Search

Serum IgA, IgG and colostrum secretory IgA prepared from specimens pooled from a large number of human beings were shown to have measurable levels of antibodies against Escherichia coli, Pseudomonas aeruginosa, Klebsiella Pneumoniae, poliovirus, Coxsackie B virus, echovirus and influenza virus. Serum IgA exerted a bacteriostatic effect in vitro on E. coli and P. aeruginosa, which increased in the presence of the iron-binding proteins lactoferrin and transferrin. This bacteriostasis was reduced when the iron-binding proteins were saturated with iron. Similar results were obtained with IgG and secretory IgA. The bacteriostatic effect of serum IgA was also shown in vivo, in the peritoneal cavity of mice. The effect was suppressed by iron. Iron-chelating substances, siderophores, excreted by E. coli diminished the co-operative bacteriostatic effect of serum IgA and transferrin. Siderophore production by E. coli was inhibited in the presence of serum IgA, but not when serum IgA was deprived of specific antibody by absorption with E. coli. These results indicate that serum IgA has a potent bacteriostatic effect in co-operation with transferrin or lactoferrin because of the inhibitory effect of the specific antibody on siderophore production by E. coli.

A plasminogen activator secreted from human kidney cells was highly purified by affinity chromatography on an anti-urokinase IgG-Sepharose column. The purified plasminogen activator was inactive and had a single-chain structure and a Mr of 50,000. It not only did not incorporate diisopropyl fluorophosphate, which reacts with active site serine residue in urokinase, but also did not bind to p-aminobenzamidine-immobilized CH-Sepharose, to which urokinase bind via its side-chain binding pocket present in active center. The plasminogen activator was converted to the active two-chain form with the same Mr by catalytic amounts of plasmin. Its potential enzymatic activity was quenched completely by anti-urokinase IgG, but not by anti-tissue plasminogen activator Ig. These results indicate that the plasminogen activator is an inactive proenzyme form of human urokinase. Therefore, the plasminogen activator was termed single-chain pro-urokinase. The cleavage of single-chain pro-urokinase by plasmin induced conformational change which followed the generation of reactive serine residue at active site, the increase enzyme activity and the reduction of its high affinity for fibrin. These findings suggest that conformational change occurs in both regions responsible for enzyme activity and affinity for fibrin upon activation of single-chain pro-urokinase.

Tubuloreticular inclusions (TRI) were induced in normal blood lymphocytes after incubation with Staphylococcus aureus Cowan 1 (STA), but they were not induced by pokeweed mitogen (PWM), as we reported previously. TRI were also induced in Raji cells when grown in the medium of STA culture. Alpha-interferon (alpha IFN) was detected only in the medium of STA culture and not in PWM culture. The cells of PWM cultures formed TRI when exposed to various concentrations of human leukocyte alpha IFN. The incidences of TRI-positive cells in the presence of 50-500 IU/ml of alpha IFN were 3-5% on day 2 and increased to 10% on day 7. On days 5-7 of the PWM cultures, plasmacytoid cells containing TRI were seen not infrequently. In the presence of a high concentration of alpha IFN (10,000 IU/ml), which was sufficient to inhibit cell growth and differentiation, the growth of the TRI region was not altered and the incidence of TRI-positive cells was 9% on day 2 and increased to 15% on day 7. Our observations suggest that the TRI formation in STA culture is attributable to the alpha IFN produced endogenously by STA-stimulated cells and that some relationship might exist between the incidences of TRI-positive cells in these mitogen-stimulated cultures and the biologic functions of IFN.

Immunoglobulin samples (HIV-Ig) were prepared by cold ethanol fractionation of human plasma containing antibody against human immunodeficiency virus (HIV). The ability to prevent viral spreading was studied using either human T-cell leukemia virus type I (HTLV-I)-carrying MT-4 cells or in a coculture system using MOLT-4 cells and virus-producing MOLT-4/HIVHTLV-IIIB cells. Treatment of HIV-infected MT-4 cells with HIV-Ig effectively blocked the appearance of antigens of HIV and the virus-induced cytopathic effect. HIV-Ig blocked multinucleated giant cell formation in the MOLT-4 and MOLT-4/HIVHTLV-IIB coculture system.

More than 10(4) plaque-forming units (pfu)/ml of HIV are inactivated during the alcohol fractionation step from plasma to fraction (Fr)-II+III, greater than 10(4) pfu/ml is inactivated from Fr-II+III to Fr-II and greater than 10(4) pfu/ml is inactivated during the polyethylene glycol (PEG) fractionation process from Fr-II+III to intravenous IgG (IVIG). The total inactivation rate from plasma to IVIG via Fr-II+III or Fr-II was calculated to be greater than 10(8) or 10(12), respectively. The PEG fractionation method produces an intact and unmodified IVIG. In addition, the PEG fractionation method at a low ionic strength was found to be effective for the elimination of greater than 10(5) units of other viruses, including hepatitis B, vesicular stomatitis and Sindbis viruses.

ヒト白血球インターフェロン(HLIF)をHBsAg陽性慢性活動性肝炎患者1名に投与し,臨床病理学的検討をおこなった.対象は26歳の男性でHLIF投与前,HBsAg持続陽性,肝腫大,中等度の血清トランスアミナーゼ値の異常を認め,肝組織像では慢性活動性肝炎であった. HBeAg, anti HBeは陰性であった.KLIFを連日総量6.4×107IU非経口的に投与した.HLIF投与開始30日目頃より肝腫大および血清トランスアミナーゼ値の改善がみとめられたが,投与中止1ヵ月目より上昇傾向を示し入院前値にもどった.経時的な血中インターフェロン値は101U/ mlと低値であった.HLIF投与前後でHBsAg, anti HBs, HBeAg, anti HBeには変動はみられなかったが,HLIF投与前後において肝組織中のHBcAgは1/3から1/10に減少した.HLIF投与後の肝組織像では慢性非活動性肝炎を呈し,HLIF投与前に比較し改善傾向をみとめた.以上の成績より,XLIFはHBsAg陽性慢性活動性肝炎にある程度効果があるものと思われる.

The coagulability of Urokinase (UK) has been assessed by recalcification time well known as “zero coagulant assay method” reported by Alkjaersig (1965). However it is quite difficult to obtain suitable substrate plasma as described by him. The platelet poor plasma routinely prepared by silicon technique does not seem to produce a good response with UK.It has been shown that there are two modes in the coagulative action of UK preparation. One is based on a contaminant of a thromboplastin like substance (TLS) from urine, the other one can be initiated or stimulated by the fibrinolytic process as reported previously by Prentice (1969) and Fujimori (1978).In this report, it was demonstrated that TLS possesses a potent supplementary effect for plasma deficient in Factors XI and XII, and also that a celite exhausted plasma (artificial deficient plasma of Factors XI and XII) which was prepared by Nossel's method, was more suitable for determining the coagulant activity of UK preparations by means of zero coagulant assay.It was demonstrated that to eliminate the influences of coagulability stimulated by the fibrinolytic process, a plasminogen free plasma obtained by treatment with celite and lysine-Sepharose was very useful for detection of TLS in UK preparations.

The relative fibrin-binding, fibrinolytic and fibrinogenolytic properties of single-chain pro-urokinase, an inactive proenzyme form of human urokinase purified from cultured human kidney cells, and urokinase were compared. The affinity of single-chain pro-urokinase for fibrin was much higher than that of urokinase. In Vitro thrombolytic studies showed that single-chain pro-urokinase is approximately three times more potent in fibrinolysis than urokinase and that it does not degrade fibrinogen in the plasma at a concentration, at which complete plasma clot lysis takes place; whereas, urokinase extensively degrades the fibrinogen in the plasma. These specific, potent thrombolytic properties of single-chain pro-urokinase seem to be due to its high affinity for fibrin and to its conversion from the inactive single-chain form to the active two-chain form on the thrombus by the catalytic amount of plasmin generated during coagulation. This single-chain pro-urokinase obtained from human kidney cells by tissue culture should prove advantageous than urokinase in thrombolytic therapy.

Kallikrein was highly purified from 1000 liters of fresh pooled urine collected from healthy men by the following procedure : silica gel adsorption, gel filtration on Sephadex G-75, DEAE-Sephadex chromatography, bentonite treatment, affinity chromatography on aprotinin-Sepharose 4B, and rapid gel filtration on a TSK Gel G-3000 SWG column. The final preparation, 17mg of human urinary kallikrein (HUK), was purified 621.6-fold from the crude preparation obtained by silica gel adsorption. One mg of HUK showed activities of 5.58 PNA units toward H-D-Val-Leu-Arg-pNA, and about 1100 kallikrein units in the dog hypotensive assay. HUK thus isolated was found to be pure by means of disc electrophoresis, immunoelectrophoresis, and gel filtration. The following enzymes and bioactive substances were not detected : kininase, urokinase, caseinolytic proteases, blood group substances, substances affecting plasma coagulation, and pyrogens. On isoelectric focusing, HUK separated into microheterogeneous components. The pI's of the dominant components were 3.5, 3.8, and 4.1, while the corresponding molecular weights were 5.4×104, 4.9×104, and 4.4×104, respectively. An aqueous solution of HUK was stable in the pH range from 6 to 12, and was also stable at 60°C for 2h at neutral pH. Michaelis constants were from 4.0×10-5 to 4.0×10-4M toward five kinds of synthetic substrates conventionally used for HUK assay. Optimal pH's for these substrates and human kininogen were in the alkaline pH range of 7.5 to 11.5.

Single-chain pro-urokinase is an inactive proenzyme form of human urokinase with a single-chain structure and a Mr of 50,000 and converted to the active two-chain form by catalytic amounts of plasmin. It was isolated from culture fluid of human kidney cells and subjected to chemical (CNBr) and proteolytic (lysyl endopeptidase) degradation. The resulting peptides were separated by reverse-phase high performance liquid chromatography and subjected to automated sequence analysis. Amino acid sequence of 85% of the 411 residues recovered in 17 peptides were found to be consistent with those of the A chain (157 amino acids) and B chain (253 amino acids) of human urokinase reported by Günzler and co-workers (Günzler, W. A., Steffens, G.J., Otting, F., Kim, S.-M., A., Frankus, E., and Flohé, L. (1982) Hoppe-Seyler's Z. Physiol. Chem. 363, 133-141; 1155-1165; Steffens, G.J., Günzler, W.A., Otting, F., Frankus, E., and Flohé, L. (1982) Hoppe-Seyler's Z. Physiol. Chem. 363, 1043-1058). It revealed the presence of Lys at position 158 in single-chain pro-urokinase through which the two polypeptide chains of human urokinase are unified into one molecule. In addition, firm evidence was found that upon activation by plasmin single-chain pro-urokinase is cleaved at the Lys-Ile bond between residues 158 and 159, resulting in the formation of a two-chain urokinase molecule held together by one disulfide linkage. These results indicate that the cleavage at the Lys-Ile bond between residues 158 and 159 is responsible for conformational change, appearance of enzyme activity and reduction of its high affinity for fibrin.

Before HBsAg screening of plasma was instituted, the preventive effect of immunoglobulin preparations (ISG) against Hepatitis B was not clearly defined. Since HBsAg screening became a requirement, all ISG has shown Anti-HBs and it further became clear that such preparations are effective in preventing Hepatitis B.Recently, cases of Hepatitis NANB associated with administration of intravenous immunoglobulin (IVIG) were reported. Since there is no NANB screening test presently available and IVIG tends to be administered in a large volume, a virus inactivation treatment should be performed on IVIG.Now that heat treatment at 60°C for 10 hours, which had previously been considered to be impossible or unnecessary for immunoglobulin, has become possible, an intravenous immunoglobulin preparation which is more ideal in stability and safety is available.

In intraduodenal administration of /sup 125/I bromelain in the dog, a blood radioactive peak was attained at 4 hr, equivalent to 9.2% of the administered radioactivity, but, over 24 hr after the administration, 0.213 to 0.263% of the dose was found in the high molecular serum fraction, 21% of which was respondible for the absorbed as intact, equivalent to 0.042 to 0.052% of the administered. The gastrointestinal absorption immunologically proved the same. In intragastric administration of enteric-coated capsules containing /sup 125/I-bromelain as much as 1/8 th of the intraduodenal dose, the blood level reached the maximum at 6 hr following a 3 hr lag phase, equivalent to 0.6% of the administered, and 0.013% of the dose was attributed to the radioactivity in the high molecular serum fraction. The presence of bromelain absorbed was not evidenced immunologically. In intravenous administration of /sup 125/I-bromelain, /sup 125/I-bromelain was eliminated from the circulation at 50 min half-life, and low /sup 125/I molecules were detected immediately after the administration; it is indicative of the rapid break-down of /sup 125/I-bromelain by serum proteases. A binding of these low /sup 125/I-molecules to serum proteins was also revealed. A 24 hr urinary collection was found 68% of the dose. As for the anti-inflammatory effect of bromelain on the carrageenin-induced edema in the rat hind paw, bromelain orally administered in enteric-coated form showed 6.6% inhibition of edema at 73.5 mg/kg dose, and 39.0% inhibition at 120 mg/kg dose. This effect highly significant and almost equivalent to that obtained with hydrocortisone given at 10 mg/kg dose, while no powdered bromelain orally administered showed an inhibitory effect on the edema at 20 - 300 mg/kg dose level. The data obtained here indicate that orally administered bromelain is absorbable from the gastrointestinal tract and exhibits fairly potent anti-inflammatory activity on the exudative phases in enteric-coated form.

A sensitive and specific assay system for anti-HBc, the passive hemagglutination (PHA) method, has been established. The reactivity of PHA cells prepared by conjugating purified recombinant HBcAg-particles with fixed sheep blood cells (SRBC) was highly specific to monoclonal- and polyclonal anti-HBc IgGs. The sensitivity of PHA method was higher than that of radioimmunoassay (RIA). However, uncertainty for the positivity of anti-HBc still remained in plasma with the PHA titers lower than 2(5). A relatively high ratio (19%, 37 of 196) of anti-HBc-positive plasma, which had been confirmed to be HBsAg negative, was demonstrated in blood donors whose bloods have been considered suitable for transfusion. The hazards of anti-HBc-positive bloods and the importance of anti-HBc detection in plasma are discussed in this paper.

It has been assumed that a mononuclear phagocyte system is related to the excretion of PFC emulsions: PFC particles are phagocytized by blood monocytes to be expelled through the lung alveoli. This monocyte-related mechanism may well explain excretion at an early stage when PFC particles are abundant in the blood stream. It does not, however, fully explain the manner by which PFC cells are released from the RES cells into the blood stream and into the adipose tissue. To explain this, the following mechanism has been proposed and discussed based on some experimental results. PFC emulsion particles taken up by the RES organs, are stripped at their surfactant layers in the cells and move across the cell membranes to the blood vessels and into other tissues such as adipose, at a rate that depends on the lipophilicity of the PFC's. In the blood stream, PFCs are delivered by lipoproteins to the lung and excreted into the expired air. Pharmacokinetical analysis with a compartmental model for the excretion also supported this proposed mechanism.

A cDNA library was constructed in pBR322 from 18S to 20S mRNA that was extracted from human kidney cells, frasctionated on oligo(dT)-cellulose column and sucrose-density gradient, and confirmed for urokinase production in Xenopus laevis oocytes. The Escherichia coli RR1 transformants were hybridized to synthetic oligonucleotide probe prepared according to the known amino acid sequence, Glu 73 to Glu 77 of human urinary urokinase chain B. The entire cloned cDNA covers a 2250-bp region, wherein the 1293-bp sequence codes for preprourokinase consisting of 431 amino acids, with the first 20 residues being a signal peptide. The 5'-untranslated region is at least 80 bp long and the 3'-untranslated region is longer than 850 bp.

Thrombolytic properties of highly purified plasminogen proactivator (PPA) isolated from culture medium of human kidney cells were compared with those of human urinary urokinase(u-UK). When 125-I-PPA or u-UK was added to whole blood perfusion medium containing preformed thrombi made from whole blood by Chandler loop method, the rates of uptake by the thrombi of PPA and u-UK for 4 hrs were only 2.5% and 2.7% respectively. Using the same thrombi and perfusion medium, an in vitro thrombolytic effect of PPA was examined over a 4 hr period. In early perfusion time, a lag phase was observed in the lysis-time curve of PPA, but not of u-UK. After the lag phase, the lysis by PPA increased linearly without early reaching a low level plateau that was characteristic of the lysis by u-UK. At 4hr, the PPA percent lysis was higher than the u-UK figure. Half lives in rabbit of 125-I-labelled PPA and u-UK iv doses were 5.1±0.2 min and 7.3±0.2 min respectively.The thrombolytic ability of PPA in vivo was evaluated in rabbit pulmonary embolism induced by iv 125-I-fibrin suspension as well as cynomolgus monkey femoral vein thrombosis produced by formation of 125-I-fibrinogen labelled clot in an isolated segment of the vein. In both models, PPA was as effective in thrombolysis as u-UK. However conversion to active UK form, consumptions of plsminogen and PI, fibrinogenolysis and prolongation of APTT in plasma were minor in the PPA group, but not in the u-UK group. It is thus suggested that PPA may cause a local activation limited on the fibrin to lead to a lysis of the thrombi without incurring systemic fibrinolysis

Three gamma-globulin (IgG) preparations were prepared by enzymatic digestion with plasmin [IgG (plasmin)] or pepsin [IgG (pepsin)] and by polyethylene glycol (PEG) fractionation [IgG (PEG)] and their efficacy was compared in terms of the ability to promote phagocytosis in vitro of three strains of pyogenic bacteria, Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus pneumoniae, by human polymorphonuclear leukocytes. IgG (plasmin) had the composition of IgG : Fab : Fc=60 : 26 : 14 ; IgG (pepsin), IgG : F (ab')2 : Fc=8 : 77 : 15 and IgG (PEG), 90% or more IgG. In the phagocytosis test, IgG (PEG) was the most efficient and IgG (plasmin) more than IgG (pepsin) though agglutinin titers of these were much the same. The F (ab')2 fragments were almost inefficient in spite of their agglutinin titer similar to those of the IgG preparations. The Fab and Fc fragments showed neither agglutinin titer nor the ability to promote phagocytosis. Since it has been confirmed by a previous study (ref. 16) on the phagocytosis mechanism that these differences in phagocytic effects result from a phagocytotic mechanism in which antigen bound to antibody at its Fab portion is phagocytized through receptors for Fc and complement present on the polymorphonuclear leukocyte surface, it may be concluded that an IgG preparation desirable for clinical use is that which contains intact IgG molecules as much as possible.

The occurrence of macromolecular change accompanying differentiation in embryonic or neoplastic development is well recognized, even if poorly understood.'+ In the course of immunochemical studies on macromolecular changes related to differentiation in the amphibian,".' we have had occasion to examine the antigenic composition of the f rog kidney and of the f rog renal adenocarcinoma, which is believed to be of viral origin.", I" The following is a report of observations on antigenic differences between the normal and tumor kidney of the frog, on the possible identity of one of these antigens, and on some differences between tumor-susceptible and resistant f rogs.1'-*3

A method is described for the isolation of lysozyme from human placenta. The procedure utilizes an initial separation of lysozyme from mucoproteins and globulins, as well as contaminating hemoproteins, in placenta, the subsequent batchwise fractionation on CM-Sephadex, and a final chromatographic purification on CM-cellulose and on Sephadex G-100. The isolated enzyme showed a purification of 1550-fold on the basis of the initial fraction precipitated with ammonium sulfate, and had a specific activity of 3.2 in comparison with 1.0 for egg-white lysozyme. Cellulose acetate electrophoresis and sedimentation behavior of the enzyme preparation indicated it to be homogeneous. The enzyme seemed to have an isoelectric point of pH 11.7 and apparently possessed a low molecular weight since the sedimentation coefficient ( s 20, w ) was 1.8S. The enzyme was found to be labile at alkaline pH.

IN 1938, Yamagata suggested that the nitrate reductase which reduces nitrate to nitrite participates in some bacterial respiration1. Recently, the mechanism of nitrate reductase and nitrate respiration has been investigated in bacteria and rat liver by Egami and his group2, but frog liver was found not to contain this enzyme3.

Fluosol-DA, a PFC emulsion has been developed by The Green Cross Corp. of Japan as the first practical artificial blood substitute. From basic and clinical studies, this preparation has been proven to be safe and effective. In many cases with massive hemorrhage, this preparation significantly improved hemodynamics and blood gas state. A major inconvenience of this preparation, however, is that it must be kept under frozen state for long-term storage due to its instability at room temperature. To improve this point, we have continued to study new PFCs from the aspects of toxicity and emulsion stability. As a main part of this project, a number of compounds were synthesized by electrochemical fluorination, purified, characterized, and used for screening test. Among these are: F-N-alkylperhydroisoindoles, F-octahydroquinolidines, F-N-methylperhydroquinoline, and F-N-cycloalkylpyrrolidines. There has been a little known about electrochemical fluorination on cyclic amines. Our findings concerning the effects of molecular structure, reaction conditions on the fluorination of these cyclic amines will be reported in detail.