Quantitative studies of in situ immune complex glomerulonephritis in the rat induced by planted, cationized antigen.

Cationized human IgG can bind to the rat glomerular basement membrane (GBM), act as planted antigen, and induce in situ immune complex formation accompanied by severe glomerulonephritis. Perfusion of highly cationized human IgG (isoelectric point {more than} 9.5) via the left renal artery resulted in preferential localization within the perfused kidney (up to 56 percent of dose injected); after intravenous administration, only 4 percent was bound to the kidneys. The planted antigen was localized along the glomerular capillary walls and was accessible for antibody administered intravenously 1 h after perfusion, when virtually no antigen remained in the circulation. Persistence of cationized human IgG in the perfused kidney was markedly prolonged when complexed with antibody; one-half the cationized human IgG was still present after 12 d. There was a difference in the disappearance rates of antigen and antibody, as cationized human IgG was removed faster from the kidney than the antibody, the binding of which remained almost unchanged during the first week. Renal perfusion of a minimum of 20 mug of cationized human IgG, followed by intravenous injection of antibody, regularly induced severe glomerulonephritis with a proteinuria of at least 100 mg/24 h. The degree and the persistence of proteinuria induced depended on the dose of cationized human IgG perfused. Experiments using radiolabeled antigen and antibody showed that after renal perfusion of 20 mug cationized human IgG, 11.1 mug was kidney bound at the time of antibody injection. At the onset of proteinuria, 4.0 mug of antigen and 31.9 mug of anti-human IgG antibody were present in the perfused kidney. Immunofluorescence revealed immune deposits consisting of cationized human IgG and rabbit IgG (anti-human IgG) along the GBM. The staining pattern was linear (confluent) during the first 2 d and became granular during the course of the disease. Electronmicroscopically, a prominent finding was the accumulation of dense deposits, mainly in the subepithelial space and beneath the slit pores.

Isolation and characterization of nephritogenic antigen from bovine glomerular basement membrane.

A method for isolation of a potent nephritogenic antigen from bovine glomerular basement membrane has been established; the glomerular basement membrane was solubilized by trypsin digestion and fractionated successively by gel filtration on Ultrogel AcA-34, concanavalin A affinity chromatography and affinity chromatography on immobilized antibodies. The antigen thus prepared was found to be highly nephritogenic; it causes glomerulonephritis in rats by a single injection of 0.1 mg per individual. Amino acid and carbohydrate analyses revealed that the antigen is a glycoprotein which contains amino acids and sugars characteristic of collagen, namely, hydroxyproline, hydroxylysine, glycine, glucose and galactose, although the relative amounts of these amino acids and sugars are less than those found in Type IV collagen of glomerular basement membrane.

A two-stage method for cross-linking antibody globulin to ferritin by glutaraldehyde. III. Size and antibody activity of the conjugates.

Immunoferritin conjugates consist of conjugates of different size. They can be separated in 4.5% polyacrylamide gel electrophoresis into at least 4 bands. The amount of the smallest 1:1 conjugate, in which one antibody molecule is linked to one ferritin molecule, is highest in coupling products with low overall yields of conjugated ferritin. Therefore relatively mild reaction conditions are recommended. With increasing size the antibody binding capacity of the conjugate is reduced.

The isolation of an optimal small size immunoferritin conjugate fraction.

A procedure is described for preparing immunoferritin conjugates consisting of one antibody molecule attached to one ferritin molecule. Results obtained with such a reagent faithfully reflect the true distribution of antigenic determinants, and allow more accurate quantification.

Immunohistological staining of antigens on semithin sections of specimens embedded in plastic (GMA-Quetol 523).

Glycol methacrylate-Quetol 523, introduced by Kushida (1977) for combined light and electronmicroscopy studies at low magnification, also permits application of immunofluorescence methods to semithin sections. To recover the antigenicity of proteins fixed with formaldehyde, abrupt dehydration before embedding and subsequent treatment of the semithin sections with protease were essential. Post-staining with suitable histological stains allows exact correlation of antigen localization with tissue structure.

An approach to postembedding staining of protein (immunoglobulin) antigen embedded in plastic: prerequisites and limitations.

A method is described for performing postembedding staining of protein (immunoglobulin) antigen embedded in styrene-methacrylate resin. Fixation of specimens in a combination of 4% paraformaldehyde and 0.2% picric acid and washing in buffer containing 7% sucrose, followed by abrupt dehydration with absolute acetone in the cold preserved the antigenicity, although in a masked form. The masked antigenicity could be reexposed by treatment with nonspecific protease. Staining with fluorescent-, peroxidase-, or ferritin-labeled antibodies on semi- and ultrathin sections resulted in specific localization of the antigen. We applied this technique to the localization of rabbit immunoglobulin in specimens of renal tissue obtained from rats with anti-glomerular basement membrane nephritis; we also localized human IgG in a renal biopsy specimen. The prerequisites for recovery of antigenicity are such that preservation of tissue structure at the light microscopic level is good, but relatively poor at the electron microscopic level.

Masking of protein antigen by modification of amino groups with carbobenzoxychloride (benzyl chloroformate) and demasking by treatment with nonspecific protease.

Cryostat sections of various substrates were treated with carbobenzoxychloride in acetone to modify antigens. By applying specific fluorescent antibodies, it could be shown that the antigenic determinants of rabbit gamma-globulin and bovine insulin were totally masked. The antigenicity of ACTH was markedly reduced, whereas the polysaccharide antigens of Salmonella typhimurium were only partially masked. After masking, antigenicity could be restored by treatment with nonspecific protease. The reversible protection of amino groups by carbobenzoxychloride may be a way to preserve protein antigens during embedding in plastics, as such materials also bind to amino groups, blocking the antigenicity of proteins.

Gastrectomy with combined resection of other organs for carcinoma of the stomach with invasion to adjacent organs: clinical efficacy in a retrospective study.

BACKGROUND: Carcinoma of the stomach invading one or more adjacent organs raises serious concerns over en bloc dissection because en bloc resection has an associated high risk and such advanced carcinoma is frequently associated with incurable factors. Thus, it is important to understand the efficacy of gastrectomy combined with other organ resection and to refine the indications for en bloc dissection. STUDY DESIGN: Seventy-seven patients with carcinoma of the stomach directly invading adjacent organs or structures were analyzed retrospectively to investigate the efficacy of en bloc resection. Forty-one patients underwent gastrectomy combined with resection of one or more invaded organs (combined resection group), while the other 36 patients underwent gastrectomy with palliative abrasion between the primary tumor and the invasion site (noncombined resection group). RESULTS: The five-year survival rate was 23 percent in the combined resection group and 0 percent in the noncombined resection group (p < .05). The 23 curative cases and 18 noncurative cases in the combined resection group had a survival rate of 41 percent and 0 percent, respectively (p < .05). The survival rate after a single organ resection was 29 percent, and after a multiple organ resection, 0 percent (p < .05). Cases of carcinoma invading either the pancreas or mesocolon had a slightly but not significantly better prognosis. In Borrmann type IV carcinoma there was no difference in survival between the curative and noncurative operation. Cases with P1 dissemination had a better prognosis than those of P2 and P3 dissemination. CONCLUSIONS: The best indication for an en bloc combined organ resection was an invasion limited to only one other organ, not more than N2, no incurable factor, and any type except Borrmann type IV. Additionally, an en bloc combined resection would be worth trying for any type of gastric carcinoma with not more than P1 dissemination and with no other incurable factor.

(1)H double-quantum filtered MR imaging of joints tissues: bound water specific imaging of tendons, ligaments and cartilage.

The (1)H double-quantum filtered (DQF) NMR and DQF MRI is applied to the joint tissues of rabbits for selective visualization of tendons, menisci and articular cartilage. The (1)H DQF NMR selectively filters double-quantum coherence arising from the (1)H dipolar interaction of the "bound" water in these tissues. The double-quantum creation time dependency of the DQF signal intensity is determined by the molecular environment of the "bound" water. Therefore, each tissue has a unique creation time at which the DQF signal reaches its maximum intensity, tau(max) (Achilles tendon: 0.46 +/- 0.02 ms, patella: 0.55 +/- 0.8 ms, anterior cruciate ligament: 0.60 +/- 0.05 ms, meniscus: 0.78 +/- 0.02 ms, skin: 0.81 +/- 0.07 ms). We have presented the creation-time-contrasted DQF images of the meniscus, patella, foot, and knee joint. Compared with conventional T(2)*-weighted gradient-echo (GRE) MR images, tendons, ligaments, menisci, and articular cartilage were more clearly seen in the DQF MR images. All these tissues were distinctly discriminated from each other by their creation times. DQF MR images of foot and knee joints can selectively demonstrated tendons, ligaments, and cartilage, which make it easier to understand the complicated anatomic structure of joints. Because the DQF NMR signal intensity and tau(max) are sensitive to the order structure of the "bound" water, it might be possible to introduce the creation-time dependent-contrast of (1)H DQF MR images as a new tool for analyzing the changes in the ordered structure of the tissue.

Autoantibody specific for the glomerular mesangium and Bowman's capsule in man.

Investigation of serum from a nominally healthy subject revealed an unusual autoantibody with specificity for the glomerular mesangium and Bowman's capsule. Immunofluorescence studies on rodent kidney sections revealed typical mesangial fluorescence and intense linear staining of Bowman's capsule. The antibody was organ, but not species, specific and did not correspond to any previously described antibody with anti-mesangial activity (e.g. anti-actomyosin or anti-fibronectin). Intravenous injection in mice resulted in heavy mesangial deposition of antibody within one hour, the antibody disappeared within 2--4 weeks, and no urinary abnormalities were seen. Retrospective analysis of clinical data revealed a history of chronic hepatitis and possible drug abuse, but no evidence for impaired renal function at any timepoint. To our knowledge this is the first report of specific anti-mesangial antibody in man, the pathogenetic relevance remains unclear.

A case of late onset primary hyperoxaluria type I (PH-I) presented with black liver.

A 63-year-old woman who had received hemodialysis therapy since she fell acute on chronic renal failure 4 years ago presented with multiple joint pain. Nephrocalcinosis was not detected by abdominal X-ray when hemodialysis therapy was initiated. Laboratory testing showed azotemia, anemia, hypoproteinemia and mild liver dysfunction but no liver cirrhosis. Biopsied bone tissue demonstrated numerous calcium oxalate crystal depositions. Laparoscopy revealed black liver in macroscopic view. Histological studies showed numerous lipofuscin-like dark brown granules were deposited in hepatocytes. The activity of alanine : glyoxylate aminotransferase (AGT) was less than 0.1 U/g in biopsied patient's liver tissue. Generally, clinical symptoms demonstrated by Japanese primary hyperoxaluria type I (PH-I) patients are milder than those of European patients. Some PH-I patients may successfully avoid urinary tract calcification unless they fall into oliguria by some other causes. The lipofuscin granules are most likely the source of the dark color. Massive deposition of the lipofuscin granules indicated that the duration of the liver metabolic abnormality had lasted for long time. Thus, black liver may be related to a mild form of PH-I.

A case of early infantile epileptic encephalopathy (EIEE) with anatomical cerebral asymmetry and myoclonus.

The authors report a case of early encephalopathy with myoclonus, tonic spasms and a suppression-burst pattern on electroencephalography (EEG) associated with unilateral cerebral hypertrophy following hemiatrophy. This patient showed frequent myoclonus in relation to a suppression-burst pattern resembling that in early myoclonic encephalopathy (EME). Moreover, the case also showed tonic spasms, from the age of 13 days, in series, as seen in Ohtahara syndrome. On the other hand, there was a previously undescribed peculiar CT scan finding, which showed hypertrophy of the right cerebral hemisphere at birth, following hemiatrophy. Neuropathological examination revealed cerebral atrophy associated with heterotopia and an ependymal hyperplasia in the right hemisphere, suggesting hemimegalencephaly. This case should be classified as Ohtahara syndrome accompanied by myoclonus, because of the spasms in series interrupting the suppression-burst pattern, and the etiological factor of brain malformation. The nosological aspects of this epileptic encephalopathy are discussed.

Investigation of effect of cortisone and endotoxin on mesangial function.

The effect of endotoxin and cortisone treatment on the mesangial handling of ferritin in rats was examined. Experimental conditions were chosen where the RES was saturated, this enabled us to test for a direct effect on the mesangium. We were unable to detect any influence of either endotoxin or cortisone on the degree of uptake, or the rate of elimination of ferritin from the mesangium. Endotoxin did appear to affect the intra-glomerular distribution of ferritin, which was seen mainly as larger nodules. The iron core of ferritin was found to persist for longer than the antigenic (protein) portion, indicating that some degree of breakdown of ferritin molecules occured within the mesangium.

Characteristics of proton NMR T(2) relaxation of water in the normal and regenerating tendon.

The molecular behavior of water in normal and regenerating tendons was analyzed using the transverse relaxation time (T(2)) measured by spin-echo proton nuclear magnetic resonance ((1)H-NMR) spectroscopy at 2.34 T (25 degrees C). A section of the Achilles tendon was dissected from an anesthetized Japanese white rabbit, and its longitudinal axis was oriented at 0, 35, 54.7, 75, and 90 degrees to the static magnetic field. In the normal tendon, the T(2) relaxation of water presented biexponential relaxation and anisotropy in both the long T(2) (5.41 to 6.21 ms) and short T(2) (0.41 to 1.43 ms) components, in which the greatest values were obtained at 54.7 degrees. However, the range of the anisotropy was much narrower than we expected from the (1)H dipolar interaction of water bound to the collagen fibers in the tendon. The apparent fractions of water proton density also varied with orientation: the fraction of the longer T(2) components was at its maximum at 54.7 degrees. These results suggest that a simple two-compartment model could not be applicable to orientational dependency of the T(2) value of the tendon, and the well ordered water in the short T(2) relaxation component may show an elongated T(2) relaxation time that falls in the range of the long T(2) relaxation component at 54.7 degrees. This hypothesis can explain both the narrower range of the T(2) relaxation time and the orientational dependency on the apparent fraction of (1)H density. Regenerating processes of the Achilles tendon were followed for 18 weeks by analyzing the T(2) relaxation time. There is only a long T(2) relaxation time component (21.8 to 28.0 ms) up to 3 weeks after transection. Biexponential relaxation is revealed at 6 weeks and thereafter, whereby (i) the T(2) relaxation times become shorter, (ii) there is anisotropy in the short and long T(2) values, and (iii) the orientational dependency of the apparent fraction of water proton density becomes evident with maturation of the regenerating tendon. From these results, the (1)H T(2) relaxation time of water might be used to monitor the healing process of collagen structures of the tendon non-invasively.

[Study on fluctuation of several parameters in varicella].

Specific immunological responses (varicella-specific IgG and IgM) and various parameters (interferon, C3, C4, platelet count, erythrocyte count, erythrocyte CR1) in varicella infection were determined over the course of the disease (days) and their relationships to severity and immunological significance evaluated. Defining the day varicella appeared as disease day 0, IgG appeared on the disease day 4 and IgG on day 5. The preceding disease days were dominated by non-specific immunological mechanisms. Interferon appearance preceded that of these antibodies but did not correlate with severity. In serious cases, C3 and C4 increased in the acute stage while platelet count declined. Erythrocyte count decreased in severe cases after disease day 5. Three patients with lower erythrocyte CR1 activity values remained critical. These findings suggested that determining platelet count is expedient in determining severity in the early stage and that the complement pathway is a major component of early immunological response.

Remnant-stump early gastric cancer following partial gastrectomy.

Eight rare cases of remnant-stump early gastric cancer (RS-EGC) were investigated, retrospectively. The incidence of RS-EGC was 0.9% (8/845) of all resected early gastric cancers. The macroscopic cancer type was elevated in 75% and mucosal in 88%. All were the differentiated type with no lymph node metastasis. The initial reconstruction involved a Billroth II in 75% of the patients. The RS-EGC was usually found during a periodic endoscopic follow-up. All patients are still alive with no postoperative complications and no cancer recurrence.

A clinicopathological study of distal advanced gastric carcinoma with duodenal invasion.

We studied retrospectively the clinicopathological features of gastric cancer with duodenal invasion and compared these with those with non-invasion, to investigate the prognostic significance of duodenal invasion. The patients were grouped into two groups of Group A (65 patients) with duodenal invasion and Group B (197 patients) without invasion who underwent gastrectomy for advanced distal gastric carcinoma according to histological findings. In Group A, many were at high age and with symptoms of pyloric stenosis. Group A (91%) had a significantly higher incidence of serosal invasion and infiltration into the pancreas head than did Group B (76%) (p < 0.01). In N3 metastasis, there was a significant difference between Group A (60%) and Group B (36%) (p < 0.01). The five-year-survival rate in Group A was 14.0% and in Group B was 43.3% (p < 0.001). In cases of duodenal invasion, patients with symptoms of pyloric stenosis demonstrated a poorer prognosis than those without. Those with longer than 20 mm invasion had a significantly higher incidence of N3 metastasis than those with less than 10 mm (p < 0.05). Lymphangitic type had longer invasive length to duodenum, and N3 metastasis of the lymphangitic type was frequent comparing to localized type (42%) and invasive type (47%) (p < 0.001) (95%) (p < 0.01). These results showed that duodenal invasion was a significant prognostic factor in cases of distal gastric carcinoma. In the therapeutic strategy for advanced gastric carcinoma with duodenal invasion, it is necessary to perform gastrectomy with regional lymph nodes dissection and with combined resection of adjacent organs.

[Detection of cytomegalovirus in urine specimens from renal transplant recipients using polymerase chain reaction].

Human cytomegalovirus (CMV) in this study, a polymerase chain reaction (PCR) assay was developed to detect human cytomegalovirus (CMV) from urine specimens taken from renal transplant patients and one congenital CMV infection. Twenty eight urine specimens of 20 renal transplant patients were analyzed by the PCR. CMV infection was detected in 11 specimens (8 recipients). The incidence (40.0%) of PCR positive in the renal transplant patients was highest among the other conventional methods including fluorescent antibody study, anti-IgM EIA and complement fixation test. In two cases, CMV infection could be identified by PCR before antibody development. The PCR procedure is more sensitive and rapid than conventional methods for the diagnosis of CMV. Rapid diagnosis of CMV infection using PCR may be useful in clinical diagnosis and have therapeutic value.

[Molecular differential diagnosis of herpes virus using common primer pairs: detection of HSV-1, HSV-2, VZV and CMV by the PCR].

Differential diagnosis of herpes virus species is often required in various clinical courses, such as transplantation and immunocompromised patients. Pairs of common primers which enabled sufficient amplifying and differential diagnosis of herpes virus species were designed and nested PCR using the common primer pairs was conducted to evaluate for its clinical application. The PCR was shown to provide amplification and differential diagnosis of HSV-1, HSV-2, VZV and CMV. The results of the PCR were consistent with those from fluorescent antibody of herpes virus culture and isolation. From clinical vesicular specimens, the PCR enabled the direct detection of HSV-1, HSV-2, and VZV. The PCR using the common primer pairs offers speed in the differential diagnosing of herpes virus.

[The significance of p21 expression in gastric cancer].

We examined the expression of p53, p21, cyclin D1, E, and PCNA in 75 cases of gastric cancer by immunohistochemical study and the expression of p21 RNA in cases by in situ hybridization. The rate of stage III, IV cases of p53(+) p21(-) group was significantly higher than that of any other groups. The apportinately 3-year survival rate of p53(+) p21(-) group was significantly lower than either that of p21(+) p53(-) or p53(-) p21(-) group. The 3-year survival rates of positive cases were significantly lower than those of negative cases on both cyclin D1 and E. The positive rate of cyclin E of the p53(-) p21(+) group was significantly lower than that of the p53(+) p21(-) group. The average PCNA Labeling. Index (LI) of the p53(+) p21(-) group was significantly higher than that of the p53(-) p21(+) group. The 3-year survival rate of cases with expression of p21 RNA was higher than that of cases without p21 RNA. Average PCNA L1 of cases with expression of mutant-type p53 was high and the number of poor prognostic cases in cases with expression of mutant-type p53 was large. In contrast, the average PCNA LI of cases with expression of p21 was low and the number of good prognostic cases with expression of p21 was large. These results suggest that p21 suppresses synthesis of DNA via cyclin E and PCNA.