A newly developed immunoliposome--an egg phosphatidylcholine liposome coated with pullulan bearing both a cholesterol moiety and an IgMs fragment.

An improved methodology for providing a more stable and targetable drug carrier has been developed. This method involves the synthesis of a newly designed immunoliposome by coating the outermost surface of large oligolamellar vesicles of egg phosphatidylcholine with the polysaccharide pullulan, modified to carry both cholesterol, as the hydrophobic anchor, and the monoclonal antibody fragment (anti-sialosyl Lewis X, IgMs) as the sensory device. Compared with the binding of pullulan-coated liposomes, that of this immunoliposome to specific cells in vitro was significantly increased by factors of 447 to PC-9 and 295 to KATO-III, but only by a factor of 148 to the less specific cell, 3LL. This strong and specific binding of the immunoliposome to the cell surface of PC-9 was also confirmed by a fluorescence-microscopic investigation using the immunoliposome, which bore the hydrophobic fluorescent probe, terbium trisacetylacetonate, in the liposomal membrane.

Sandwich enzyme immunoassay of tumor-associated antigen sialosylated Lewisx using beta-D-galactosidase coupled to a monoclonal antibody of IgM isotype.

Enzyme conjugates with antibody of IgG type have been used extensively in immunohistochemistry, but conjugates with antibody of IgM type have not been reported. This paper describes the beta-D-galactosidase (Gal) labeling of a monoclonal IgM antibody designated CSLEX1 (for cytotoxic sialosylated Lewisx), which is directed against a tumor-associated antigen sialosylated Lewisx (S-Lex). The antibody was first acylated with a heterobifunctional agent N-(gamma-maleimidobutyryloxy)succinimide (GMBS) to introduce the maleimide groups into the molecule; excess reagent was removed by gel filtration and then the activated antibodies were crosslinked to the thiol groups of Gal. The conjugates were partially purified of free Gal by DEAE-Toyopearl column chromatography with an increasing linear concentration of NaCl. The conjugates thus prepared retained almost full enzyme activity and were demonstrated to be free of CSLEX1 by affinity chromatography using anti-galactosidase antibody bound to Sepharose 4B. The conjugates were used as a label in a sandwich enzyme immunoassay (SEIA) to detect the antigen at concentrations as low as 0.2 U/well. The SEIA was used to measure serum S-Lex levels in both healthy subjects and lung cancer patients and mean concentrations of 70 U/ml and 198.6 U/ml were detected respectively.

Increase of plasma adrenocorticotrophin and cortisol in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated dogs.

Neuroendocrine abnormality of the hypothalamic-pituitary-adrenal axis was investigated in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated dogs as a model of Parkinson's disease. Blood samples were collected every 4 h for 3-4 days before MPTP treatment, around 2 and 4 weeks after the treatment of MPTP (2.5 mg/kg). Adrenocorticotrophin (ACTH) and cortisol concentrations in plasma were determined by radioimmunoassay. The concentrations of plasma ACTH and cortisol increased by 40% and 60% after MPTP treatment, respectively. Circadian rhythms of neither plasma ACTH nor cortisol concentrations were observed in both MPTP-treated and control dogs. This high hypothalamic-pituitary-adrenal function in MPTP-treated dogs suggests that MPTP may influence the activity of the hypothalamic neurons of the dog.

Disappearance of circadian rhythms in Parkinson's disease model induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine in dogs.

Administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) to dogs produces clinical, pathological and neurological features in dog resembling human Parkinson's disease. Using this animal model, we studied the changes in diurnal rhythms of urine volume, creatinine in urine, and vasopressin, aldosterone and renin activity in plasma. Before MPTP treatment, urine volume showed a peak between 17.00 and 1.00 and plasma vasopressin concentration also showed a clear circadian rhythm with a peak at 13.00 and a minimum level at 5.00. Two weeks after MPTP treatment (2.5 mg/kg i.v.), the rhythm of urine volume disappeared and that of vasopressin became less clear. Plasma renin activity increased 2 and 4 weeks after MPTP treatment. The increase was, however, not enough to change the concentration of plasma aldosterone. We examined the effect of L-3,4-dihydroxyphenylalanine (levodopa), on the circadian pattern of urine volume and vasopressin attenuated by MPTP. Levodopa (4 mg/kg/day) was administered orally every day from the first week after MPTP treatment. The circadian rhythms of urine volume and vasopressin reappeared within one week after the start of levodopa administration.

Separation of aromatic isomers on cyclophane-bonded stationary phases.

A cyclophane (CP66)-bonded silica gel stationary phase (CP66-SP) was prepared and the retention of water-insoluble hydrophobic compounds on it was investigated in comparison with that on the CP44-bonded stationary phase (CP44-SP) reported previously. Like CP44-SP, it retained aromatic compounds more strongly than the corresponding alicyclic compounds, as was expected by the cavity size of the cyclophane. The CP66-SP also showed isomer-selectivity for monosubstituted and disubstituted naphthalenes, but its selectivity was perfectly reversed to that of the CP44-SP. On the CP66-SP, isomers having methyl and ethyl groups at beta-position were eluted prior to those having groups at alpha-position, whereas on the CP44-SP beta-substituted naphthalenes were retained more strongly than alpha-substituted ones. Isomers of three- and four-ring aromatic compounds were also separated on these cyclophane-bonded stationary phases. The retention order on the CP66-SP was almost opposite to that on the CP44-SP; on the CP66-SP, the retention order was phenanthrene > anthracene, and chrysene > 1,2-benzanthracene > 2,3-benzanthracene, whereas on the CP44-SP, anthracene > phenanthrene, and 2,3-benzanthracene > chrysene > 1,2-benzanthracene. The retention mechanism of aromatic compounds is discussed on the basis of the structure of the cyclophane-involved complex.

Effect of alkyl substitueras in hydrophobic 8-quinolinol on the extraction of gallium(III) and applications to the separation of gallium(III) from aluminum(III).

The extraction of gallium(III) with newly prepared 5-alkyloxymethyl-8-quinolinol derivatives with alkyl substituent at the 2-position in 8-quinolinol moiety has been studied. The Ga(III)-5-octyloxymethyl-8-quinolinol (HO(8)Q), Ga(III)-2-methyl-5-octyloxymethyl-8-quinolinol (HMO(8)Q), Ga(III)-2-methyl-5-hexyloxymethyl-8-quinolinol (HM-O(6)Q), and Ga(HI)-2-n-butyl-5-hexyloxymethyl-8-quinolinol (HNBO(6)Q) complexes extracted in heptane from a perchloric acid medium were Ga(O(8)Q)(3), Ga(OH)(H(2)O)(MO(8)Q)(2), Ga(OH)(H(2)O)(MO(6)Q)(2) and Ga(OH)H(2)O)(NBO(6)Q)(2), respectively. The 2-tert-butyl-5-hexyloxymethyl-8-quinolinol did not exhibit any reactivity toward gallium(III). The extraction constants for Ga(O(8)Q)(3) (K(ex) = [Ga(O(8)Q)(3)](org) [H(+)](3)/[Ga(3+)][HO(8)Q](org)(3)), Ga(OH)(H(2)O)(MO(8)Q)(2) (K(ex) = [Ga(OH) (H(2)O)(MO(8)Q)(2)](org) [H(+)](3)/[Ga(3+)][HMO(8)Q](org)(2)), Ga(OH)(H(2)O)(2)(MO(6)Q)(2) and Ga(OH)(H(2)O)(NBO(6)Q)(2), which were extracted in heptane from an acidic solution, are 10(3.21 +/- 0.12), 10(-4.24 +/- 0.16), 10(-3.84 +/- 0.16) and 10(-4.07 +/- 0.07), respectively at I = 0.1 M and 25 degrees C. HNBO(6)Q exhibited very high selectivity toward gallium(III) in the presence of aluminum(III). Even in the presence of a 100 fold excess of aluminum(III) to gallium(III) (1.43 x 10(-5) M), gallium(III) was completely extracted and the distribution ratio of aluminum(III) was found to be less than 2.0 x 10(-3).

[Studies on defence effects of recombinant human granulocyte colony-stimulating factor (G-CSF) to infections. I. Induction of neutrophil polarization by G-CSF].

It is well known that recombinant human granulocyte increases dramatically the number of granulocytes in peripheral blood. It is interesting to see if G-CSF enhances the functions of neutrophil since the neutrophil is one of the most important factors of host defence mechanism against infections. In this study, we have demonstrated that G-CSF induced the polarization of neutrophil while it could not be chemoattractant for the cell. Polarization is considered to be the initial reaction of chemotaxis. Therefore, G-CSF might enhance the defence effect of the neutrophils+ to infections in this point.

[Effect of erythromycin on neutrophil function in chronic respiratory tract diseases with Pseudomonas infection].

The "low dose and long term" erythromycin (EM) therapy has been reported as effective (or useful) in chronic respiratory tract disease with pseudomonas (P.) infection including diffuse panbronchiolitis (DPB), however the mode of action is still obscure. Therefore in this study we have examined the effect of EM on the interaction between P. aeruginosa and human polymorphonuclear leukocyte (PMN) in vitro. The efficiency of intracellular killing and the ability of superoxide production were employed to evaluate the PMN functions. For the first step, the following results were obtained; 1) Pretreatment of PMN with 20 micrograms/ml EM did not affect the killing ability of PMNs against opsonized P. aeruginosa of standard and clinical isolate from DPB patient. 2) Superoxide production from PMNs was observed by phagocytosis of P. aeruginosa in the presence of serum. This was not affected by exposure of PMNs to 20 micrograms/ml EM even with increased ratios of bacteria to cells. 3) Pretreatment of P. aeruginosa with 20 micrograms/ml EM before opsonization enhanced the killing ability of PMNs in both standard and clinical isolate. 4) Pretreatment of P. aeruginosa with 20 micrograms/ml EM resulted in no effect on superoxide production from PMNs by phagocytosis of the bacteria. These results indicate that EM may modify a certain step of the interaction between bacteria and intracellular host defence mechanisms. Therefore for the second step, we have investigated the susceptibility of EM-exposed bacteria to killing by the cell free (glucose oxidase-glucose) system, which will detect the enzymatic generation of hydrogen peroxide (H2O2). The following results were observed; 1) EM-exposed bacteria was more susceptible to killing than control bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)

[A study of bronchoalveolar lavage analysis in patients with diffuse panbronchiolitis--the results of low dose and long term erythromycin treatment].

Cellular analysis including lymphocyte surface markers in BALF in 17 patients with DPB (14 male; 36 female) was performed. The total number of cells increased remarkably, especially the percentage of neutrophils (72.9 +/- 14.1%). On the contrary the percentage of alveolar macrophage decreased (15.4 +/- 8.9%). The CD4/CD8 ratio in DPB was also significantly decreased to 0.73 +/- 0.38% when compared with normal healthy nonsmokers. Five patients with low dose and long term EM treatment were analyzed. Total cell counts as well as the neutrophil percentage (7.3 +/- 4.0%) decreased significantly. On the other hand the percentage of alveolar macrophage increased to 76.6 +/- 0.6%, which was almost similar to those obtained from normal healthy volunteers. Therefore it is concluded that both clinical and BALF findings improved concordantly after EM treatment.

[Analysis of cellular and biochemical contents of broncho-alveolar lavage fluid from patients with pulmonary tuberculosis].

To evaluate the local immune response in lung and activity of pulmonary tuberculosis. Broncho-alveolar lavage (BAL) was performed on 15 patients with pulmonary tuberculosis. Patients consisted of nine in the active stage and six in the inactive stage. Acid-fast bacilli (AFB) in BAL fluid (BALF) were found in six out of nine active stage patients (BAL-AFB positive group). The results obtained were as follows; 1) The number of total cells in BALF increased significantly in BAL-AFB positive group. BALF from two patients of this group, who were in the very early stage, revealed that the lymphocytes increased predominantly whereas neutrophils were dominant in BALF from the other four patients. 2) No case showed increase of B cells in BALF. Lymphocytes surface markers were compared between BAL-AFB positive and negative groups. No difference was found in the numbers of OKT3, OKT4 and OKT8. The positive rates of OKIal, and OKT4/8 ratio were both increased in BALF compared to the BAL-AFB positive group. 3) The numbers of OKT4 and OKT4/8 ratio in peripheral blood, which were taken simultaneously with BAL, were decreased in patients in the active stage. 4) Protein, albumin, IgG, IgA, lysozyme and alpha 1-antitrypsin levels in BALF were all increased in tuberculous patients. No correlation between the activity of tuberculosis and levels of protein, albumin, IgG and IgA was observed. Lysozyme level increased in the inactive stage of patients. On the other hand alpha 1-antitrypsin level increased in the active stage. Fibronectin level in BALF from patients was lower than that from normal controls. 5) Smoking habit had no influence on the above results. 6) Only one case showed the appearance of new lesions after BAL procedure as a complication.

[Two cases of tuberculous peritonitis and clinical significance of serum CA-125].

We had recently experienced two cases of tuberculous peritonitis. One was suspected of ovarian cancer but finally diagnosed as tuberculosis by the exploratory laparotomy. The other was found out casually at the operation of benign ovarian tumor. Both cases accompanied with pulmonary tuberculosis. Tuberculous peritonitis should be considered in the differential diagnosis in patients with abdominal disorders. Remarkable elevation of serum CA-125 related to ovarian cancer was found in both cases. It was suggested that level of serum CA-125 might be useful for diagnosis and monitoring of the disease.

[Three cases of pulmonary atypical mycobacteriosis associated with lung cancer and adult T-cell leukemia].

Three cases of pulmonary atypical mycobacteriosis (AM) were reported. Two cases were associated with lung cancer in which the diagnosis of malignancy was difficult and delayed by the coexistence of AM. The third was a case of adult T-cell leukemia (ATL) which manifested during the course of AM. In case 1 (73 years, male) and case 2 (86 years, male), chest roentgenogram abnormalities as well as clinical symptoms were considered to be caused by mycobacteriosis because of positive smear of acid-fast bacilli in sputa on admission. Therefore it took four months and three months respectively for final diagnosis of lung cancer. The autopsy of case 1 revealed a poorly differentiated adenocarcinoma with coexisting foci of squamous cell carcinoma in right lower lung, and granulomatous inflammations with caseating necroses in right mid and lower lungs. M. avium complex was cultured from sputum on admission, and also a high titer of HTLV-I antibody was demonstrated. In case 2 malignant cells were detected in sputa (class V), however his general condition did not allow an aggressive anticancer chemotherapy and he died of malignancy with complication of thromboangiitis obliterans on right lower leg. Case 3 was a 76-year-old male who had been diagnosed as lung AM for more than two years. His chest radiography showed bilateral infiltrative shadows with frequent positive cultures of M. avium complex (more than 100 colonies) from sputum. A generalized lymphadenopathy including right hilar lymph node on chest X-ray film was followed by the presence of atypical lymphocytes in peripheral blood and the elevation of HTLV-I antibody in serum. Four months later he died with hypercalcemia and renal failure in spite of chemotherapy (CPM + VCR + ADR + PLS). The above cases suggest that AM as well as tuberculosis should be considered when pulmonary infiltrates were observed in malignant patients, especially in patients with retrovirus infections.

[Activation of neutrophil function by MDP-Lys(L 18)].

The study was carried out to confirm the effect of MDP-Lys (L 18) on the neutrophil number and function in the patients with lung cancer. The patients were enrolled only in case of leukopenia (equal or less than 3,000/mm3 after anticancer chemotherapy) and subcutaneously given 200 mcg of MDP-Lys (L 18) for 7 days. Mean leukocyte counts were found to be 3,900/mm3 and 5,500/mm3 one day and one week after MDP-Lys (L 18) treatment, respectively. The rate of "markedly effective" and "effective" was 53% (8/15). Chemiluminescence by zymosan showed a significant increase after MDP-Lys (L 18) treatment. However, no change of neutrophil function was observed in the tests of chemiluminescence by phorbol myristate acetate, superoxide (O2-) production, hydrogen peroxide (H2O2) production, chemotaxis, bacterial killing and opsonization. Transient fever was observed in 6 cases and eruption accompanied with one fever case. It was concluded that MDP-Lys (L 18) could prevent infection in cancer-bearing immunocompromised patients by increasing the neutrophil number and function.

[Sialylated Lewis(x) and sialylated Lewis(a) as tumor-associated carbohydrate antigens in sera of patients with gastric cancer--usefulness of combination assay of these two antigens].

Sialylated Lewis(x) and sialylated Lewis(a) antigens, which are structural isomer each other, were serologically tested to determine the clinicopathological differences and correlation between two antigens, and were also compared with those of serum CEA in 141 patients with gastric cancer. Percent positives in sialylated Lewis(a), CEA and sialylated Lewis(x) showed 26.9%, 21.8% and 17.0% respectively. Percent positive of sialylated Lewis(x) correlated with degree of liver metastasis and peritoneal dissemination. Therefore, it is emphasized that the role of sialylated Lewis(x) is characteristics of detecting a presence of liver metastasis and/or peritoneal dissemination. On the other hand, sialylated Lewis(a) showed high percent positive in cases which had lymph nodes metastasis and mucinous or papillary adenocarcinomas. These two carbohydrate antigens indicated no correlation (r = 0.062) in the spectrum of sera. It is suggested that combination assay of sialylated Lewis(x) and sialylated Lewis(a) is useful in detecting many gastric cancer patients including CEA-negative one.

[The effect of prostacyclin (PGI) on denervated tissues--especially, about denervation supersensitivity].

It is well known that contractive agents of smooth muscle have denervation supersensitivity. But, the existence of denervation supersensitivity in relaxed ones does not become so clear. We investigated these supersensitivity, especially prostacyclin (PGI2). We made the denervated artery by lumbar sympathetic ganglionectomy in mongrel dogs. Namely, saphenous and dorsal pedal arteries were denervated by this ganglionectomy. And we studied the changes of these arterial tensions by magnus apparatus. Dose-response (D-R) curves of denervated arteries for KCl and noradrenaline (NE) shifted to left against control arteries. Next, we studied effects of PGI2. At first, we measured ED50 of KCl and NE contraction. And, we produced the half contraction on each artery by using ED50 of KCl and NE. Lastly, PGI2 was added on the half contractive arteries to study relaxed changes. D-R curves of denervated arteries for PGI2 shifted to left, too. These denervated arteries acquired the supersensitivity for PGI2. Clinically, if we use relaxants after organ transplantation, these drugs may be very effective for the perfusion of the grafts.

[MRI findings of muscular sarcoidosis].

MRI characteristics of number lesions were studied in eight patients with sarcoidosis proved by Ga scintigraphy. T2 weighted image in spin echo method and low flip angle method were employed. The localization of lesion detected by Ga scintigraphy was quite similar to that of lesions found by MRI, but lesions were more clearly detectable in MRI than Ga. MRI could identify anatomical localization of muscular lesions. Two characteristic MRI findings were observed; one was high intensity in the whole lesion, and another was high intensity in periphery with low intensity in central lesion. MRI is a useful tool for more accurate diagnosis of muscular lesions in sarcoidosis.