A monoclonal antibody that defines rostrocaudal gradients in the mammalian nervous system.

Spinal cord axons display a rostrocaudal, positional bias in their innervation of sympathetic ganglia and intercostal skeletal muscles. In an effort to examine the molecular basis of this positional specificity, we used the cyclophosphamide immunosuppression method to produce monoclonal antibodies that bind preferentially to rostral ganglia. The staining distribution of one of these antibodies, ROCA1, has been analyzed using a novel histological method. A graded decline in binding is observed along the chain of adult rat sympathetic ganglia, as well as in the nerves innervating intercostal muscles. The antigen is identified on immunoblots as a 65 kd protein, whose distribution corresponds to the pattern found histologically. Surprisingly, ROCA1 appears to bind to glial cells, implying rostrocaudal, molecular differences in their surfaces.

Cell processing protocol for allogeneic peripheral blood stem cells mobilized by granulocyte colony-stimulating factor.

Although there is a growing body of information available regarding restoration of hematopoiesis with peripheral blood stem cell (PBSC) autografts, few studies have explored this procedure using allografts. In this study with healthy donors, we investigated the feasibility of a protocol for mobilizing PBSC using recombinant human granulocyte colony-stimulating factor (G-CSF) and subsequent bulk depletion of T cells from apheresis-harvested cells. Nine informed healthy donors were given G-CSF subcutaneously at two different dosing schedules (5 micrograms/kg/d in five donors and 2 micrograms/kg/d in four) for 5 consecutive days, and serial changes in blood components, including hematopoietic progenitor cells, were monitored. After 5 days of stimulation with G-CSF, PBSCs were collected by apheresis, and yields were compared. The number of white blood cells (WBC) reached a plateau level on either day 2 (5 micrograms) or 3 (2 micrograms), but the numbers of red blood cells and platelets were not affected. Circulating colony-forming unit-granulocyte/macrophage (CFU-GM) levels started to increase 1 or 2 days after the increase in the WBC count. By performing a 3L apheresis, the number of CFU-GM harvested was 4.6 +/- 3.3 x 10(6) (mean +/- standard error of the mean [SEM]) in the 5-micrograms group and 1.8 +/- 0.7 x 10(6) in the 2-micrograms group. Different procedures for depleting T cells, including the use of L-phenylalanine methyl ester (PME) and flasks coated with anti-CD5/CD8 monoclonal antibodies or neuraminidase-treated sheep red blood cells (SRBC), were also tested on the harvested cells. We found that cell lysis with PME before selective removal of T cells was very effective in reducing the number of cells that required further processing and was suitable for routine use. However, our current procedure resulted in unsatisfactory depletion of T cells (99.5% removal) while retaining hematopoietic progenitor cells (7.5% recovery). Further research is required in this area.

Quantitative analysis of the effect of colony-stimulating factors on human marrow progenitor growth in liquid-suspension cultures: application of limiting dilution assay.

Proliferation of human marrow progenitors in liquid cultures can be quantitated by limiting dilution clonal analysis (LDA) of progenitors in microwells. In this study, we have used LDA to study the effect of purified native or recombinant granulocyte colony-stimulating factors (G-CSFs) and recombinant granulocyte-macrophage CSF (GM-CSF) on progenitor growth. These results were compared to those of simultaneous cultures in methylcellulose. In LDA, single-hit kinetics were obtained with up to 500 U/ml of the recombinant preparation. In LDA with recombinant GM-CSF, progenitor growth conformed to single-hit kinetics from 100 to 2000 U/ml with maximum progenitor frequency at 500 U/ml. In simultaneous methylcellulose cultures with recombinant GM-CSF, colony formation reached a plateau at 100 U/ml. In LDA, purified native G-CSF was shown to support progenitor growth with single-hit kinetics at 100 U/ml, but at greater concentrations (greater than 150 U/ml), it suppressed progenitor growth with almost complete inhibition at a concentration of 200 U/ml. However, this dose-response effect was not observed in either simultaneous methylcellulose culture with G-CSF or in LDA with a purified recombinant preparation of the corresponding G-CSF. In methylcellulose cultures, colony formation reached a maximum at 100 U/ml and maintained a plateau up to 1000 U/ml. Hence, liquid culture allowed detection of contaminating suppressive activity in the G-CSF preparation that was not detected by methylcellulose assay. LDA may be more sensitive than methylcellulose culture for screening factors regulating human hematopoietic cell growth.

Engraftment potential of peripheral and cord blood stem cells evaluated by a long-term culture system.

The experiment was performed in an attempt to seek a suitable indicator of allogeneic transplantation using cord blood cells. The number of colony-forming cells (CFC) recovered after 5 weeks of culture represents that of primitive progenitors present in the initiating cell population. In this study of nine children who underwent autologous peripheral blood stem cell transplantation (PBSCT), we measured the amount of colony-forming units-granulocyte/macrophage (CFU-GM) and total CFC output after 5 weeks in long-term culture (LTC-CFC) contained in the infused grafts and compared them to the speed of hematopoietic recovery after marrow ablation. In this patient population, we found a significant negative correlation between the number of infused CFU-GM/kg body weight and the time to achieve an absolute granulocyte count (AGC) of 0.5 x 10(9)/L (r = -0.867, p < 0.01), but not with the time to achieve a platelet count of 50 x 10(9)/L (r = -0.700, p = 0.05). Although the number of infused LTC-CFC/kg did not correlate with the early phase of granulocyte recovery, a significant correlation was observed with platelet recovery speed (r = -0.930, p < 0.01) and with the time to regain an absolute granulocyte count of 2 x 10(9)/L after the nadir of the transient decrease in the number of AGC, which occurred 3 to 7 weeks following PBSCT (r = 0.967, p < 0.01). On the other hand, CFU-GM/kg showed no significant correlation with this late engraftment speed. Thus, LTC assay may reflect the kinetics of immature progenitor cells which are capable of reconstituting stable hematopoiesis after marrow ablative therapy. The data were then used in a comparative analysis of the transplantation potential of peripheral vs. cord blood stem cells. The numbers of LTC-CFC and CFU-GM per mononuclear cell (MNC) from cord blood were 2.5-fold and two-fold greater than those from peripheral blood cells, respectively. These results suggest the advantage of cord blood as an additional stem cell source in transplantation.

Effects of progenitor cell dose and preleukapheresis use of human recombinant granulocyte colony-stimulating factor on the recovery of hematopoiesis after blood stem cell autografting in children.

The effects of progenitor cell dose on the recovery kinetics of hematopoiesis were assessed in 34 children who underwent marrow-ablative chemotherapy with peripheral blood stem cell (PBSC) autografting. One patient was not engrafted, probably because of a low dose of reinfused cells. In 25 patients whose PBSCs were harvested after intensive chemotherapy without recombinant granulocyte colony-stimulating factor (rG-CSF), we found a significant correlation between the colony-forming unit for granulocyte-macrophage (CFU-GM) infused per kilogram of the patient's body weight and the time to achieve an absolute granulocyte count (AGC) of > 0.5 x 10(9)/L (r = -0.817, p < 0.001) or a platelet count of > 50 x 10(9)/L (r = -0.703, p < 0.001). No association was seen between the number of burst-forming units for erythroid (BFU-E) infused and the speed of hematopoietic recovery. In the other 8 patients with low progenitor yields (< 1 x 10(5) CFU-GM/kg), in vivo induction of PBSC was attempted by administering rG-CSF (250 to 1000 micrograms/m2/day). Seven-day administration of rG-CSF before each leukapheresis increased the committed (CFU-GM: 37.7-fold; BFU-E: 45.6-fold) as well as multipotent (CFU-mix: 6.8-fold) progenitors. These collected cells were cryopreserved and then reinfused. Using a model for simulating hematopoietic recovery kinetics, we suggest that cells induced by rG-CSF could speed the recovery of granulocyte or platelet counts after PBSC autografting.

Effects of rhG-CSF (filgrastim) on the recovery of hematopoiesis after high-dose chemotherapy and autologous peripheral blood stem cell transplantation in children: a report from the Children's Cancer and Leukemia Study Group of Japan.

In a nonrandomized study, hematopoietic recovery kinetics were evaluated in 98 consecutive patients who underwent high-dose chemotherapy without total body irradiation (TBI) and autologous peripheral blood stem cell transplantation (PBSCT). Fifty-three patients received recombinant human granulocyte colony-stimulating factor (rhG-CSF) (filgrastim) therapy after PBSCT, and the data were compared by actuarial analysis to those of 45 historic controls. The number of days required to achieve a white blood cell count (WBC) of 1 x 10(9)/L, an absolute granulocyte count (AGC) of 5 x 10(8)/L, and a platelet count (PLT) of 5 x 10(10)/L were, respectively, 12.8 +/- 6.4 (mean +/- standard deviation [SD]), 13.4 +/- 6.4, and 49.2 +/- 78.2 in treated patients vs. 12.8 +/- 4.6, 14.4 +/- 10.3, and 31.4 +/- 38.8 days in historic controls, with no significant differences. There was no significant difference between the average number of days with fever in the treated group (6.0 +/- 6.6) and that in the control group (4.0 +/- 2.8). All febrile episodes responded promptly and successfully to parenteral antibiotic therapy. Thus, the data may suggest the possibility that therapy with filgrastim has only a limited ability to enhance hematopoietic recovery after PBSCT. To confirm this notion, we initiated a prospective randomized trial by recruiting a larger number of patients.

Studies of antitumor agents. 1. Resolution of racemic 1-(tetrahydro-2-furanyl)-k-fluorouracil into the R and S isomers and examination of the biological activities of the isomers.

1-(Tetrahydro-2-furanyl)-5-fluorouracil (Thf-FU), which is named Ftorafur or FT-207 and is used clinically as an antitumor agent, was conveniently synthesized by condensation of the trimethylsilyl derivative of 5-fluorouracil with 2-acetoxytetrahydrofuran using NaI as a catalyst. This optically inactive Thf-FU was resolved into optically active (R)-(+)- and (S)-(-)-Thf-FU in high optical purity and excellent yield by formation of diastereoisomers with brucine. 13C NMR data were obtained on Thf-FU and related compounds and the antibacterial activities and in vivo antitumor activities of these isomers were tested. The degradations of these isomers to 5-fluorouracil by liver microsomes were also examined. No significant differences were found in any of these properties of these isomers.

A pharmacological study on respiratory rhythm in the isolated brainstem-spinal cord preparation of the newborn rat.

An in vitro brainstem-spinal cord preparation of the newborn rat was used to examine the effects of neurotransmitters and transmitter candidates on respiratory frequency. Spontaneous periodic depolarization of the spinal ventral roots of the 4th or 5th cervical segment was observed at a frequency of 5-15 min-1 constantly for more than 5 h. The frequency of this depolarization was monitored as an index of the respiratory frequency. An elevation of the concentration of Ca2+ or Mg2+ caused a decrease in the respiratory frequency, whereas an elevation of K+ concentration caused an increase. The frequency was also increased by a reduction of pH. The highest frequency was observed at 27-28 degrees C. Dopamine, 5-hydroxytryptamine, histamine, acetylcholine, glutamic acid, substance P, and thyrotropin releasing hormone accelerated the respiratory frequency when applied by perfusion to the brainstem, whereas noradrenaline, gamma-aminobutyric acid, glycine, and [Met5] enkephalin and [Leu5] enkephalin slowed the frequency. Experiments with antagonists suggested that the stimulant effect of acetylcholine on respiratory frequency was mediated mainly by muscarinic receptors and the depressant effect of noradrenaline was mediated by alpha-adrenoceptors.

Regeneration of immunity and varicella-zoster virus infection after high-dose chemotherapy and peripheral blood stem cell autografts in children.

We assessed recovery of the immune system in 41 children who underwent high-dose chemotherapy (without total body irradiation) and autologous peripheral blood stem cell transplantation (PBSCT) for acute leukemias or non-Hodgkin's lymphoma. The analysis was in two parts. Firstly, we performed serial monitoring of regenerating subsets and blastogenesis of lymphocytes. We then reviewed the incidence of varicella-zoster virus (VZV) infection, based on the belief that this served as a clinical indication of immunological recovery. The CD4/CD8 ratio markedly decreased in all patients, with a nadir at 3 months, due to both abnormally low levels of CD4+ cells and sustained higher levels of CD8+ cells. These abnormalities were sustained for > 12 months post-graft. Within 6 months after PBSCT, all patients showed a decreased in vitro response to mitogens including PHA, Con A and PWM but these responses gradually recovered during the subsequent 6 months. All patients had a previous history of chicken pox. The actuarial incidence of VZV was 45% at 6 months and 67% at 12 months. All patients were treated with intravenous acyclovir with relief of pain and cutaneous healing within 10 days. No patient developed visceral dissemination. These findings suggest that at least in children, no major difference is apparent between immunological reconstitution in bone marrow transplantation and PBSCT. The development of minor and reversible VZV is a common event in this group of patients.

Experience with peripheral blood stem cell collection for autografts in children with active cancer.

This report examines laboratory and clinical experience with peripheral blood stem/progenitor cell harvest for autografts in 71 consecutively referred children with various types of cancer from one institution. The age of the patients ranged from 7 months to 17 years with a median of 8 years. Ten of the 71 referred children were removed from the collection procedure because of failure to induce clinical remission (nine) or deteriorating chemotherapy-induced liver failure (one). A total of 215 aphereses were performed on a CS 3000 cell separator in 61 patients, including 18 children aged 4 years or less. The methylcellulose progenitor assay was used as an index of stem cell collection. A minimum of greater than or equal to 3 x 10(5) colony-forming units-granulocyte/macrophage (CFU-GM)/kg body weight were collected in 31 of 71 children (44%) with a mean of 3.1 (range, 1-5) aphereses, and greater than or equal to 1 x 10(5) CFU-GM/kg in 46 children (65%). The incidence of serious morbidity was low. When apheresis or chemotherapy was repeated, the progenitor yields decreased rapidly. By multivariate analysis, the age of the patients was the only significant predictor of CFU-GM yield (p = 0.009). In our experience with 12 children with acute leukemia or non-Hodgkin's lymphoma, regimens containing high-dose cytarabine (2.0 g/m2 x 10) were effective for mobilization. Our results extend the earlier findings that harvesting PBSCs for autografts in children with active cancer is a safe and reliable procedure with a low incidence of serious morbidity.

Comparative analysis of engraftment after cryopreservation of peripheral blood stem cell autografts by controlled- versus uncontrolled-rate methods.

Peripheral blood stem/progenitor cells (PBSC) were cryopreserved by different methods and their clonogenic viabilities were compared. The traditional cryopreservation method involves controlled-rate freezing with 10% dimethyl sulfoxide (DMSO) and a programmed freezer (PF). The alternative method incorporates 6% hydroxyethyl starch and 5% DMSO without PF. In this non-randomized study, we analyzed the data of 24 patients (aged 1-17 years) who were in their first complete remission and who had undergone PBSC autograft (PBSCT) for high-risk acute lymphoblastic leukemia, to determine the effects of these two different methods of cryopreservation on time to engraftment and transfusion requirements after PBSCT. In all patients, PBSC were collected within 5 months of diagnosis and all had been conditioned with the high-dose MCVAC regimen (MCNU, cytosine arabinoside, etoposide and cyclophosphamide) without total body irradiation. Twelve patients received PBSC that had been cryopreserved by the uncontrolled method without PF and the data were compared by actuarial analysis to those of 12 historical controls whose cells had been frozen by PF. No difference was observed in the time to engraftment of granulocytes and platelets or the transfusion requirements between the two groups. Our results indicate that, in terms of preserving engraftment potential, the simplified uncontrolled-rate cryopreservation method is at least as effective as the traditional controlled-rate freezing procedure with PF.

Mouse fetuses in late gestation maintained in vitro by a transplacental perfusion method and their physiological activities.

The present study demonstrated that live mouse fetuses in late gestation can be kept in vitro and their physiological activities maintained for over 24 h. Fetuses together with uterus were isolated from pregnant mice at 11.5-17.5 days gestation and transplacentally perfused with a physiological solution through a cannula inserted into the uterine artery. Under these conditions, various physiological activities including heartbeats and spontaneous and stimulation-induced fetal movements were observed.

Physiological activities of late-gestation rat fetuses in vitro.

A novel method for the in vitro maintenance of rat fetuses was used to study their physiological properties. Rat fetuses together with the uterus were isolated from anesthetized pregnant rats of 14.5-21.5 days gestation, placed in a chamber, perfused through a cannula inserted into the uterine artery with a physiological solution, and decerebrated. In this condition, it was possible to maintain heartbeats of rat fetuses for 24 h. The in vitro fetuses showed various spontaneous body movements including those that resembled respiration. Various body movements were also observed when mechanical or electrical stimulation was applied to the skin. These body movements were abolished by application of general anesthetics. It was shown that reflex-like electrical activities were retained in spinal cords isolated from fetuses which had been maintained in vitro for 10 h. These results show that in vitro rat fetuses retain abilities to generate various body movements that are presumably mediated by the electrical activities of the central nervous system. The presence of various neural activities, some of which are known to be particularly susceptible to poor psychological conditions, suggests that the condition of the in vitro fetuses is good and that the method may be applied to various studies on late-gestation mammalian fetuses.

Movements of mouse fetuses in early stages of neural development studied in vitro.

The transplacental perfusion method enables the in vitro maintenance and close observation of live mouse fetuses under conditions free of maternal influences. In the present study, this method was used to detect spontaneous movements of mouse fetuses in early developmental stages. When mouse fetuses at embryonic day (E) 12.5 were isolated together with the uterus and were maintained in vitro, they displayed periodic body movements that occurred every few minutes. Fetal movements were abolished after the application of drugs that depress neural activities. The present results obtained in in vitro mouse fetuses suggest that fetal movements and neural activities may be present during the early stages of motor system development and may play a role in the normal maturation of the motor systems.

Opiate analgesics and endorphins inhibit rat dorsal root potential in vitro.

The effects of endorphins and opiate analgesics on the dorsal root potential (DRP) were studied in vitro using the isolated spinal cord of newborn rat. Bath-applied beta-endorphin and [D-Ala2]-Met-enkephalinamide (D-Ala) greatly depressed the DRP and the depressant effects were abolished by prior perfusion with naloxone. The potency of Met-enkephalin, Leu-enkephalin and alpha-endorphin was much weaker than that of D-Ala. Morphine and levorphanol depressed the DRP and these effects were also antagonized by naloxone. The isolated rat spinal cord appears to be a convenient in vitro preparation for analysing the effects of opiates on synaptic transmission in the central nervous system.

Actions of vasopressin, gastrin releasing peptide and other peptides on neurons on newborn rat spinal cord in vitro.

The actions of various peptides were studied using isolated spinal cord preparation of newborn rat. Vasopressin, substance P, thyrotropin releasing hormone, bombesin, gastrin releasing peptide, oxytocin, neurotensin, cholecystokinin-octapeptide and angiotensin II produced marked depolarizing responses of motoneurons with threshold concentrations of 5 X 10(-10)--8 X 10(-9) M. After the elimination of transsynaptic action by tetrodotoxin, the actions of these peptides were depressed to various extents, the former 5 peptides producing relatively large responses. Somatostatin and enkephalin depressed the dorsal root potential and produced slight hyperpolarization of dorsal root fibers. It is suggested that many of these peptides play important roles in synaptic transmission in mammalian spinal cord.

Application of biological samples treated by wet-digested mineralization to cation-exchange high-performance liquid chromatography with post-column reaction by 4-(2-pyridylazo)-resorcinol.

To quantify metals in biological samples, we tried to find good conditions for wet-digested mineralization of the samples and for separation of metal ions in chromatography. A 500 microliters volume of aliquot was transferred to a glass tube, and was evapolated at 100 degrees C for 2 hours. A 5.5 ml volume of a mixture of concentrated nitric acid-70% perchloric acid (10: 1, v/v) was added and heated, consecutively, at 80 degrees C for 12 hours, at 140 degrees C for 2 hours, at 180 degrees C for 2 hours, and finally at 190 degrees C for 1 hour to evapolate the residual acids. After addition of 500 microliters of 10 mM nitric acid, the metals were extracted by a suspension mixer (32 r.p.m., 1 hour). One hundred microliters of the extracted solution was applied to the chromatographic system: cation-exchange column, TSKgel IC-Cation SW (Tosoh Co.); eluent, 0.35 M lactic acid-0.35 M sodium lactate (pH 3.0); flow rate, 0.7 ml/minute; column temperature, 30 degrees C. After adding a color-forming reagent (100 mg/l 4-(2-pyridylazo)-resorcinol in 40 g/l Na2CO3; flow rate, 0.7 ml/minute) to the effluent, five different metal ions of Cd2+, Co2+, Cu2+, Ni2+ and Zn2+ were detected at 520 nm. The peaks were separated in approximately 25 minutes, and were quantified even at the 1-10 ppb levels. The present procedures were considered to provide simultaneous detection and accurate quantitation of the above five metals in the biological samples.

Urinary albumin determination by gel-filtration high-performance liquid chromatography.

To determine urinary albumin in a minute amount, a gel-filtration high-performance liquid chromatographic procedure was newly established. When urine of a normal rat was eluted isocratically at 0.6 ml/min by 100 mM Na sulfate in 20 mM Na, K-phosphate (pH 7.4), approximately 6 hrs for complete elution of urinary peak-forming substances was needed. Retention time of albumin was found to be 22.9 min. To shorten the analytic time, 100 mM Na sulfate in 20 mM Na, K-phosphate (pH 7.4) was first used during a 30 min period for separation of albumin. A mixture of acetonitrile/the above solvent = 3/7 (v/v) was then flushed to wash away the peak-forming substances. By this elution mode, the analytic time could be reduced to 3 hrs. When the validity of this procedure was tested, the detection limit of albumin was 0.04 microgram/injection, and a linearity was observed between 0.2 and 50 micrograms/injection. Rats then received single subcutaneous injections of puromycin aminonucleoside, which is a nephrotoxic agent. The plasma albumin concentrations fell at 5, 10 and 15 days after the administration, and the urinary excretions of albumin rose from the 1st day up to the 15th day. The results denoted that our procedure could be a good evaluative tool for nephrotoxicity studies where albuminuria was manifested.

[Clinical studies on panipenem/betamipron in pediatrics].

Panipenem/betamipron (PAPM/BP) was given by 30 minutes drip infusion to 15 children with acute bacterial infections including 11 with acute pneumonia, 2 each with staphylococcal scalded skin syndrome and urinary tract infections. Good to excellent clinical responses were obtained in all of the 15 patients and bacterial eradications were obtained for all 12 strains identified in these cases. Urticaria considered to be drug related was observed in 1 patient. Slight elevations of GOT and GPT and eosinophilia were observed in 1 case each. From the above clinical results, it appears that PAPM/BP is a useful antibiotic for treatment of pediatric patients with various bacterial infections.

[Successful treatment of a case of hepatitis-associated severe aplastic anemia by anti-lymphocyte globulin (ALG)].

A 7-year-old girl was admitted to our hospital because of fever and multiple petechiae following non-A non-B hepatitis. Peripheral blood on admission showed pancytopenia, and she was diagnosed to have hepatitis-associated severe aplastic anemia. She was treated with oxymetholone, prednisolone, 2 courses of bolus methylprednisolone, 2 courses of high dose gamma-globulin therapy and ALG (Pressimmun) without success. Nineteen months after diagnosis. ALG (Lymphoser) and bolus methylprednisolone followed by oxymetholone and prednisolone were tried. Hematologic conditions improved gradually, and have become normal except mild thrombocytopenia over a year. This case suggests that there is a difference in clinical efficacy between ALG preparations, and this observation gives the basis of repeating ALG therapy using a different preparation even if the preceding one has failed.