Okayama Igakkai Zasshi (Journal of Okayama Medical Association)

Extraction of tumor membrane protein and it's use in a skin test for immunological monitoring

Part 1, Comparison of macrophage migration inhibition activity between DOC- and 3MKCL-solubilized-tumor membrane extract

Yasuhei KUROSE

1983 Volume 95 Issue 3-4 Pages 185-193
Published: April 30, 1983
Released on J-STAGE: March 30, 2009

DOIhttps://doi.org/10.4044/joma1947.95.3-4_185

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Ehrlich ascites cancer cells and MHl34 cells were extracted with Deoxycholate(DOC) or 3MKCL. The antigenicity of these extracts was studied with the macrophage migration inhibitory test (MI-test), and the results can be presented briefly as follows:
1. The extract from DOC solubilized Ehrlich ascites cancer cells had about 50 times the antigenic titer of the extract from 3MKCL solubilized cells.
2. The extract from DOC solubilized MHl34 cells had an antigenic effect at 500ug/ml but the extract from 3MKCL solubilized cells had no effect at any concentration.
3. These extracts were fractionated with Sephadex G-200 and the antigenic sites were determined with the MI-test.

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Extraction of tumor membrane protein and it's use in a skin test for immunological monitoring

Part 2. Skin tests using autologous tumor membrane extracts

Yasuhei KUROSE

1983 Volume 95 Issue 3-4 Pages 195-203
Published: April 30, 1983
Released on J-STAGE: March 30, 2009

DOIhttps://doi.org/10.4044/joma1947.95.3-4_195

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Autologous skin tests with DOC-solubilized-tumor extracts including 20 cases of colon cancer and 25 cases of gastric cancer revealed the following:
1. There were 9 positive reactions and 11 negative reactions in the colon cancer cases. On the other hand, there were 16 positive reactions and 9 negative reactions in the gastric cancer cases.
2. In gastric cancer cases, the time span of autologous skin tests revealed that curable cases with positive skin reactions had negative reactions after operation and uncurable cases remained negative after operation.
3. There was no relationship of the clinical stage and nonspecific skin test (PPD skin test) to the autologous skin test.

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Induction of blastogenesis of peripheral lymphocytes by various mitogens and suppression of blastogenesis by sera in renal allograft recipients

Yojiro ORITA

1983 Volume 95 Issue 3-4 Pages 205-223
Published: April 30, 1983
Released on J-STAGE: March 30, 2009

DOIhttps://doi.org/10.4044/joma1947.95.3-4_205

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It has been reported that a decrease in cellular immunity occurs and a serum immunosuppressive factor exists in uremic patients. In this study, we attempted to examine the change in cellular immunity after renal transplantation, the serum immunosuppressive factor and the usefullness of mitogenic blastogenesis for immunological monitoring.
Peripheral blood lymphocytes were isolated by the Ficoll-Conray gradient from 10 ml of fresh heparinized blood obtained from renal allograft recipients and healthy individuals. Serum was isolated from 10 ml of fresh nonheparinized blood and added in culture after inactivation and sterilization. Pooled AB serum was used for control. Lymphocytes were adjusted to a concentration of 5×10⁵/ml in RPMI, supplemented with 60 μg/ml Kanamycin and 20% patient's serum or AB serum, and then to 200 μl of this suspension, phytohemaggultinin, pokeweed mitogen and concanavalin A were dispensed. The culture was incubated for 72 hrs in a humidified atomosphere of 5% CO₂ and air, after which IμCi tritiated thymidine was added to each well and the incubation continued for 24 hrs. The contents of each well were transfered to glassfiber filters, and the amount of isotope incorporated was determined in a liquid scintillation counter.
PHA and PWM blastogenesis of patients treated with hemodialysis were significantly decreased. PHA, PWM and Con A blastogenesis of renal allograft patients were significantly decreased, but turned to recover I year after renal transplantation. Sera of renal allograft patients suppressed all kinds of blastogenesis of healthy individuals, but only PHA and PWM blastogenesis of renal allograft patients themselves. In renal allograft patients with good renal function I year after transplantation, Con A autosuppression was very weak and negative suppression increased significantly. There were correlations between preoperative PHA blastogenesis and acute rejection, and between postoperative PWM blastogenesis and chronic rejection.
From these findings, it is concluded that clear elavation of PHA, PWM blastogenesis and suppression of Con A blastogenesis are slightly usefull for immunological monitoring of acute and chronic rejection.

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Immunological study of lupus nephritis

Demonstration of nuclear ribonucleoprotein(RNP) in diseased kidney from patients with systemic lupus erythematosus

Noriyuki KURATA

1983 Volume 95 Issue 3-4 Pages 225-232
Published: April 30, 1983
Released on J-STAGE: March 30, 2009

DOIhttps://doi.org/10.4044/joma1947.95.3-4_225

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Renal tissue from 50 patients with systemic lupus erythematosus (SLE) and 25 with primary glomerulonephritis were tested for the presence of nuclear ribonucleoprotein (RNP) by an immunofluorescent technique. Antibody to RNP was obtained from a patient with mixed connective tissue disease. Fluorescent isothiothianate (FITC) was labelled on purified IgG from the serum. Bright speckled nuclear staining was obtained when the tissue section of mouse kidney was incubated with this conjugate. The staining was completely inhibited when the section was preincubated with non labelled antibody to RNP eluted from RNP immune complex formed in vitro.
Granular deposition of RNP antigen in glomeruli was found in 7 of 50 SLE kidneys. The antigen was distributed mainly in the subendothelial site of the glomerular basement membrane and mesangium. The staining was completely inhibited by preincubation of the section with non-labelled antibody to RNP. Deposition of RNP was also found along the tubular basement membrane in 3 of 7 SLE. None of the kidneys from primary glomerulonephritis patients showed positive staining of RNP irrespective of the presence of immunoglobulins and complements in glomeruli.
The present observations support the hypothesis that antibody to RNP besides antibody to double stranded DNA might also be of importance in the pathogenesis of renal disease in SLE.

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Function of peripheral blood lymphocytes in patients with systemic lupus erythematosus

Part 1. Characteristics of spontaneously proliferating cells

Kimiaki MORIMASA

1983 Volume 95 Issue 3-4 Pages 233-242
Published: April 30, 1983
Released on J-STAGE: March 30, 2009

DOIhttps://doi.org/10.4044/joma1947.95.3-4_233

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Increased ³H-Thymidine(³H-TdR) uptake was noticed when peripheral blood mononuclear cells(MNC) from systemic lupus erythematosus(SLE) patients were cultured without any mitogenic stimulation. The peak of ³H-TdR uptake was found immediately after transfering the cells to in vitro culture. DNA synthesizing cells were observed directly by means of autoradiography by pulsing with ³H-TdR for 4 hours. Therefore, it was presumed that there were some MNC in the peripheral blood from SLE patients which were already triggered to proliferate in vivo.
These spontaneously proliferating MNC were found to fractionate into a nonadherent, non-E-rosette forming and surface immunoglobulin negative cell population, the so called null cell population.
These results suggest that there were activated MNC in the peripheral blood from patients with SLE which fractionated into a null cell population.

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Function of peripheral blood lymphocytes in patients with systemic lupus erythematosus

Part 2. Significance of null cells in B cell activation

Kimiaki MORIMASA

1983 Volume 95 Issue 3-4 Pages 243-251
Published: April 30, 1983
Released on J-STAGE: March 30, 2009

DOIhttps://doi.org/10.4044/joma1947.95.3-4_243

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Activation of B lineage cells in systemic lupus erythematosus(SLE) has been indicated by the synthesis of IgG without mitogenic stimulation.
In the course of this study, a cell population in SLE peripheral blood which synthesized DNA spontaneously and which fractionated into a null cell population was noticed. It was found that cells responsible for hyperproduction of IgG without any stimulation in vitro belonged to the null cell population, not to the sIg(+) cell population. The data suggested that both spontaneous IgG production and DNA synthesis might be performed by the same cell population in SLE.
To test whether or not secreted IgG was synthesized by preactivated SLE peripheral blood lymphocytes, these cells were stained with FITC-labeled anti-human Ig antibodies. Significantly more cytoplasmic Ig(+) staining in SLE cells than in normal cells was found.
These observations suggest that there was a small group of cells of B cell lineage in the SLE peripheral blood which was stimulated to proliferate and differentiate into a sIg(-) plasmablast line able to secrete IgG.

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Studies on peripheral monocytes in diabetes mellitus

Part I. Monocyte chemotaxis in diabetes mellitus

Kiyoshi MIYAHARA

1983 Volume 95 Issue 3-4 Pages 253-262
Published: April 30, 1983
Released on J-STAGE: March 30, 2009

DOIhttps://doi.org/10.4044/joma1947.95.3-4_253

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Chemotaxis of diabetic monocytes was studied. The use of 0.25×10⁶ monocytes, 20% Zymosan activated serum and incubation at 37°C for 90 minutes were found to be the optimal assay conditions for the Boyden chamber method using a nuclepore filter. Under these conditions, monocyte chemotaxis was tested in 48 diabetics and 27 normal controls. The results were as follows:
1) Diabetics had low monocyte chemotaxis, especially in aged patients. The normal controls showed no difference with aging.
2) Poorly controlled diabetics showed significantly lower chemotaxis than both normal controls(P<0.005) and good controlled diabetics(P<0.05). The mean values were 40.1±32.4, 71.3±29.9 and 61.4±36.2 monocytes/HPF, respectively.
3) There was no difference in monocyte chemotaxis between diabetic groups treated with diet alone, oral agents or insulin.
4) Six of nine poorly controlled diabetics showed increased monocyte chemotaxis after being good controlled with insulin.

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Studies on peripheral monocytes in diabetes mellitus

Part II. Lysosomal enzyme activity of monocyte in diabetes mellitus

Kiyoshi MIYAHARA

1983 Volume 95 Issue 3-4 Pages 263-270
Published: April 30, 1983
Released on J-STAGE: March 30, 2009

DOIhttps://doi.org/10.4044/joma1947.95.3-4_263

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Lysosomal enzyme activities of diabetic monocytes were studied using cytochemical staining of beta-galactosidase and non-specific esterase. The enzyme activity of each monocyte was graded from (-) to (++++) depending on the staining strength. The results were as follows:
1) The beta-galactosidase(β-GAL) activity of monocytes from 23 normal controls was 40.3±20.2% and that of 49 diabetics was 42.6±20.1%, with no difference between diabetics and normal controls in graded β-GAL activity.
2) The degree of diabetic control and the way of therapy did not affect the β-GAL activities.
3) Non-specific esterase(NSE) activities of 37 normal controls and 41 diabetics were 94.0±6.3% and 95.9±6.9%, respectively. The enzyme activities of each grade were different between the two groups, that is, (+++) NSE activity was significantly lower in diabetics than in normal controls (28.7±14.1% vs 47.4±17.9%, P<0.001). On the contrary, (+) and (++) NSE activities of diabetics were significantly higher than those of normal controls.
4) There was no difference in NSE activities depending on the way of therapy. Poorly controlled diabetics had significantly lower (+++) and (++++) NSE activities than others.
5) Further evaluation of NSE activity using the scoring method by Kaplow revealed no difference between diabetics and normal controls.
6) The total monocyte NSE activity, which was calculated from the NSE score multiplied by the absolute monocyte count was lower in diabetics than in normal controls, although there was no difference in the absolute peripheral monocyte count between the two groups.

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Brain active oxygen, free radicals, lipid peroxide and the redox state of glutathione in the Fe³⁺ induced epileptic focus of the rat

Hiroyoshi KHOCHI

1983 Volume 95 Issue 3-4 Pages 271-282
Published: April 30, 1983
Released on J-STAGE: March 30, 2009

DOIhttps://doi.org/10.4044/joma1947.95.3-4_271

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Epileptic discharges were induced 15 minutes after 0.1M FeCl₃ was injected into the rat sensory motor cortex and continued for 6 months after the injection. In order to study the Fe³⁺ induced seizure mechanism, Fe³⁺ bound methemoglobin, free radicals, active oxygen, malondialdehyde and reduced and oxidized glutathione were measured in the foci of rats after the FeCl₃ injection. It was found that Fe³⁺ induced a significant increase in Fe³⁺ bound methemoglobin, active oxygen and malondialdehyde and accelerated the glutathione redox reaction 5 minutes after the injection. Fe³⁺ was thought to induce lipid peroxidation by free radical reaction and thus produce the peileptic focus.

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Immunopathological studies on lupus nephritis; Detection of antibodies to nuclear ribonucleoprotein(RNP) and Sm in kidney eluates

IKUO HARA

1983 Volume 95 Issue 3-4 Pages 283-294
Published: April 30, 1983
Released on J-STAGE: March 30, 2009

DOIhttps://doi.org/10.4044/joma1947.95.3-4_283

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For Immunological and immunopathological studies on lupus nephritis, kidneys from nine autopsied and one nephrectomized patients with systemic lupus erythematosus(SLE) were subjected to immunofluorescence(IF) and elution study.
Six patients had diffuse proliferative lupus nephritis, showing granular or lumpy glomerular deposits by IF. Three had chronic renal failure with scanty glomerular deposits. One had no clinical renal disease but showed glomerular mesangial deposits. The serum level of antibodies to native(n)-DNA were slightly or moderately elevated in six patients. Hemolytic activity of serum complement(CH50) was markedly decreased in seven.
Elution was performed as follows: the renal cortex was homogenized and washed repeatedly with cold saline, then the sediment containing glomeruli was disrupted by ultrasonication, and then suspended in 0.02 M glycine buffer, pH 2.8, in phosphate buffered saline for the control study.
Antibodies to RNP were detected in six instances, and those to Sm in five. The hemagglutinating titers of antibodies to RNP and Sm in acid eluates were 8 to 128 times and 32 to 256 times higher than those in controls, respectively. However, when compared with the hemagglutinating titers in sera, specific enrichment of antibodies to RNP and Sm in acid eluates was less than 8 times.
Three of ten acid eluates were demonstrated to contain antibodies to n-DNA which were determined by counterimmunoelectrophoresis(CIE). Two of these showed a positive precipitin reaction against denatured DNA. Minimum amounts of IgG producing positive reaction by CIE were measured in kidney eluates and sera obtained from the same patients. The ratio of the minimum concentrations of IgG in sera to those in eluates ranged between 33 and 125 with n-DNA system. Similarly, the ratio was between 33 and 62 with denatured DNA system.
The results suggest that not just DNA immune complex but RNP and Sm immune complexes could be involved in the pathogenesis of lupus nephritis.

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Model studies on the alteration of the phospholipid composition of Staphyloccous aureus in response to the lack of the cell wall

Reiko KARIYAMA

1983 Volume 95 Issue 3-4 Pages 295-303
Published: April 30, 1983
Released on J-STAGE: March 30, 2009

DOIhttps://doi.org/10.4044/joma1947.95.3-4_295

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In order to elucidate why cardiolipin increases markedly in Staphylococcus aureus cells which lack cell walls, the phase transition temperature of cardiolipin (CL) was determined and compared with that of a major phospholipid, phosphatidylglycerol (PG). CL composed of a fatty acid with a given length was synthesized from dimyristoyl PG and dipalmitoyl PG with the aid of phospholipase D prepared from cabbages and was purified by chromatography. Analysis by differential scanning calorimetry showed that the phase transition temperatures of dimyristoyl PG, tetramyristoyl CL, dipalmitoyl PG and tetrapalmitoyl CL were 25.0, 47.0, 40.5 and 62.2°C, respectively. A mixture of the two phospholipids showed a higher phase transition temperature than PG alone, but lower than CL alone. In the presence of divalent cations, especially Ca²⁺, the phase transition temperature of CL increased more than that of PG. These results clearly indicate that cardiolipin can increase the membrane rigidity and suggest that S. aureus may increase cardiolipin content of the membrane to compensate for the loss of mechanical protection due to the lack of the cell wall.

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Assessment of mediastinal involvement of esophageal carcinoma using computer tomography (CT)

Motoomi OKAWA

1983 Volume 95 Issue 3-4 Pages 305-315
Published: April 30, 1983
Released on J-STAGE: March 30, 2009

DOIhttps://doi.org/10.4044/joma1947.95.3-4_305

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To assess the extent of mediastinal spread, especially to the left main bronchus and the descending aorta, CT findings were analyzed in 42 patients with mid-thoracic(Im) esophageal carcinoma.
The minimum distance between the anterior margin of the vertebral body and the left main bronchus, the minimum inner diameter of the left main bronchus and the maximum size of the tumor were measured and used as diagnostic criteria. CT was capable of evaluating the invasion to the left main bronchus when the minimum distance between the anterior margin of the vertebral body and the left main bronchus was more than 21 mm and the minimum inner diameter of the left main bronchus was less than 8 mm, and to the descending aorta when the tumor was more than 31 mm.
CT accurately outlines the extent of lesions and is helpful in determining the type of surgical procedure and in planning radiation therapy.

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Inhibitory effect of histamine on the potentials recorded from the VPM and RF evoked by stimulation of the tooth pulp

Ikuo OKADA

1983 Volume 95 Issue 3-4 Pages 317-334
Published: April 30, 1983
Released on J-STAGE: March 30, 2009

DOIhttps://doi.org/10.4044/joma1947.95.3-4_317

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The effect of histamine (Hi) on the evoked potentials recorded from the nucleus ventralis posteromedialis thalami (VPM) and the midbrain reticular formation (RF) was studied in unanesthetized rabbits. Under pentobarbital anesthesia, the recording electrodes were implanted stereotaxically into the VPM and RF, and stimulating electrodes were inserted into the tooth pulp of both incisors of rabbits. To maintain a high electrical resistance between the stimulating electrodes, attention was paid to selecting a suitable material for protecting the soldering point. When the tooth pulp was stimulated by the repetition of square wave pulses either ipsilaterally or contralaterally, similar evoked potentials were recorded at the VPM and RF. The potentials were biphasic consisting of an initial negative deflection followed by a positive deflection. Contralateral stimulation induced larger amplitude waves with shorter latencies than those of ispilateral stimulation. Intraventricular administration of Hi decreased the amplitudes of evoked potentials in a dose-dependent fashion. The effect of 200 μg of Hi corresponds to that produced by intravenous administration of 2 mg/kg of morphine. When the same dose of Hi was given together with 20 μg of pyrilamine, the depression of the potentials was eliminated remarkably, but visible alteration was not brought about by simultaneous administration of cimetidine.

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Studies on the determination of urinary protein

part III. A new semiquantitative method for determining minute amount of urinary protein by staining with Coomassie Brilliant Blue R-250 of a urine spot on filter paper

Yukiko TANAKA

1983 Volume 95 Issue 3-4 Pages 335-345
Published: April 30, 1983
Released on J-STAGE: March 30, 2009

DOIhttps://doi.org/10.4044/joma1947.95.3-4_335

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A simple and sensitive method for rapid semiquantitive determination of urinary protein was devised.
Five μl of the urine to be measured is dropped on the 2 cm² filter paper, and dried at room temperature. The paper is immersed in a mixture of ethanol, acetic acid and distillated water (35:10:55) which contains 0.2% Coomassie Brilliant Blue R-250 (CBB), and washed with water until the waste water becomes colorless. The paper is again immersed into a mixture but without CBB, after which the paper is rinsed with water again. The protein concentration in the urine sample is determined by comparing the hue of the urine spot with a standard violet hue chart, as well as by the color intensity of the extract of the spot with 3% hydrogen chloride in ethanol at 595 nm by spectrophotometry.
This method was applied to the determination of protein in urine from junior college girls immediatly after arriving at school and after cooking study, from adults in a factory at the time of their regular medical examination, and from high school boys before and after physical exercise.
The results were compared with that of the TONEIN-TP method. With the SPOT-CBB-DYE method, samples can be kept as colored spots on the filter paper, and the protein in urine can be preserved as a spot on the filter paper and measured at any time.

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CSF monoamine metabolites and cyclic nucleotides in depression

Kazuko MIYAUCHI

1983 Volume 95 Issue 3-4 Pages 347-367
Published: April 30, 1983
Released on J-STAGE: March 30, 2009

DOIhttps://doi.org/10.4044/joma1947.95.3-4_347

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Monoamine metabolites(5HIAA, HVA and MHPG) and cyclic nucleotides(c-AMP and c-GMP) were determined in lumbar CSF from 16 healthy controls and 36 patients hospitalized for depression. Determination of monoamine metabolites in the depressed patients was performed before and after treatment with antidepressants. The diagnosis was made according to the DSM-III(1980).
The following results were obtained:
1) 5HIAA in patients with major depression was significantly lower than in controls regardless of the severity of clinical symptoms, while it was unchanged in patients with the bipolar affective disorder. It is suggested that major depression may differ biologically from the bipolar disorder.
2) The low CSF-5HIAA values in patients with major depression may not be dependent on the clinical state, but rather on a genetic trait, because they were not changed by clinical improvement or treatment with antidepressants.
3) As the initially low CSF-HVA levels in the depressed patients increased along with clinical improvement of the affective retardation, low CSF-HVA levels seem to be clinical state dependent rather than trait dependent in such patients.
4) CSF-MHPG was unchanged in the untreated depressed patients, while it decreased significantly after treatment with any of antidepressants.
5) After treatment both CSF c-AMP and c-GMP levels in completely recovered patients were significantly higher than those in control and un-improved patients. As an increase in c-AMP was found to be related to clinical improvement of depression, it seems to be state dependent in the depressed patients.

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Changes in local blood flow of the brain-stem in acute intracranial hypertension

Norio SUNAMI

1983 Volume 95 Issue 3-4 Pages 369-385
Published: April 30, 1983
Released on J-STAGE: March 30, 2009

DOIhttps://doi.org/10.4044/joma1947.95.3-4_369

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This study was carried out to evaluate the effect of supratentorial mass lesions on the local cerebral blood flow (1-CBF) of the brain-stem with special reference to the following three points: the level of the intracranial pressure (ICP) at which the 1-CBF of the brain-stem begins to decrease, alterations in the 1-CBF of the upper brain-stem at the beginning of uncal herniation, and alterations in the 1-CBF of the medulla oblongata during the Cushing's vasopressor response. Platinum electrodes were placed stereotaxically in the thalamus (Th), inferior colliculus (IC) and medulla oblongata (MO) of 40 cats for measurement of 1-CBF by the hydrogen clearance method. The 1-CBF of the brain-stem was serially measured before and after intermittent increases in ICP produced by the inflation of an extradural balloon. Arterial blood pressure, supra- and infratentorial extradural pressure were continuously recorded. The mean control 1-CBF in the Th, IC and MO were 37.5±9.9, 42.1±8.6 and 30.7±4.9 ml/100g/min, respectively. At 20 to 30 mmHg of supratentorial pressure (STP), the 1-CBF of the Th started to decrease, and at 20 to 30 mmHg of infratentorial pressure (ITP), the 1-CBF of the IC started to decrease. Finally at 40 to 60 mmHg of ITP, the 1-CBF of the MO was affected. At the beginning of uncal herniation, indicated by anisocoria, the mean 1-CBF of the IC abruptly decreased from 33.7 to 19.6 ml/100g/min (n=16). In this stage, the pressure gradient between the supra- and infratentorial spaces was 24.6±11.4 mmHg. The Cushing's vasopressor response was evoked at the STP of 93.4±14.6 mmHg and ITP of 49.9±6.8 mmHg (n=16). The blood pressure significantly increased from 121.5 to 140.0 mmHg, however, immediately before and during vasopressor response, there was only a slight, 0.6 mmHg change in the mean cerebral perfusion pressure (CPP) of the posterior fossa. The 1-CBF of the MO also did not show a significant change. The data suggest that in supratentorial mass lesions, the 1-CBF of the thalamus decreases first, followed by the 1-CBF of the inferior colliculus, and then the medulla oblongata, in that order. At the beginning of uncal herniation, the 1-CBF of the upper brain-stem markedly decreased. During the Cushing's vasopressor response, the 1-CBF of the MO did not change significantly.

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Partial purification and properties of Ca²⁺-binding protein from rat liver mitochondrial matrix

Masaaki TOKUDA

1983 Volume 95 Issue 3-4 Pages 387-393
Published: April 30, 1983
Released on J-STAGE: March 30, 2009

DOIhttps://doi.org/10.4044/joma1947.95.3-4_387

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A 193-fold purification of Ca²⁺-binding protein from rat liver mitochondrial matrix was achieved. The Ca²⁺-binding protein consisted of 3 polypeptide subunits whose respective molecular weights by SDS polyacrylamide gel electrophoresis were 62K, 49K and 37K. The molecular weight of the protein was 150K to 220K. The Kd for Ca²⁺ was 1.3×10^-5M and lower for Mn²⁺ and Mg²⁺. The protein was inactivated by heat treatment at 100°C for 1 min, though stable against treatment with 0.5% w/v trypsin at 37°C for 30 min. Ruthenium red did not inhibit Ca²⁺-binding.

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