A novel immunohistochemical method for in vivo detection of endotoxin using horseshoe crab factor C.

We developed a novel immunohistochemical method for in vivo detection of endotoxin (LPS) localization in relation to the biologically active region, by use of factor C, an initiation factor in the Limulus clotting system which is mediated by LPS, as a specific affinoligand to LPS, and using rabbit anti-factor C IgG. The competitive inhibition of various LPS, lipid A, anti-LPS factor, or polymyxin B to factor C binding indicates that the immunohistochemical reaction is specific to LPS. Investigating the time course of LPS distribution during 6 hr after IV injection of 5 mg/kg to rats, the greatest uptake of LPS was evident in the reticuloendothelial system (RES), particularly in Kupffer cells, 5 min after injection, and in adrenocortical cells 3 hr after the injection. Shortly after the injection, LPS also appeared in platelet thrombi, intravascular monocytes, and a few neutrophils, and on the surface of endothelial cells in liver, kidney, spleen, lung, and aorta. Both smooth and rough forms of LPS were detectable and there was no apparent difference in their localization. This approach facilitates immunohistochemical analyses of the mechanisms involved in development of endotoxemia.

Doxorubicin-heparin complex: reduction of cardiotoxicity of doxorubicin.

We have compared the antitumor activity and cardiotoxicity of free doxorubicin (Dox) and doxorubicin-heparin complex in vivo and in vitro. Dox and Dox-heparin complex equally inhibited the DNA synthesis of leukemic cells and showed a similar anticancer activity against tumor-bearing mice. Acute toxicity of Dox at the dose of 20 mg/kg or 30 mg/kg was significantly more profound than that of the Dox-heparin complex, which was demonstrated by survival rate (P < 0.01). Chronic toxicities of Dox and the Dox-heparin complex were compared by giving the respective reagent (2 mg/kg) weekly for 20 weeks. The weight gains of the mice given Dox-heparin complex were greater than those of the mice given Dox alone (P < 0.01). The pathological damage to the cardiac tissue in mice treated with Dox-heparin complex was significantly less severe than that of mice treated with Dox. Thus, the present study indicates that complexing with heparin diminished the acute and chronic toxicity of Dox without reducing its antitumor activity in mice, and suggests a possible clinical application of Dox-heparin complex in humans.

The localization of lipopolysaccharide in an endotoxemic rat liver and its relation to sinusoidal thrombogenesis: light and electron microscopic studies.

The distribution of lipopolysaccharide (LPS) and sequential thrombus formation in the liver was investigated by immunohistochemical and cytochemical techniques in endotoxemic rats, using horseshoe crab factor C, a specific ligand for biologically active LPS, a monoclonal antibody against it, and rabbit anti-rat fibrinogen IgG. One hour after the intravenous administration of LPS (5 mg/kg), LPS was localized in the secondary lysosomes of Kupffer cells and in the vesicles of endothelial cells, mainly at the peripheries of the hepatic lobules. Small necrotic foci of hepatic tissue were scattered close to the LPS-containing Kupffer cells, and were frequently associated with infiltration of neutrophils and deposition of fibrin. Three hours after the administration of LPS, the immunohistochemical reaction of LPS became stronger and was mostly confined to Kupffer cells. Strands of polymerized fibrin were frequently observed on both the surface of the LPS-containing Kupffer cells and on endothelial cells. These findings suggest that the activation of the coagulation cascade in plasma is first initiated, even though only transiently, by hepatic necrosis which is probably caused by LPS-activated leukocytes, and then by the procoagulant activity expressed on the surface of both Kupffer cells and endothelial cells. Fibrinogen-related antigens were also immuno-ultrastructurally detected in the lysosomes of Kupffer cells three hours after the injection of LPS, which suggested that the Kupffer cells phagocytozed and degraded fibrin. Therefore Kupffer cells in endotoxemia may closely participate in both the sinusoidal thrombogenesis and degradation of fibrin.

High-dose methylprednisolone therapy for nephrotic syndrome in Henoch-Schoenlein nephritis.

Twelve patients with nephrotic syndrome (NS) in Henoch-Schoenlein (HS) nephritis were treated with a high dose of intravenous methylprednisolone (MP) on each of nine alternate days followed by oral prednisolone for 4 to 6 months. Renal biopsy was performed on 10 of the 12 patients. The glomerular change in 5 patients, which was accompanied by crescents and/or sclerosis, with NS and acute nephritic syndrome (ANS) at onset, was more severe than that of the other 5 patients with NS and hematuria at onset. The renal insufficiency or hypertension in these 5 patients with NS and ANS improved within 2 weeks on this MP therapy. After a mean follow-up period of 40.5 months, all patients except 2 revealed normal physical findings and renal function as well as urinary findings. Repeated biopsies in the 2 patients with NS and ANS at onset demonstrated an improved renal pathology in comparison with their initial biopsies. No severe side effects related to high-dose intravenous MP followed by oral prednisolone therapy were encountered in any of the patients. Our results suggest that high-dose intravenous MP therapy can lead to a favorable outcome in patients with NS in HS nephritis.

The natural history of screening detected IgA glomerulonephritis in children.

The clinical course of 43 children with IgA glomerulonephritis detected by mass urine screening was followed for a mean period of 8.1 years. Histological findings were graded according to the severity of glomerular and tubulointerstitial lesions. There was no correlation in the severity of histological grade and clinical outcome between subjects with microscopic hematuria and those with microscopic hematuria and proteinuria nor between those with and without one or more episodes of macroscopic hematuria during the follow-up period. None of the 35 children with proteinuria less than or equal to 1 g/m2/day had severe histological findings or developed renal impairment. In contrast, the 8 children with proteinuria greater than 1 g/m2/day had moderate and severe histological findings. Four of these 8 children developed hypertension or renal insufficiency during the follow-up period. Our study indicates that the outcome of screening detected IgA glomerulonephritis in children correlates with the level of proteinuria and the severity of renal pathology.

Asymptomatic isolated microhaematuria: natural history of 136 children.

In a mass screening programme, 251 children with isolated microhaematuria were detected. Of these 251 children, 115 were excluded from the study because of microhaematuria secondary to a specific cause. The remaining 136 children were diagnosed as having asymptomatic isolated microhaematuria (ASH). Of these 136 children, 23 had evidence of urinary abnormalities in their family members. Red blood cell casts were evident in 31 children at their initial visit or during the follow-up period. Ten children had one or more episodes of macrohaematuria during the study. Renal biopsy was performed in 19 children because of indications of glomerular disease, and 13 of these 19 children had mild to moderate glomerulonephritis. None of these 136 children developed hypertension or renal impairment after a mean period of 7.4 years (range 6-13 years). Thirty-five children had normal urinary findings within 6 years of their initial visit, and 100 have had persistent microhaematuria without proteinuria throughout the follow-up period. The other child had microhaematuria with proteinuria greater than 1 g/m2 per day at the end of the study. This study suggests that the prognosis of ASH is good, and that renal biopsy is not indicated for children with ASH.