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There has been a long-standing need for guidelines on the diagnosis and treatment of keloids and hypertrophic scars that are based on an understanding of the pathomechanisms that underlie these skin fibrotic diseases. This is particularly true for clinicians who deal with Asian and African patients because these ethnicities are highly prone to these diseases. By contrast, Caucasians are less likely to develop keloids and hypertrophic scars, and if they do, the scars tend not to be severe. This ethnic disparity also means that countries vary in terms of their differential diagnostic algorithms. The lack of clear treatment guidelines also means that primary care physicians are currently applying a hotchpotch of treatments, with uneven outcomes. To overcome these issues, the Japan Scar Workshop (JSW) has created a tool that allows clinicians to objectively diagnose and distinguish between keloids, hypertrophic scars, and mature scars. This tool is called the JSW Scar Scale (JSS) and it involves scoring the risk factors of the individual patients and the affected areas. The tool is simple and easy to use. As a result, even physicians who are not accustomed to keloids and hypertrophic scars can easily diagnose them and judge their severity. The JSW has also established a committee that, in cooperation with outside experts in various fields, has prepared a Consensus Document on keloid and hypertrophic scar treatment guidelines. These guidelines are simple and will allow even inexperienced clinicians to choose the most appropriate treatment strategy. The Consensus Document is provided in this article. It describes (1) the diagnostic algorithm for pathological scars and how to differentiate them from clinically similar benign and malignant tumors, (2) the general treatment algorithms for keloids and hypertrophic scars at different medical facilities, (3) the rationale behind each treatment for keloids and hypertrophic scars, and (4) the body site-specific treatment protocols for these scars. We believe that this Consensus Document will be helpful for physicians from all over the world who treat keloids and hypertrophic scars., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s) 2019.)

Lambert-Eaton myasthenic syndrome (LEMS) is an autoimmune disease of the neuromuscular junction caused by autoantibodies binding to P/Q-type voltage-gated calcium channels. Breakdown of the blood-brain barrier and diffusion of cerebellar granule/Purkinje cell-reactive autoantibodies into the CNS are critical for the pathogenesis of paraneoplastic cerebellar degeneration (PCD) with Lambert-Eaton myasthenic syndrome. We recently found evidence that glucose-regulated protein 78 (GRP78) autoantibodies in the plasma of patients with neuromyelitis optica promote the CNS access of AQP4 autoantibodies. In the present study, we investigated whether the GRP78 autoantibodies in PCD-LEMS IgG boost the brain uptake of cerebellar cell-reactive antibodies across the blood-brain barrier and facilitate cerebellar dysfunction. We first evaluated the effects of purified IgG from PCD-LEMS or PCD patients on the blood-brain barrier function in human brain microvascular endothelial cells using a high content imaging system with nuclear factor κB p65 and intracellular adhesion molecule 1 (ICAM1) immunostaining. Next, we identified GRP78 autoantibodies causing blood-brain barrier permeability in PCD-LEMS IgG by co-immunoprecipitation and the living cell-based antibody binding assays. Exposure of brain microvascular endothelial cells to IgG from PCD-LEMS patients induced nuclear factor κB p65 nuclear translocation, ICAM1 upregulation, reduced claudin-5 expression, increased permeability and increased autocrine IL-1β and IL-8 secretion; the IgG from patients with Lambert-Eaton myasthenic syndrome did not have these effects. We detected GRP78 autoantibodies in the IgG of LEMS-PCD (83.3%, n = 18), but observed fewer in patients with LEMS (6.6%, n = 15) and none were observed in the control subjects (n = 8). The depletion of GRP78 autoantibodies reduced the biological effect of LEMS-PCD IgG on brain microvascular endothelial cells. These findings suggest that GRP78 autoantibodies play a role beyond neuromyelitis optica and that they have direct implications in the phenotypic differences between PCD-LEMS and LEMS., (© The Author(s) (2019). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: In glioblastoma (GBM), Id1 serves as a functional marker for self-renewing cancer stem-like cells. We investigated the mechanism by which cyclooxygenase-2 (Cox-2)-derived prostaglandin E2 (PGE2) induces Id1 and increases GBM self-renewal and radiation resistance., Methods: Mouse and human GBM cells were stimulated with dimethyl-PGE2 (dmPGE2), a stabilized form of PGE2, to test for Id1 induction. To elucidate the signal transduction pathway governing the increase in Id1, a combination of short interfering RNA knockdown and small molecule inhibitors and activators of PGE2 signaling were used. Western blotting, quantitative real-time (qRT)-PCR, and chromatin immunoprecipitation assays were employed. Sphere formation and radiation resistance were measured in cultured primary cells. Immunohistochemical analyses were carried out to evaluate the Cox-2-Id1 axis in experimental GBM., Results: In GBM cells, dmPGE2 stimulates the EP4 receptor leading to activation of ERK1/2 MAPK. This leads, in turn, to upregulation of the early growth response1 (Egr1) transcription factor and enhanced Id1 expression. Activation of this pathway increases self-renewal capacity and resistance to radiation-induced DNA damage, which are dependent on Id1., Conclusions: In GBM, Cox-2-derived PGE2 induces Id1 via EP4-dependent activation of MAPK signaling and the Egr1 transcription factor. PGE2-mediated induction of Id1 is required for optimal tumor cell self-renewal and radiation resistance. Collectively, these findings identify Id1 as a key mediator of PGE2-dependent modulation of radiation response and lend insight into the mechanisms underlying radiation resistance in GBM patients., (© The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Glioblastoma multiforme (GBM) is a highly heterogeneous malignant tumor. Recent data suggests the presence of a hierarchical organization within the GBM cell population that involves cancer cells with stem-like behavior, capable of repopulating the tumor and contributing to its resistance to therapy. Tumor stem cells are thought to reside within a vascular niche that provides structural and functional support. However, most GBM studies involve isolated tumor cells grown under various culture conditions. Here, we use a novel three-dimensional organotypic "explant" system of surgical GBM specimens that preserves cytoarchitecture and tumor stroma along with tumor cells. Notch inhibition in explants results in decreased proliferation and self-renewal of tumor cells but is also associated with a decrease in endothelial cells. When endothelial cells are selectively eliminated from the explants via a toxin conjugate, we also observed a decrease in self-renewal of tumor stem cells. These findings support a critical role for tumor endothelial cells in GBM stem cell maintenance, mediated at least in part by Notch signaling. The explant system further highlighted differences in the response to radiation between explants and isolated tumor neurospheres. Combination treatment with Notch blockade and radiation resulted in a substantial decrease in proliferation and in self-renewal in tumor explants while radiation alone was less effective. This data suggests that the Notch pathway plays a critical role in linking angiogenesis and cancer stem cell self-renewal and is thus a potential therapeutic target. Three-dimensional explant systems provide a novel approach for the study of tumor and microenvironment interactions.

Introduction: Dry ejaculation with loss of seminal emission is reported in patients who have been administered silodosin, an alpha1A-adrenoceptor antagonist., Aim: We investigated the impact of dry ejaculation caused by orally administered silodosin on orgasmic function., Methods: In a double-blind crossover study, 50 healthy volunteer men were randomly assigned to receive either a single dose of 4-mg silodosin or placebo with 3 days of washout before crossover. Subjects masturbated 4 hours after administering agents., Main Outcome Measures: Numerical rating scale (NRS) score from 0 (highest) to 10 (lowest) for subjective quality of orgasm, the subjective number of contractions of the bulbocavernosus/pelvic floor muscles, and the amount of semen were examined. Results. After the administration of silodosin, the NRS score worsened by 1.3 points (P = 0.003), the number of contractions of the bulbocavernosus/pelvic floor muscles decreased by about 1 (P = 0.003), and there was a decrease of 1.8 mL in the amount of semen produced (P < 0.0001). Eleven men overall (22%) on silodosin administration had less than a 50% decrease from baseline in the amount of semen., Conclusions: Silodosin may adversely affect the subjective orgasmic function by causing an abnormal ejaculation with decreased (or no) semen discharge and a decrease in the number of bulbocavernosus/pelvic floor muscle contractions. Semen passing through the urethra and sufficient rhythmic contraction of the muscle of the pelvic floor may contribute to the subjective pleasure of orgasm.

Background: The introduction of prostate-specific antigen (PSA) testing has not only shortened the time required to make diagnosis but changed the treatment strategies of localized prostate cancer. We conducted the decision analysis on its treatment focusing on patients with biochemical failure., Methods: We developed a Markov model to calculate life expectancy (LE) and quality-adjusted life expectancy (QALE) stratified by age, comorbidity and tumor characteristics in patients with newly diagnosed prostate cancer or biochemical failure after curative therapy. For newly diagnosed patients, three treatment strategies were considered as primary managements: radial prostatectomy (RP), external beam radiotherapy (EBRT) and watchful waiting (WW). Managements considered for biochemical failure were: after RP, salvage radiotherapy (SRT), salvage hormonal therapy (SHT) and WW; and after EBRT, SHT and WW. Transition probabilities in the Markov model were derived from published studies. Quality of life (QOL) data to estimate QALE score were derived from 323 patients with prostate cancer., Results: For patients with Gleason 2-6 cancer at diagnosis, WW yielded the greatest number of QALE. For patients with Gleason 7 cancer, it was controversial whether curative therapy was the preferred strategy. For patients with Gleason 8-10 cancer, curative therapy yielded the greatest number of QALE in younger patients without severe comorbidity. Patients' benefit from salvage therapy for biochemical failure after curative therapy depended on age, comorbidities, tumor characteristics and QOL effect., Conclusions: Our findings support the need for various treatment options, taking into consideration the patient's age, comorbidity and the QOL effect in the aging society.

Background: Ultrasensitive prostate-specific antigen (PSA) is a significant serum biomarker for identifying the PSA nadir and early biochemical failure after radical prostatectomy (RP). We assessed the efficiency of ultrasensitive PSA assay in the follow-up after RP., Methods: We generated longitudinal ultrasensitive PSA data using a computer program assuming that patients experienced biochemical failure after RP. The simulation experiments, based on several different scenarios, were performed to assess the sensitivity and specificity in the diagnosis of biochemical failure using ultrasensitive PSA values and to estimate the lead time, which is the time advantage of detecting positivity for biochemical failure using the ultrasensitive PSA values compared with conventional PSA assay. We validated the sensitivity, specificity and lead time using actual follow-up data of 182 patients receiving RP., Results: It was suggested that the sensitivity obtained from the actual data was more similar to that obtained using ultrasensitive PSA with an exponential increase than with a linear increase in the simulation experiments. Diagnosing biochemical failure based on two consecutive increases in the ultrasensitive PSA values was not recommended. Of non-biochemical failure patients, 9.4% showed four consecutive increases in their ultrasensitive PSA values. Average lead time in the actual data was 11.2 months (SD: 10.1)., Conclusions: For an accurate diagnosis of biochemical failure, our findings suggest the importance of a certain duration of follow-up and exclusion of false-positive results afterwards.

Background: It is suggested that IgA nephropathy (IgAN) manifests differently in children vs adults on the basis of biopsy findings. However, this has been difficult to establish owing to the uncertainty of the timing of disease onset in adult IgAN. We addressed this question by comparing both histology and leucocyte accumulation in biopsies of recently diagnosed childhood and adult IgAN., Methods: Biopsies taken within 2 years from the onset of renal abnormalities in 33 childhood (10 +/- 3 years of age) and 38 adult (35 +/- 6 years) cases of IgAN were examined for histological changes (cellularity in mesangial, endocapillary and extracapillary areas, matrix expansion, adhesions/crescents and interstitial damage), glomerular deposition of immunoglobulin and complement, and the presence of macrophages, activated macrophages and T cells by immunohistochemistry., Results: Glomerular hypercellularity owing to increased cells in mesangial area was prominent in paediatric IgAN and significantly greater than in adult IgAN. In contrast, glomerular matrix expansion, crescent formation and interstitial damage were more severe in adults compared to paediatric IgAN. Indeed, glomerular hypercellularity correlated with proteinuria in paediatric but not in adult IgAN, whereas glomerular matrix correlated with proteinuria and renal function in adult but not in paediatric IgAN. The degree of C3c deposition was significantly greater in paediatric IgAN, while deposition of fibrinogen was greater in adult IgAN. Glomerular and interstitial CD68+ macrophages and a subset of sialoadhesin (Sn)+ activated macrophages were identified in both paediatric and adult IgAN, being significantly greater in number in adult IgAN. Glomerular leucocyte infiltration correlated with proteinuria while interstitial leucocyte infiltration correlated with interstitial damage in both groups. However, only the subset of Sn+ macrophages gave a significant correlation with renal function, glomerular hypercellularity and glomerular matrix., Conclusions: This study has demonstrated significant differences in the early glomerular lesions of IgAN in children vs adults. Furthermore, Sn+ activated macrophages are implicated in the pathogenesis of IgAN in both patient groups. The prognostic significance of these findings warrants further study.

Background: Sialoadhesin (Sn; CD169) is a lectin-like receptor whose expression is restricted to subsets of tissue and inflammatory macrophages. We have previously identified accumulation of Sn+ macrophages as an important marker of disease progression versus remission in rat mesangial proliferative nephritis. The current study examined the significance of Sn+ macrophages in human proliferative glomerulonephritis., Methods: Frozen kidney sections from normal adult human kidney (n = 4) and pediatric nephropathy (n = 40) were stained for total macrophages (CD68+ cells), Sn+ macrophages, CD3+ T-cells and collagen type I by immunofluorescence. Leukocyte infiltration and the severity of glomerular lesions and interstitial damage were scored. A second protocol biopsy was performed in 27 cases and clinical and biopsy-based data obtained., Results: Sn+ macrophages were absent from glomeruli in normal adult human kidney and in thin basement membrane disease (n = 4), but were detected in 4 of 9 cases of purpura nephritis; 7 of 17 IgA nephropathy; 5 of 5 membranoproliferative glomerulonephritis, and 5 of 5 lupus nephritis. Sn+ macrophages were localized in areas of focal glomerular and interstitial damage. Two-colour immunostaining confirmed that Sn+ cells are a subset of total CD68+ macrophages. The number of glomerular Sn+ macrophages correlated with the degree of proteinuria and glomerular lesions (r = 0.44, P = 0.0045 and r = 0.82, P<0.0001; respectively), while interstitial Sn+ macrophages correlated with the degree of proteinuria and interstitial damage (r = 0.59, P<0.0001 and r = 0.75, P<0.0001; respectively). Combined immunostaining revealed that interstitial Sn+ macrophages and CD3+ T-cells co-localized in areas of tubulointerstitial damage with increased type I collagen deposition. There was significant correlation between the number of interstitial Sn+ macrophages and CD3+ T-cells (r = 0.74, P<0.0001). Most patients responded to a 2 year period of glucocorticoid therapy with a reduction in proteinuria and glomerular lesions and this correlated with the reduction in the number of glomerular Sn+ macrophages., Conclusion: This study has identified Sn+ cells as a macrophage subset whose accumulation in the kidney correlates with proteinuria and histologic damage. These results, together with recent findings from animal studies, suggest that Sn+ macrophages may play an important role in progressive renal disease.

Background: At present, there are few available animal models of progressive renal failure originating from mesangial proliferative glomerulonephritis (GN). In the current study, we examined the usefulness of anti-Thy-1 monoclonal antibody (mAb) 1-22-3-induced GN in uninephrectomized rats as a model of progressive renal failure by analysing the similarities to human disease., Methods: GN was induced by intravenous injection of mAb 1-22-3 into uninephrectomized male Wistar rats. The natural course of the disease was analysed in this model for 47 weeks. The effect of treatment with the angiotensin-converting enzyme inhibitor, captopril, on renal functional outcome was also examined in this model for 23 weeks, beginning from 1 week after antibody injection., Results: Injection of mAb 1-22-3 induced a persistent proteinuria during the entire study period. Animals showed a progressive decline in renal function and 63% died by week 47. Severe glomerular and tubulointerstitial lesions were consistently observed. Treatment with captopril significantly inhibited increases in proteinuria and blood pressure, and attenuated renal injury. Captopril also retarded the progression of renal failure, and decreased mortality. Finally, the level of proteinuria was significantly correlated with the rate of decline in renal function, and the reduction in proteinuria by captopril was accompanied by a slower progression of renal failure., Conclusions: The mAb 1-22-3-induced GN in a uninephrectomized rat model simulates the clinical manifestations of human disease, indicating that this model may be useful for studying progressive renal failure and for investigating new therapeutic strategies against renal failure.

Background: How podocytes respond to injury is poorly understood, although podocyte injury in the glomerulus has been proposed as the crucial mechanism in the pathogenesis of proteinuria and focal segmental glomerulosclerosis. An increase in sodium/myo-inositol co-transporter (SMIT) transcripts, an osmoprotective gene, has been demonstrated in a variety of brain injury models. In the present study, we investigated SMIT expression in podocytes in experimental nephrosis., Methods: Two types of nephrosis were induced in rats: puromycin aminonucleoside (PAN) nephrosis and monoclonal antibody (mAb) 5-1-6 nephropathy. Podocyte injury was morphologically distinct in the former type of nephrosis and limited to a minimum in the latter. SMIT expression in isolated glomeruli was estimated by ribonuclease protection assay. Localization of SMIT-expressing cells in glomeruli was examined by in situ hybridization., Results: SMIT transcripts in glomeruli increased conspicuously in the nephrotic stage of PAN nephrosis, whereas the transcripts in cortices and medullae did not show significant changes. In situ hybridization revealed that podocytes were predominant cells expressing SMIT in the glomerulus. Significant increase of SMIT mRNA in the glomeruli was detected before the onset of massive proteinuria. In contrast, up-regulation of SMIT expression was not observed in mAb 5-1-6 nephropathy, whose urinary protein levels were comparable with those in the nephrotic stage of PAN nephrosis., Conclusions: These findings suggest that SMIT expression in podocytes is not provoked by an effect of massive proteinuria but by extensive cellular injury.

Nephrin is identified as a product of the gene mutated in a patient with congenital nephrotic syndrome of the Finnish type. To analyse the function of nephrin and the relationship of nephrin and the target antigen of nephritogenic anti-rat slit diaphragm monoclonal antibody (mAb) 5-1-6, the rat homologue of nephrin was cloned. The deduced amino acid sequence of rat nephrin shows 82.2% homology to human nephrin. Signal sequences of rat nephrin are 36 amino acids, whereas those of human nephrin are reported to be 22 amino acids. The localization of rat nephrin always coincided with that of mAb 5-1-6 antigen. The specific anti-nephrin antibody recognized the mAb 5-1-6-antigen complex. mAb 5-1-6 reacted with COS cells transfected with rat nephrin cDNA. These results demonstrate that nephritogenic mAb 5-1-6 identifies the extracellular domain of rat nephrin, thereby documenting that nephrin is a functional protein of the slit diaphragm. The staining pattern of nephrin shifted from a linear-like pattern to a discontinuous coarse granular pattern not only in mAb 5-1-6-induced nephropathy, but also in puromycin aminonucleoside nephropathy, adriamycin nephropathy and passive Heymann nephropathy. Rat nephrin is detected first on the basal and lateral side below the junctional complex at the S-shaped body stage. With the interdigitation of foot processes, nephrin becomes concentrated in the slit pore, and finally restricted in the site of the slit diaphragm bridging two adjacent foot processes.

Because of the widespread use of aluminium- and calcium-containing phosphate binders for the control of hyperphosphataemia in patients with end-stage renal failure, an iron(III) chitosan complex was synthesised and fed to rats to measure its effect on serum phosphorus and calcium, intestinal phosphate binding and phosphate absorption. Thirty-six Wistar rats were randomly selected and distributed into a baseline group (n = 6), a control group (n = 8 (days 0-15), n = 8 (days 16-30)) and a treatment group (n = 8 (days 0-15), n = 8 (days 16-30)). The control groups ingested AIN-76 diet mix with a 1% w/w fibre content; however, the treatment groups had the fibre content completely substituted with iron(III) chitosan. The mean weights of the treated rats were slightly lower from 15 days (not significant); but overall, rat growth was not stunted in the treatment groups. The serum phosphorus levels of the treated group (n = 8) were significantly reduced after 15 days (P = 0.004; control: 5.7+/-0.9 mg dL(-1); treatment: 4.4+/-0.5 mg dL(-1); 95% CI of difference: 0.5-2.2) and 30 days (P = 0.002; control: 5.5+/-0.9 mg dL(-1); treatment = 4.1+/-06 mg dL(-1); 95% CI of difference: 0.6-2.3) as compared with the respective control group. The serum calcium-phosphorus product was 62.0+/-12.1 mg2 dL(-2) for the control and 45.1+/-6.6 mg2 dL(-2) for the treatment group after 30 days (P = 0.004). The serum iron concentration of the treatment group did not differ from the baseline value after 15 and 30 days, but the treatment group was significantly higher than the control group (P<0.05) after 30 days. The faeces phosphorus levels (mg day(-1)) were higher (P<0.01) and its iron content was much higher (P<0.01) for the treated group. The urine phosphorus (mg kg(-1)) was not significantly reduced for the treated group, but the mean was consistently less. The kidney and liver weights of both groups were similar, but the phosphorus content of the kidney (mg (g kidney)(-1)) was higher for the treated group after 30 days (P = 0.041; control, 4.2+/-1.2 mg g(-1) vs treatment, 5.6+/-1.4 mg g(-1). Because iron(III) chitosan had a high phosphorus-binding capacity of 308 (mg P) per gram of Fe3+ for both the in-vitro (pH 7.5) and in-vivo studies, which is greater than nearly all commonly used phosphate binders, and a small net phosphorus absorption difference of 3.7 mg day(-1), it is an efficient phosphate binder for lowering serum phosphate levels without increasing serum calcium levels.

Background: Sustained proteinuria is reported to be very harmful to the tubulointerstitium, leading to severe interstitial injury. However, it remains unclear whether sustained proteinuria itself is responsible for severe interstitial injury because, in the previously reported models, the development of factors other than proteinuria in tubulointerstitial lesions could not be excluded completely., Methods: After treatment to induce immune tolerance to mouse immunoglobulin, 20 rats were injected with anti-rat slit diaphragm monoclonal antibody (mAb) 5-1-6 twice a week for 6 months and were then sacrificed., Results: mAb 5-1-6 induced massive proteinuria in 11 rats. In nine rats with mild proteinuria, no histological alteration could be detected with light microscopy and immunofluorescence. In nephrotic rats, light microscopy showed minor glomerular abnormalities, with interstitial oedema, tubular epithelial cell degeneration and interstitial cell infiltration. Immunofluorescence revealed increased expression of vimentin and an increased number of OX1-, OX19- and ED1-positive cells. However, we could not detect any accumulation of type I and IV collagen or laminin in the tubulointerstitium. RT-PCR showed that the expression of mRNA for type I collagen was not increased, compared with that in control rats., Conclusions: We succeeded in developing a model of persistent nephrosis without severe glomerular abnormalities, nephrectomy or other manoeuvres known to induce disturbed haemodynamics, using an agent without tubulointerstitial toxicity, and considered it to be suitable for investigating the direct toxicity of proteinuria. In this model, isolated massive proteinuria induced interstitial injury. However, the degree of injury was suggested to be much less than that observed in other previously developed models.

Glycophosphatidylinositol (GPI)-linked Thy-1 molecules, well known cell surface markers of murine T cells, are present on the glomerular mesangial cells of the rat kidney. The administration of anti-Thy-1.1 MoAbs 1-22-3 and OX-7 to rats induces severe and mild complement-dependent mesangial proliferative glomerulonephritis, respectively. In order to determine whether protein-tyrosine kinase (PTK) activity is associated with Thy-1 molecules on rat mesangial cell surface, we performed an immune complex kinase assay, using anti-Thy-1 MoAbs 1-22-3 and OX-7, followed by reimmunoprecipitation with anti-phosphotyrosine, anti-fyn, anti-lck and anti-lyn antibodies. Physical association of PTK, p59fyn and p56/53lyn with Thy-1 molecules was demonstrated in cultured rat mesangial cells. The activities of these kinases detected in MoAb 1-22-3 precipitates were higher than those in MoAb OX-7 precipitates. These results suggest that Thy-1 molecule transduces some signals also in rat mesangial cells.

Histone can mediate the binding of both free DNA and DNA complexed to anti-DNA antibody to the glomerular capillary wall. We tested whether performed histone-DNA-anti-DNA immune complexes (IC) could bind to the glomerular capillary wall. The immune complex, generated with anti-DNA antibody derived from an SLE patient and excess of 125I-DNA followed by digestion with DNase, was mixed with histones. The complex containing 4 micrograms DNA was injected via the aorta into the left kidney of rats. At 15 min, 1-3% of the histone-DNA-anti-DNA antibody complex bound (measured as 125I-DNA), when histone was omitted less than 0-1% of the DNA-anti-DNA antibody complex bound. By immunofluorescence human immunoglobulins and histones, representing the IC, could be observed in a capillary pattern; but no complement deposition was detected. Electron microscopy revealed discrete, electron dense deposits in a subendothelial, subepithelial and mesangial localization at 15 min. These results provide direct evidence that antibodies from serum of SLE patients can form soluble histone-DNA-anti-DNA immune complexes that bind to the glomerular capillary wall in vivo.

A new animal model of progressive glomerulosclerosis was developed by administering a single i.v., injection of MoAb 1-22-3 to unilaterally nephrectomized rats. Renal morphological analysis revealed that glomerular lesions characterized by mesangial cell proliferation and mesangial matrix expansion were induced in about 95% of the glomeruli. Approximately 20% of the glomeruli of the unilaterally nephrectomized rats showed sclerosis or segmental sclerosis by week 6 after MoAb injection and crescent formation was observed in some glomeruli (ca 4%). Cellular infiltration was also noted in some parts of the interstitium. Increased expression of transforming growth factor-beta (TGF-beta) was observed in the unilaterally nephrectomized rats treated with MoAb 1-22-3, but we could not demonstrate pathological involvement of platelet-derived growth factor (PDGF), even though early-stage mesangial cell proliferation was observed. The mechanism of mesangial cell proliferation in this model remains to be elucidated. The relatively short period of time needed to induce the sclerotic changes in considered to be a great advantage of this model for clarifying the mechanisms involved in the chronic progression of mesangial proliferative glomerulonephritis.

Susceptibility to the development of MoAb 5-1-6-induced proteinuria was investigated in four different rat strains, i.e. Brown-Norway (BN), Lewis (LEW), Sprague-Dawley (SD) and Wistar. An intravenous injection of 5 mg of MoAb 5-1-6 to female 7-week-old rats of a given strain induced massive proteinuria in BN, LEW and Wistar rats. However, SD rats developed almost no proteinuria. A similar tendency was observed in the second experiment, in which the injected dose of MoAb was adjusted according to the body weight of each rat (3 mg/100 g body weight). Immunofluorescence (IF) and immunoelectron microscopy (IEM) revealed no differences between the binding patterns of the MoAbs to normal rat kidneys derived from each strain. Quantitative study using 125I-labelled MoAb showed that there was no significant difference in the amount of antibody bound to the kidney 1 h and 5 days after injection between two rat strains, LEW and SD. Localization of 5-1-6 in vivo and its kinetics were investigated. In IF a linear-like pattern along capillary walls was observed 2 h after injection in both LEW and SD strains. This linear-like pattern was shifted to a granular pattern in proteinuric LEW rats 6 days after injection, whereas it remained linear-like in non-proteinuric SD rats. IEM confirmed this difference in the localization of injected MoAb 6 days after injection to LEW and SD rats also at the ultrastructural level. We conclude that there is a clear-cut strain difference in the development of proteinuria induced by MoAb 5-1-6. SD rats were less susceptible to MoAb-induced glomerular injury than BN, LEW and Wistar rats. Although the exact reason for strain variation in susceptibility to MoAb-induced proteinuria remains to be clarified, the movement of bound MoAb, presumably together with corresponding antigenic molecule along the glomerular epithelial cell surface followed by endocytosis into the epithelial cell, seems to be closely related to the induction of proteinuria.

A cDNA encoding the 14-kDa subunit of vacuolar ATPase was cloned from human fetal brain. The sequence was composed of 680 nucleotides containing an open reading frame of 357 nucleotides. The deduced peptide sequence consisted of 119 amino acid residues with a calculated molecular weight of 13,369 Da and a pI of 5.19. Overall, this amino-acid sequence was respectively 69% and 70% identical to those of Manduca sexta and Drosophila melanogaster 14-kDa subunits, although the two representatives of Class Insecta were remarkably similar to one another (91% identity). Three regions in particular (the N-terminal, amino acids 5-36; the middle, residues 58-68; and the C-terminal, residues 88-118) were highly conserved. Hence, we think that the 14-kDa subunits have evolved from the same ancestral gene, and that the three conserved regions are important for the structure and function of vacuolar ATPase. A single 0.8-kb band was detected in various human tissues by Northern blot analysis. Since the human 14-kDa subunit is expressed ubiquitously, it might be a housekeeping protein. A separate transcript found in the cDNA library lacked a 6-bp segment in the 5' non-coding region (nucleotides -40 to -35) and also carried a 23C to T (8Thr to Ile) point mutation in the coding region; these minor differences likely reflected normal polymorphism.

A single i.v. injection of 3 mg of the F(ab')2 fragment of MoAb 5-1-6 into rats induced immediate proteinuria (128.1 +/- 80.7 mg/24 h on day 1) which lasted 1-2 days. In contrast, rats administered 10 mg of the corresponding Fab fragment did not develop abnormal proteinuria even though an equivalent dose of the intact MoAb 5-1-6 far exceeded the nephritogenic dose. The total kidney binding of 125I-Fab fragment was 209.5 +/- 34.3 micrograms/2 kidneys. This exceeded that obtained by injection of 3 mg MoAb 5-1-6 IgG1 (58.9 +/- 12.5 micrograms/2 kidneys at 1 h) and was similar to that obtained following injection of 3 mg F(ab')2 fragment (235.3 +/- 16.9 micrograms/2 kidneys). Immunofluorescence (IF) showed a linear pattern along the glomerular capillary wall at 1 h after the administration of MoAb 5-1-6 IgG1, F(ab')2 or Fab fragment. On day 5, fine to coarse granules were observed scattered in F(ab')2-injected rat glomeruli, whereas granules were densely localized in Fab-injected rat glomeruli. Complement-depleted rats injected with 3 mg of MoAb 5-1-6 IgG1 developed proteinuria with the same time course as non-depleted rats. This observation, together with the ability of F(ab')2 to induce proteinuria, indicates that proteinuria induced by MoAb 5-1-6 is complement-independent. This study suggests that MoAb 5-1-6-induced proteinuria is initiated by cross-linking of the epitopes by divalent MoAb 5-1-6 and is independent of complement activity.

Murine MoAb 1-22-3 has already been reported to bind to the mesangial cell surface and to cause transient proteinuria and mesangial morphological changes characterized by mesangiolysis, subsequent mesangial cell proliferation and mesangial matrix increase by a single i.v. injection. In this study, MoAb-induced glomerulopathy was quantitatively analysed. No correlation between the severity of mesangial morphological changes and the degree of proteinuria was detected (r = 0.190). The minimum dose injected to induce abnormal proteinuria was 25 micrograms. This dose corresponded to 1.79 micrograms/2 kidneys 30 min after MoAb injection. The highest average level of proteinuria was observed in rats injected with 500 micrograms of MoAb, and less proteinuria was observed in rats injected with 10.0, 5.0 and 2.0 mg. Although the amounts of kidney-fixing MoAb and the subsequent deposition of rat C3 in the high-dose-injected group were larger than in the 500 micrograms injected group, the numbers of infiltrating inflammatory cells were the same in both groups. No correlations between the degrees of such mediators and proteinuria were observed.

We have investigated the effects of various extracellular matrix (ECM) components on the behaviour of human mesangial cells (HMC) in a gel culture system using a modified MTT assay method. When cultured on a reconstituted basement membrane, Matrigel (M gel), HMC aggregated and formed isolated colonies initially, then extended an array of cell processes to form a dendritic network structure and proliferated very slowly as the culture period progressed. On type I collagen gel (CI gel), however, HMC developed elongated bipolar shapes, migrated into the gel, and showed rapid cell growth. Next, separate ECM components, such as type III and IV collagens, laminin, heparin and heparan sulphate, were incorporated into CI gel and HMC proliferation was assessed. Although attachment of HMC to each gel did not differ significantly, HMC proliferation was inhibited markedly on gels containing type III collagen, heparin and heparan sulphate; type IV collagen suppressed HMC proliferation slightly; and laminin had no significant effect. These data suggest that interstitial type I and III collagens, which are often observed in diseased glomeruli, as well as the basement membrane components, may play important roles in the regulation of HMC proliferation under pathophysiological conditions in vivo. We conclude that HMC behaviour is affected by the surrounding ECM constituents, which appear to function as a refined modulator.

The effect of chlorpromazine, one of several calmodulin antagonists that inhibit cytoskeletal movement, on the local kinetics of injected proteinuria-inducing MoAb 5-1-6 was examined to test the hypothesis that proteinuria is inhibited if the antigen recognized by MoAb 5-1-6 or injected MoAb remains on the surface of epithelial foot processes. MoAb 5-1-6 was injected into both chlorpromazine-treated (5 mg/100 g body weight) and untreated rats. As a positive control for the chlorpromazine treatment, anti-Fx1A serum was also injected into other chlorpromazine-treated and untreated rats. Chlorpromazine inhibited neither the change in localization of injected MoAb 5-1-6 nor proteinuria, although it showed an inhibitory effect on redistribution of immune complex and the fixation of complement in passive Heymann glomerulonephritis induced by injection of anti-Fx1A serum. We conclude that the kinetics of bound MoAb 5-1-6 are regulated by a system different from that operating in passive Heymann glomerulonephritis.

By immunization with rabbit immunoglobulins and the injection of a subnephritogenic dose of rabbit nephrotoxic serum (NTS), accelerated-type nephrotoxic serum nephritis (NTN) was induced in heterozygous (rnu/+) rats but not in athymic nude (rnu/rnu) rats. By transferring rat antibody against rabbit immunoglobulins, marked proteinuria was induced also in nude rats (202.0 +/- 98.4 mg/day on day 3) as in rnu/+ rats (122.6 +/- 35.3 mg/day on day 3). No marked differences in histological findings could be found between both groups. The most marked increase in the number of intraglomerular infiltrating cells was observed in heterozygous rats indicating that the presence of thymus-derived cells leads to the accumulation of more cells in glomeruli. We conclude that humoral immunity alone is enough to accelerate the pathogenic mechanism which induces glomerular injury with heavy proteinuria in this model.

Irreversible mesangial changes with persistent proteinuria were induced in rats given two consecutive injections 2 weeks apart of a MoAb 1-22-3 to rat mesangial cell. The characteristics of the resulting lesions were investigated and compared with those of the reversible change induced by a single injection. At 24 h after the second injection, mesangiolytic changes similar to those after a single injection were evident. The accumulation of macrophage-like cells in glomeruli observed at 1 week after the first injection was not evident during the experimental period after the second injection. Hypercellularity with the characteristics of intrinsic mesangial cell and increased mesangial matrix were already present 1 week after the second injection. And mesangial sclerotic change progressed up to 6 months. Deposition of collagen type I and type III and accumulation of collagen fibril at the ultrastructural level were evident in rats 6 months after the second injection. Proteinuria started immediately and continued for more than 6 months after the second injection. The mesangial sclerotic change with persistent proteinuria described here is considered to be a better model for investigating the mechanism of chronic progression of human mesangial proliferative glomerulonephritis.

A MoAb 1-22-3 (IgG3) was produced in mice immunized with rat glomeruli. A blotting study indicated the antigen molecule recognized by this MoAb has a molecular weight of about 25 kD, which is the same as that of the Thy 1.1 molecule. This MoAb is capable of inducing the morphological changes similar to those induced by anti-thymocyte serum or the anti-Thy 1.1 MoAb, ER4 and massive proteinuria in rats by a single i.v. injection. Proteinuria started immediately after MoAb 1-22-3 injection and peaked on day 5. Reaction products were detected by immunoelectron microscopy in vitro on the limited mesangial cell surface facing endothelial cells and in vivo in partially lysed mesangial cells 30 min after injection. Unlike the proteinuria-inducing MoAb ER4, reactivity of MoAb 1-22-3 was detected neither on endothelial cells, epithelial cells, nor along the glomerular basement membrane in vivo and in vitro. There is a difference in reactivity toward guinea-pig and rabbit materials between MoAb 1-22-3 and the commercial anti-Thy 1.1 MoAb (OX7). The antigenic determinant of MoAb 1-22-3 is concluded to be a new epitope and only the binding of MoAb 1-22-3 to this epitope proved to lead to an abnormal increase of glomerular capillary wall permeability.

Murine monoclonal antibody (MoAb) 5-1-6 was already reported to bind to epithelial cell foot processes and to cause proteinuria in rats. In vivo kinetics of the injected MoAb 5-1-6, relationship between the quantity of kidney-binding antibody and proteinuria, and changes in the amount of antigenic molecule recognized by this MoAb in the proteinuric state were studied. The amount of total kidney-binding antibody (TKAb) as determined 1 h after a 2 mg administration was 50.8 +/- 10.4 micrograms/2 kidneys, and TKAb declined to 1.9 +/- 0.4 at day 15. The minimum dose of MoAb required to induce proteinuria was 125 micrograms as the injected dose. This dose corresponded to 12.8 micrograms of TKAb at 1 h and 0.34 micrograms of TKAb at day 5. The amount of MoAb 5-1-6 binding to isolated normal glomeruli was also shown to exceed 147.7 micrograms/76,000 glomeruli, indicating proteinuria to be induced provided more than 8.7% (= 12.8/147.7) of the critical epitopes is specifically occupied by MoAb. The total amount of MoAb 5-1-6 bound to glomeruli in vivo and in vitro was assayed with [125I]-labelled anti-mouse IgG. The amount of [125I] anti-mouse IgG bound to glomeruli was 6.93 +/- 0.45 micrograms/10,000 glomeruli from rat 5 days after this MoAb injection and 26.58 +/- 0.66 micrograms/10,000 control glomeruli, indicating the decrease in the number of MoAb 5-1-6-recognized antigen molecules in glomeruli isolated from the rat in proteinuric state induced by this MoAb. Thus, the MoAb 5-1-6-recognized molecule itself may principally function to regulate the permeability of the glomerular capillary wall and the decrease of the molecule may lead to proteinuria.

A description is made of renal lesions in rats induced by heterologous (rabbit) nephrotoxic serum with or without subsequent host immune reactions against it and the effects of immune reactions on the course of classical nephrotoxic serum (Masugi) nephritis are discussed. The disease was induced by injecting congenitally athymic ACI nude rats (rnu/rnu) and their normal heterozygous littermates (rnu/+) with rabbit anti-rat glomerular basement membrane (GBM) antiserum. In the autologous phase, rat IgG and immunoglobulins were localized in a linear pattern along capillary walls only in nephritic heterozygous rats. In the indirect plaque-forming cell (PFC) assay against rabbit immunoglobulins in the autologous phase, significantly more PFC could be detected in nephritic heterozygous rats than in nephritic nude rats. The nude and heterozygous rats were essentially the same with respect to the amount of urinary protein, histological change and clinical course. At least in classical nephrotoxic serum nephritis in rats, host immune reactions against GBM bound heterologous nephrotoxic serum were concluded to have no effect on the course of the disease.

The present study was conducted to determine the manner in which monocytes increase mesangial matrices, particularly glycosaminoglycans (GAGs) which interact with various other matrix components such as collagens, laminin, fibronectin and lipoproteins. A supernatant of human peripheral blood monocyte cultures activated by lipopolysaccharide (LPS) contains stimulating factors for glycosaminoglycan synthesis in rat mesangial cells (MCs). The culture supernatant in this study was concentrated and fractionated by gel chromatography and the GAG-stimulatory factor was found to have a molecular weight of 10-17 kD. This factor was shown to be present in fractions different from that of IL-1. Gel and ion-exchange chromatography studies of GAGs synthesized by MCs indicated the elution patterns of GAGs in the presence and absence of the monocyte culture supernatant to be essentially the same. Local infiltration of monocytes into the glomerulus, often seen in various types of glomerular injury, may be an important factor in the accumulation of the mesangial matrix.

Conditioned media from human peripheral blood leucocytes treated with lipopolysaccharide (LPS) induced a marked increase in the 3H-thymidine incorporation of cultured mesangial cells at low serum concentration (four to six times higher than control). Two sizes (100-70 and 8-12 kD) of monocyte-derived mesangial cell proliferating factors (MDF) were separated by column chromatography. Their peaks were distinct from those of thymocyte proliferating activity. The addition of anti-human interleukin-1 (IL-1) or anti-recombinant human interleukin-6 (IL-6) antibody to the fractionated MDF failed to have any effect on the mitogenic activity toward mesangial cells. The addition of anti-human platelet-derived growth factor (PDGF) antibody to the low molecular weight fraction decreased mesangial cell mitogenic activity (40-60% of control), but addition to the higher fraction did not (80-100% of control). From these data it seems that a large portion of the monocyte-derived mesangial cell growth factor was not comprised of IL-1 or IL-6 but of PDGF-like molecules; and that there is an unknown mesangial cell proliferating factor (or factors) besides IL-1, IL-6 and PDGF.

Experimental immune complex glomerulonephritis was induced with human IgG (HIgG) in two strains of rats, Wistar-King-Aptekman (WKA) and spontaneously hypertensive rats (SHR). The rats were pre-immunized s.c. with 1 mg of HIgG in Freund's complete adjuvant, and 8 weeks later were given a daily i.v. 2 mg of HIgG injections for 4 weeks. Renal tissue, obtained at weekly intervals after the beginning of i.v. injections, was examined by light, immunofluorescence and electron microscopic tests. SHR developed an endocapillary proliferative glomerulonephritis with heavy proteinuria after the administration of HIgG for 4 weeks. They had massive depositions of HIgG, rat IgG, and rat C3 both in the mesangial area and along the capillary wall. On the other hand, in WKA rats, the proliferative lesions were scarcely seen and the immune deposits were observed almost exclusively in the mesangium. Moreover, urinary protein excretion of these rats was within normal range. In comparison with bovine serum albumin (BSA) nephritis in SHR, HIgG-induced glomerular lesion was relatively mild. This difference seemed attributable to the nature of the HIgG, such as its molecular weight and immunogenicity.

IgM and IgG class rheumatoid factor (RF) containing glomerular immune complex (IC) deposits were detected in renal biopsy material from 10 out of 123 patients with various forms of glomerulonephritis (GN) by an immunofluorescent assay using FITC conjugated aggregated human IgG(FAHIgG). In lupus GN, six out of 28 cases (23.5%), showed such activity. Solubilization of glomerular IC deposits in frozen sections of lupus GN was observed after a 5 h incubation with native 7S IgG. The binding of FAHIgG to glomerular IC deposits and solubilization of deposits by excess HIgG were also confirmed in sections of two types of rat experimental GN in which HIgG acted as exogenous antigen; in situ IC formation type GN in cationized HIgG planted kidneys and chronic serum sickness type GN. These results suggest a possible antigen excess mechanism contributing to the solubilization of glomerular IC deposits via RF and IgG interactions in lupus GN.

In vivo studies in rats demonstrated that the binding of a highly cationic antigen (cationized human IgG, pI greater than 9.5) to glomerular polyanion could be significantly reduced by prior application of a small polycation, protamine sulfate. The degree of inhibition was dose dependent and the highest dose used, 4 mg/100 g body weight, reduced antigen binding by approximately 70%. In further experiments the ability of protamine sulfate to enhance elimination of cationic antigen-antibody immune complexes from the glomerular capillary wall was examined. Daily treatment with protamine sulfate, starting after induction of nephritis, produced a significant but only moderate reduction in the persistence of the antigen, without having any effect on proteinuria. Proteinuria could only be prevented when protamine sulfate was given immediately before induction of nephritis. Protamine sulfate had little influence on the course of established renal disease in the model employed. These results do not substantiate the concept of charge competition as a potentially useful therapeutic strategy.

We have developed hybridoma cell lines, each of which secretes a monoclonal antibody (MoAb) to rat renal and hepatic tissue antigens, from Lewis rat with Heymann's nephritis. Three antibodies bind to the brush border of proximal tubular epithelium (BB), one in a fine net-like pattern (no. 3-11), another one in a coarse granular pattern (no. 1-8) and the third one in a typical pattern (no. 3-9). Three antibodies bind to glomerulus in characteristic patterns but not to BB. After repeated intravenous injections of MoAb (no. 3-11), granular mesangial deposits of rat IgG were observed and of MoAb (no. 1-8), fine granular deposition along capillary walls. These monoclonal autoantibodies should be of value in research on the mechanism of autoimmune membranous nephropathy.

Viral and bacterial infections may produce abnormalities in carbohydrate metabolism in normal subjects and profound changes in glucose homeostasis in insulin-dependent diabetics. Using an RIA with [125]porcine insulin, the effect of rat interferon (IFN) on the production/secretion of immunoreactive insulin (IRI) in cultured cells was investigated. We found that, after incubation of rat insulinoma cells with rat IFN, a decrease in the production of IRI was observed. The decrease correlated with the concentration of IFN and the length of the incubation. IFN also caused a decrease in the production/secretion of IRI from rat pancreatic beta-cells in primary culture. This IFN has all of the properties of IFN including species specificity, and the insulin inhibitory effect cannot be dissociated from the antiviral activity of IFN. These data suggest that one possible explanation for abnormalities in glucose metabolism associated with viral or bacterial infections is that virus-or bacteria-induced production of IFN may alter the concentration of insulin.

The role of epitope density in cationic antigen was investigated in an active model of in situ immune complex glomerulonephritis (ICGN) using the hapten-carrier system. Trinitrophenol (TNP) was conjugated with variable density to cationic human immunoglobulin (C-HIgG) to yield TNP6.2-C-HIgG (low-valency antigen) and TNP31.3-C-HIgG (high-valency antigen). In rats preimmunized with TNP17.3-bovine serum albumin (BSA), endocapillary proliferative GN with proteinuria developed in rats receiving high-valency antigen. In contrast, no significant abnormalities in renal histology or urinalysis were observed when a low-valency antigen was injected. These results indicate that glomerular injury produced by hapten-specific immune reaction is affected by the number of haptenic groups conjugated to the carrier molecule (epitope density) in active in situ ICGN.

A new model of experimental glomerulonephritis (GN) in which a cell-mediated (delayed-type) reaction predominated was established. Cationized TNP-BSA conjugates were planted in the kidney of rats by perfusion via the renal artery. Rats which had been sensitized with the TNP hapten 7 days previously revealed marked exudative and proliferative changes in their glomeruli, accompanied by transient proteinuria. A cellular influx was seen without deposition of any autologous antibody. The resulting data are compatible with the idea that cell-mediated immune reactions can participate in tissue damage in glomerular disease.

Immunization of rats with Engelbreth-Holm-Swarm tumour induced a nephropathy immunohistopathologically similar to Heymann nephritis except that IgG was deposited along the tubular basement membranes. Autoantibodies against both brush border and basement membranes of proximal tubules were found in the sera. Immunoperoxidase staining of the tumour cells and absorption studies suggested that the tumour cells produced the FX1A-related antigens. Immunodiffusion analysis suggested that the antigens which induced this nephropathy differed from gp 330 and gp 600 which have been reported to be responsible for Heymann nephritis.

A close relationship was assumed between the developments of nephropathy and thymoma in the previous study, in which the effect of introduction of the rat nude gene was studied in high thymoma BUF/Mna rats. In this paper, the effect of neonatal thymectomy on the development of nephropathy was examined to clarify the relationship between these two lesions in BUF/Mna rats. The average amount of urinary protein excreted from sham-operated and thymectomized BUF/Mna rats was 30.8 +/- 17.1 and 40.0 +/- 20.0 mg/day, respectively, and the number of affected glomeruli per 100 glomeruli 4.9 +/- 1.0 and 5.3 +/- 2.0, respectively. There were no significant differences in the urinary protein content, the number of the affected glomeruli, and immunofluorescence findings. In a control group, ACI/NMs rats exhibited 8.9 +/- 2.6 mg/day protein in urine and 0.8 +/- 0.4% affected glomeruli, which were significantly different from that of sham-operated and thymectomized BUF/Mna rats. These results indicate that nephropathy in BUF/Mna rats results neither from thymoma itself nor from the immunological abnormality secondary to it, and suggest that this lesion might be ascribed to a genetic factors.

Insulin radioreceptor assays were developed using splenic leukocytes and peripheral erythrocytes from individual mice. Splenic leukocytes were prepared using an NH4Cl buffer which did not alter insulin binding, but gave much higher yields than density gradient methods. Mouse erythrocytes were isolated from heparinized blood by three passages over a Boyum gradient, and a similar buffer was used to separate cells from free [125I]iodoinsulin at the end of the binding incubation. Insulin binding to both splenic leukocytes and peripheral erythrocytes had typical pH, temperature, and time dependencies, and increased linearly with an increased number of cells. Optimal conditions for the splenic leukocytes (6 X 10(7)/ml) consisted of incubation with [125I]iodoinsulin at 15 C for 2 h in Hepes buffer, pH 8.0. In cells from 20 individual mice, the specific [125I]iodoinsulin binding was 2.6 +/- 0.1% (SEM), and nonspecific binding was 0.3 +/- 0.04% (10.6% of total binding). Erythrocytes (2.8 X 10(9)/ml) were incubated with [125I]iodoinsulin at 15 C for 2 h in Hepes buffer, pH 8.2. In cells from 25 individual mice, the specific [125I]iodoinsulin binding was 4.5 +/- 0.2%, and nonspecific binding was 0.7 +/- 0.03% (13.6% of total binding). In both splenic leukocytes and peripheral erythrocytes, analysis of equilibrium binding data produced curvilinear Scatchard plots with approximately 3500 binding sites/leukocyte and 20 binding sites/erythrocyte. These data demonstrate that adequate numbers of splenic leukocytes and peripheral erythrocytes can be obtained from individual mice to study insulin binding in a precise and reproducible manner. This should facilitate direct comparisons of these cells with classical target cells in a variety of mouse model diseases.