrombopoietin in patients with these diseases as well as in normal controls and that the serum thrombopoietin level may regulate the function of circulating platelets in vivo.
Enzyme-Linked Immunosorbent Assay↗
Pulmonary arterial impedance analysis by the use of the oscillated assist flow.
Pulmonary arterial impedance is an important and interesting characteristic that can be used to evaluate the physiological properties of the pulmonary vessel. However, power spectrum analysis of the pulmonary artery pressure and flow pattern have suggested that peak power in the relatively high frequency range (> 10 Hz) is significantly low; thus, we cannot analyze the vessel properties in the high frequency range. In this study, we used the newly developed vibrating flow pump (VFP), which can generate oscillated blood flow with a relatively high frequency (10-50 Hz) for right heart bypass, to evaluate the pulmonary arterial impedance pattern in the high frequency range. Acute animal experiments of the right heart bypass from the right atrium to the pulmonary artery using 6 healthy adult goats were performed. The flow pattern and pressure of the pulmonary artery, electrocardiograms (ECGs), and arterial and right atrial pressures were continuously monitored during the experiments. Spectral analysis of the hemodynamic parameters using the fast Fourier transform (FFT) method was performed to evaluate the spectral properties. The coherence function, transfer function, and phase patterns were calculated to analyze the impedance pattern in the relatively high frequency area. Previously, various investigators had tried to analyze the impedance patterns of the pulmonary artery; however, they could not analyze the impedance patterns over 10 Hz because the spectral patterns of the pulmonary flow do not have high power at high frequencies. These physiological analyses may be useful in designing the optimal pulmonary circulation.
Animals↗
Early calcium signaling and calcium requirements for the IL-2 receptor expression and IL-2 production in stimulated lymphocytes.
Two kinds of lymphocyte populations were prepared from human resected tonsils: one was a mixed population consisting of T cells, B cells, other lymphocytes, and a few macrophages, and the other was a T-enriched population obtained by removing adherent cells from the mixed population with a nylon wool column. A transient rise in cytosolic free calcium ion concentration ([Ca2+]i) was observed in both the populations within a few minutes of stimulation with concanavalin A (Con A). However, emergence of cells which had high [Ca2+]i after 4 min of Con A stimulation was observed practically only in the mixed population. The [Ca2+]i elevation occurring within a few minutes of stimulation in both populations was interpreted as being due to a release from the intracellular Ca-storing organelles, whereas the high [Ca2+]i in a group of cells after 4 min of Con A stimulation in the mixed population was caused by an influx of extracellular calcium that probably corresponds to a transient enhancement of Ca2+ uptake observed only in the mixed population during early stimulation. Con A induced both interleukin 2 receptor (IL-2R) expression and interleukin 2 (IL-2) production. A nonmitogenic lectin, wheat germ agglutinin (WGA), also induced a rise in the [Ca2+]i within a few minutes. WGA induced IL-2 production, but did not induce IL-2R expression. Chelation of extracellular calcium with EGTA at the time of Con A addition resulted in a decrease in IL-2R expression, IL-2 production, and DNA synthesis, but not when CaCl2 equimolar to EGTA was present in the culture medium. Chelation of calcium 12 hr after Con A stimulation decreased IL-2R expression, but had no effect on IL-2 production. These results indicate that IL-2 production required Ca2+ only in the early (G0) stage and that IL-2R expression was dependent on Ca2+ in both the G0 and the G1 stages.
Calcium↗
Effects of sugars and polymers on crystallization of poly(ethylene glycol) in frozen solutions: phase separation between incompatible polymers.
PURPOSE: This study examined the effect of third components (low-molecular-weight saccharides and polymers) on the crystallization of poly(ethylene) glycol (PEG) in frozen solutions, focusing on the relationship between their crystallization-inhibiting ability and molecular compatibility. METHODS: Effects of sugars and polymers on the crystallization of PEG 3000 in frozen solution were monitored by differential scanning calorimetry (DSC). Pulsed-NMR was employed to monitor the molecular mobility of water and solutes in the frozen solutions. Miscibility between PEG and third components in aqueous solution was estimated from the lowering of cloud point of PEG 20,000. Thermal analysis of frozen solutions containing some non-crystallizing solutes was used to examine the possibility of phase separation in frozen solutions. RESULTS: Some sugars and polymers inhibited the crystallization of PEG and formed practically stable amorphous phases among ice crystals. The mobility of solute molecules in the amorphous phase increased above the softening temperature of maximally concentrated solutions (Ts), whereas that of water molecules appeared at a lower temperature. Mono- and disaccharides that are relatively less miscible with PEG in solution inhibit PEG crystallization to a lesser degree. Two Ts regions were observed in frozen solutions containing both polyvinylpyrrolidone (PVP) and dextran, at much lower concentrations than those causing aqueous two-phase separation at ambient temperatures. CONCLUSIONS: Ice crystallization raises the concentration of solutes in the remaining solution, which can lead to phase separation in the amorphous phase. Molecular compatibility between components is an important factor determining their propensity to phase separate and crystallize.
Calorimetry, Differential Scanning↗
Increased stabilizing effects of amphiphilic excipients on freeze-drying of lactate dehydrogenase (LDH) by dispersion into sugar matrices.
PURPOSE: The stabilizing effect of amphiphilic excipients and sugars against protein inactivation during freeze-drying was studied in relation to their physical states in freeze-dried cakes. METHODS: Physical states of amphiphilic excipients were studied by powder X-ray diffractometry and differential scanning calorimetry (DSC). Stabilizing effects of excipients were studied using lactate dehydrogenase (LDH) as a model protein. RESULTS: Although poly(ethylene glycols) (PEGs) 1000 to 2000 crystallized when freeze-dried alone, the addition of sugars decreased their crystallinity by dispersing PEG into sugar-dominant matrices. Sugars species, molecular weight of PEGs, and buffer concentration also affected the crystallinity of PEGs. Sugars also dispersed some of other amphiphilic excipients, which tended to crystallize or become "microscopically liquid" when freeze-dried without sugar. Only the amphiphilic excipients that remained amorphous solid state protected the enzyme during freeze-drying in the absence of sugars. However, combinations of sucrose and all the amphiphilic excipients studied increased the stabilizing effects markedly. The remaining activities were greater than the sum of their individual ones. CONCLUSIONS: Various amphiphilic excipients are good stabilizers for freeze-drying of proteins when dispersed into sugar-dominant matrices.
Dose-Response Relationship, Drug↗
Distribution of a Ca2+ storing site in PtK2 cells during interphase and mitosis. An immunocytochemical study using an antibody against calreticulin.
To study the distribution of a major Ca(2+)-sequestering site in PtK2 cells, a rat kangaroo kidney epithelial cell line, during interphase and mitosis, we prepared an affinity-purified polyclonal antibody against bovine liver calreticulin (CRT), a major Ca(2+)-binding protein of the endoplasmic reticulum (ER). Immunofluorescence microscopy and immunoperoxidase electron microscopy showed that the anti-CRT antibody labeled a continuous reticular network of the ER and the nuclear envelope in interphase PtK2 cells. The same PtK2 cells double-stained with DiOC6 (3) and the anti-CRT antibody revealed labeling of identical reticular membranes. In contrast to the localization in the ER localization, the mitochondria and the Golgi apparatus were not labeled. These results confirm the exclusive localization of CRT in the ER and that this organelle is a major site for Ca2+ storage in non-muscle cells. In mitotic cells, marked changes of the labeled structure began at prophase-prometaphase and persisted throughout all phases of mitosis. The cytoplasm of the mitotic cells showed diffuse fluorescence, this being more intense around, but not inside, the mitotic spindle. Confocal microscopy and immunoelectron microscopy demonstrated that the CRT-containing membranes changed to segmented tubuloreticular structures, which were concentrated around the mitotic spindle. The ER containing CRT could be responsible for the sequestration of Ca2+ and for the regulation of the concentration of this cation during mitosis, as well as during interphase.
Animals↗
Application of accelerated testing to shelf-life prediction of commercial protein preparations.
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Drug Stability↗
Replication sites as revealed by double label immunofluorescence against proliferating cell nuclear antigen (PCNA) and bromodeoxyuridine (BrdU) in synchronized CHO cells and vincristine-induced multinucleate cells.
Double label immunofluorescence against PCNA and BrdU clearly revealed several characteristics of DNA replication sites in synchronized CHO cells. We observed that the distribution of replication sites changed from early to late S phase, particularly in the nucleolar and perinuclear regions, and the amount of PCNA in each replication site markedly decreased or disappeared with the progression of S phase. Although co-localization of PCNA and BrdU was usually seen, the intensity of fluorescence occasionally differed between the labeled PCNA and BrdU, particularly in late S phase. Based on the assumption that such a difference may reflect a different configuration of the chromatin, we propose that the brightly granular fluorescent staining of PCNA or BrdU in early S phase indicates a condensed segment of euchromatin. In the late S phase nucleus, we clearly observed a chromatin-like structure in the late replicating segments of heterochromatin in anti-PCNA stained material. In vincristine-induced multinucleate cells, a discrepancy between PCNA-distribution and BrdU-incorporation in sister nuclei was sometimes seen. Such observations indirectly support the following two mechanisms for premature chromosome condensation proposed by others: asynchronous initiation of DNA synthesis; and a difference in the rate of DNA synthesis. In addition, the finding that BrdU was not incorporated into sites that had PCNA deposits suggests a third mechanism: the local disturbance of replication sites. Furthermore, unusual distribution patterns for replication sites suggest that the nucleation process in multinucleate cells differs from that in normal cells.
Animals↗