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We studied the relative role of gp70-antigp70 complexes in the pathogenesis of murine lupus nephritis, and we used as an index of its contribution, the association between attenuation, or progression, of renal disease and decrease or increase in concentration of gp70 complexes and IgG in the plasma and renal eluate of NZB/W mice. The arrest or attenuation of nephritis, by immunosuppression, was associated with decreased concentrations in the plasma and renal eluates of IgG and gp70 complexes, and the relative concentration of these reagents in the eluates better reflected the extent of renal disease than did their concentration in the plasma. Neither the relative concentration of Clq-reactive materials, nor the quantity of antiDNA antibodies in the plasma, distinguished the mice whose nephritis was arrested by immunosuppression from those with terminal renal failure. The findings further support that the gp70-antigp70 system plays an important role in the pathogenesis of murine lupus nephritis.

Endothelial prolifertion was studied in sites of acute inflammation induced by necrotizing (60 C for 20 seconds) or mild (54 C for 20 seconds) thermal injury to the skin of rsts. DNA synthesis in endothelial cells was assayed 6 hours to 10 days following injury by quantitation of the (3)H-thymidine labeling indices on 2-mu Epon section autoradiographs. In lesions induced at 60 C for 20 seconds, increase in DNA synthesis in small vessels around the necrotic tissue began at 1 day and became significant at 2 and 3 days (10 to 12% for endothelial cells, 9% for perivascular cells). This increased endothelial replication resulted in the formation of new blood vessels by 5 to 7 days. Endothelial labeling diminished progressively after 3 days, as the epidermis regenerated. Foci completely covered by new epidermis consistently showed lower labeling indices than those which were not reepithelialized. Mild thermal injury (54 C for 20 seconds) also resulted in significant increases in endothelial labeling (6%), but the labeling was present mainly in superficial vessels and was not followed by neovascularization. The findings with mild injury are consistent with data that vascular leakage from superficial vessels is due to direct, albeit delayed, endothelial damage. Electron microscopic studies confirmed labeling in endothelial cells and indicated that ultrastructural alterations that were previously ascribed to activation, recovery, or regenerative transformation of endothelium represent, in the main, endothelial proliferation.

Glomerular epithelial slit alterations and their relation to proteinuria have not been studied in detail in New Zealand Black/White (NZB/W) mice. The kidneys of proteinuric and nonproteinuric female NZB/W mice and normal Swiss albino mice were perfusion-fixed with tannic acid-glutaraldehyde and studied by light and electron microscopy. Semiquantitative studies were performed on full montages of glomeruli enlarged 10,000 times. Fine structural alterations of the epithelial slits, with emphasis on the slit diaphragm, were studied on semiserial thin sections. Proteinuric NZB/W mice with features of membraneous nephropathy exhibited: (1) wedging of electron-dense deposits below the slit diaphragm, (2) enlargement and distortion of interpedicel spaces, (3) displacement, folding, and stacking of slit diaphragms, (4) formation of occluding junctional complexes in residual slits, and (5) variable loss of foot processes. Similar alterations were not observed in controls or nonproteinuric NZB/W mice, including animals having complexes inglomerular mesangia but not in epithlialslits. These studies show that in NZB/W mice, abnormal protein excretion is associated with structural modification of the slit pore and suggest a role for such a component in the process of protein ex

We studied the relative role of anti-DNA antibodies in pathogenesis of murine lupus nephritis, and we used, as index of their contribution, the association between attenuation, or progression, of renal disease and decrease or increase in concentration of anti-DNA antibodies in the plasma and renal eluate of NZB/W mice. The concentration of anti-DNA antibodies in plasma did not discriminate mice with incipient or drug attenuated nephritis, who had normal renal function, from mice with progressive nephritis, who developed renal failure. Although the quantity of IgG eluted from kidneys reflected the extent of renal disease (mice with progressive nephritis greater than mice with attenuated nephritis greater than mice with incipient nephritis), the anti-DNA activity of such antibodies was negligible. The low anti-DNA activity could not be attributed to either excess DNA or DNAase in renal eluates. In fact, the eluates contained a factor that inhibited the interaction between anti-DNA antibody and DNA. The results indicate that immune complex systems other than, or in addition to, DNA-anti-DNA are likely to play a role in the pathogenesis of murine lupus nephritis.

Cytopathic effects of cytomegalovirus infection were studied in human cell cultures at various time intervals. Cells derived from human embryonic thyroid, skin-muscle, and lung were infected with five different strains of cytomegalovirus at multiplicities of infection of approximately 5 plaque forming units per cell. Under these conditions, cell rounding and early cytoplasmic inclusions were first apparent at 5 hours postinfection, whereas nuclear inclusions were first observed as a homogenous eosinophilic bead at 24 hours postinfection. Cytoplasmic and nuclear inclusions underwent extensive morphogenesis through 96 to 120 hours postinfection. Development of nuclear inclusions included the formation of distinctive beadlike subunits, which increased in size from their first appearance at 48 to 72 hours postinfection and underwent apparent contraction and breakup after 96 hours postinfection. While the cytopathology induced by various cytomegalovirus strains studied was generally similar, the kinetics of their development was different and independent of both the multiplicities of infection and the source of the fibroblastic cells. Such cytomegalovirus strain-associated differences in cytopathology could result from variances in biologic characteristics of the strains studied.

Detailed immunopathologic studies of early or silent renal alterations in systemic lupus erythematosus have been sparse. The renal biopsies of 16 lupus patients with normal renal function, including 8 with hematuria and/or proteinuria of recent onset, and 8 without clinically detectable renal disease were investigated by light, immunofluorescence, and electron microscopy. Immunoglobulins, complement components, and electron-dense deposits were detected in glomeruli of all patients, regardless of morphologic appearance or lack of clinical evidence of renal involvement. Features of membranous glomerulonepritis were observed in 4 patients with substantial proteinuria. In the remaining 12 patients, including 3 with hematuria and 4 with slight proteinuria, either minimal glomerular alterations or features of mesangial proliferative glomerulonephritis were seen. Transformation of the original disease was demonstrated in 3 of 3 patients rebiopsied within 2 years. The significance of these findings is discussed in relation to a) the spectrum of clinical and immunopathologic alterations in lupus nephritis and b) transformation of the original disease.

To study surface charge characteristics of small blood vessels and perivascular components in vivo, rat cremaster vessels exposed to serotonin or mild thermal injury were labelled with systemically injected cationized ferritin and studied by electron microscopy. Leaky vessels showed increased density of anionic sites on the luminal endothelial plasma membrane, compared to controls. Binding of cationized ferritin and the increased density of anionic sites in leaky vessels occurred in the absence of serum factors; albumin diminished both phenomena. Anionic sites were also demonstrated a) on the surface membranes of open interendothelial junctions, b) on the attachment surface of endothelial cells, c) along the vascular basal lamina, perimysial membrane, and interstitial collagen. The biological significance of these findings is considered in relation to ligand-anionic sites interaction, inflammation, vascular permeability, and thrombosis.

We investigated the effects of methylprednisolone on immunoreactants of plasma and kidney to determine factors that might be relevant to the arrest of murine lupus nephritis. At the onset of nephritis, at about 5 months of age, the mice were divided in two groups and received either methylprednisolone or saline injections for 12 weeks. Before and after therapy (or saline injections), we determined the concentrations of plasma IgG, complement (C3), anti-DNA antibodies, Clq-reactive materials, creatinine, and urea nitrogen; in the kidneys, we assessed the relative distribution of IgG, IgM, and C3 in glomeruli, and we determined the concentration of IgG and anti-DNA activity of the eluted proteins. Our results indicated that methylprednisolone administered at the onset of nephritis preserved glomerular structure and function by decreasing the amount of tissue-bound immunoreactants and by inducing a preferential localization of immunoreactants in mesangia. Of the immunoreactants studied in plasma, a decreased concentration of IgG, but not the concentrations of anti-DNA antibodies, C3, and Clq-reactive materials, was associated with the arrest of nephritis. The anti-DNA activity in the renal eluates was very low and comparable in treated and untreated mice. Immune complex systems other than, or in addition to, DNA-anti-DNA likely play a role in the pathogenesis of murine lupus nephritis.

Behçet's syndrome is a complex multisystem disease that, due to the absence of a pathognomonic laboratory test, must be diagnosed using clinical criteria. Clinical pathergy testing, the induction of a sterile pustule 24 hours after cutaneous trauma, has been proposed as a useful adjunct to diagnosis. We have expanded this concept by showing the usefulness of examining pathergy lesions by routine and immunofluorescence microscopy in the diagnosis of nine patients with Behçet's syndrome. Furthermore, histopathologic pathergy assessments correlated with clinical disease activity and/or response to experimental oral thalidomide therapy in five of six patients with Behçet's syndrome who were retested.

We investigated the effects of methylprednisolone on the glomerular permeability of mice with lupus nephritis. At the onset of nephritis, the mice were divided into two groups: one group was injected with methylprednisolone and the other with saline; treatment continued for 12 weeks. We determined the protein concentration in specimens of urine collected every 2 weeks, and pre- and posttherapy urine samples were analyzed by electrophoresis. After 12 weeks, we injected anionic or cationized ferritin systemically into mice of each group and studied kidney samples by electron microscopy. At the onset of nephritis (5 months of age), proteinuria (5.2 mg/day) was selective and albumin was the predominant (87%) protein excreted. Electron-dense deposits were then confined to mesangial areas. In untreated mice, protein excretion values doubled at 5.5 months of age, tripled at 6 months of age, and increased 5-, 7-, 9-, and 11-fold at subsequent intervals of 2 weeks, to reach 55.0 mg/day at 8 months of age. Proteinuria was poorly selective, and protein excretion values correlated inversely with survival rate (35% at the end of study). The glomerular basement membranes were studded with electron-dense deposits and were depleted in anionic sites, as judged by decreased binding of cationized ferritin molecules. The anionic ferritin molecules permeated the basement membrane at the vicinity of electron-dense deposits and reached the urinary space through residual slit pores. In methylprednisolone-treated mice, by contrast, protein excretion values were low, proteinuria was selective, and remained virtually unchanged at 5.3 mg/day by 8 months of age. There were no deaths in this group. Most of the electron-dense deposits were present in mesangia. Anionic sites of the glomerular basement membranes were largely preserved and anionic ferritin molecules were mostly limited to the luminal aspect of the basement membrane. These studies suggest that methylprednisolone therapy preserved glomerular permeability characteristics by decreasing the localization of immunoreactants in glomeruli and by interfering with factors that favor the localization of immune complexes in the capillary wall.

The pathogenesis of proteinuria associated with immune complex disease is incompletely understood. A quantitative electron-microscopic study was undertaken to determine the relative contribution of lesions in capillary loops and mesangial basement membrane areas and their possible correlations to urinary protein excretion data. Pathologic features including the loss of foot processes (and slit diaphragms), the formation of junctional complexes in visceral epithelium, and the distribution of immune complexes in basement membrane were assessed in glomeruli of mice with lupus nephritis. Swiss albino mice served as control animals. In control animals the distribution of split pores per unit length of basement membrane was approximately 60% higher in capillary loop compared to mesangial basement membrane areas. In mice with lupus nephritis, the reduction in the number of slit pores per unit length of basement membrane to 30% or less of normal, the formation of epithelial junctions, and the relative distribution of immune complexes were not statistically different in capillary versus mesangial basement membrane areas. Animals with murine lupus showed poorly selective proteinuria, but the correlation between features studied and extent of protein excretion was poor. The results of these studies 1) establish the relative distribution of slit pores in mesangial and peripheral loop basement membrane, 2) demonstrate that glomerular changes associated with immune complex deposition are comparable in capillary and mesangial basement membrane areas, and 3) are consistent with a focal and nonuniform alteration in glomerular permeability properties associated with immune complex disease.

Patients with progressive systemic sclerosis (PSS, scleroderma) exhibit a variety of immunologic abnormalities. To verify whether the renal vascular lesions of such patients might be mediated by an immunologic mechanism, kidney tissues of 16 patients with PSS were investigated by means of fluorescence, light, and electron microscopy; elution of tissue-bound antibody; and fixation of heterologous (guinea pig) complement. Controls consisted of 12 nonsclerodermatous patients with similar levels of hypertension with no evidence of associated immunologic abnormalities. Diffuse vascular deposits of immunoglobulins (predominantly IgM) and/or complement (predominantly Clq) were found in all 16 patients with PSS. These deposits were bound to the intima of intralobular and arcuate arteries which, by light microscopy, often exhibited typical fibromucinous alterations. Elution of antibody and heterologous complement fixation studies suggested that such reactants may represent the interaction of complement-fixing antibody and antigen. Electron microscopies studies demonstrated abundant fibrillar and ground substance material in the arterial intima but features of deposited (circulating) immune complexes were not found. By contrast, in the hypertensive (control) group, deposits of immunoglobulin (s) and/or complement were rare and, when present, were mostly confined to the arterioles. As judged by the results of elution and heterologous complement fixation, these arteriolar deposits appeared to represent trapped rather than specifically bound serum proteins. The possible signficance of these findings are discussed in relation to immunologic mechanisms which might be implicated in the pathogenesis of the renal vascular disease of PSS.

Occasional patients with nephrotic syndrome and minimal histologic change demonstrate glomerular deposition of small amounts of immunoglobulin and complement. Some consider this a disease distinct from MCNS. To investigate the clinical importance of immune deposits and mesangial hypercellularity in the initial biopsy, the clinical records, follow-up data, and renal biopsies of 68 patients (ages 6 months to 16 years) with MCNS by light microscopy were reviewed. Among 68 patients followed a mean of 6.2 years, eight of 25 patients with immune deposits on initial renal biopsy were steroid nonresponsive. Only one of 43 patients without immune deposits was steroid nonresponsive (P = 0.00005). Of 44 patients with normal mesangial cellularity, 31 experienced fewer than three relapses a year, whereas of 15 patients with mesangial hypercellularity, only six experienced fewer than three relapses a year (P = 0.035). The data suggest that immune deposits and increased mesangial cellularity in children with NS and minimal light microscopic change may predict the clinical course.

The glomerular lesions induced in 10 chimpanzees infected with variable numbers of Schistosoma japonicum cercariae were studied by means of light and electron microscopy and fluorescent antibody technic. Ten animals served as controls; 5 were uninfected and 5 were only lightly infected. The animals were observed for periods ranging from 3 to 17 months, and by the time of sacrifice, all had developed advanced liver fibrosis. In general, the degree of glomerular injury was related to infection intensity and degree and duration of portal liver fibrosis. Some animals had terminal BUN elevation and slight proteinuria. By light and electron microscopy, in the initial stages, only part of the glomeruli were involved and exhibited mesangial matrix expansion and mesangial cell proliferation with intracellular hyaline droplets. At later stages, a larger number of glomeruli were affected and exhibited diffuse hypercellularity, glomerular basement thickening, mesangial sclerosis and less often, focal necrosis, crescent formation, synechiae and global hyalinization. In addition, there were discrete electron-dense deposits localized in the mesangial area in some glomeruli. Immunofluorescent studies utilizing antisera to chimpanzee gamma-globulin and complement (C3) and to human properdin disclosed only faint deposits of C3, apparently in mesangial areas. The association of hepatosplenic schistosomiasis and nephropathy, the possible role of schistosomal antigen and the mechanism(s) of such glomerular injuries are reviewed and compared with the disease in humans and other host species infected with Schistosoma.

To probe the mechanism of spontaneous formation of anti-DNA antibodies, we studied the isotype distribution of anti-DNA antibodies in the plasma of NZB/W mice with incipient, chronic, and drug-attenuated nephritis. The concentration of anti-DNA antibodies in plasma did not discriminate between the various groups of mice, and the anti-DNA activity in renal eluates was very low and did not reflect the course of nephritis. Progression of incipient to chronic nephritis was associated with increase, and drug attenuation of nephritis with decrease, in plasma concentration of all isotypes tested. Anti-DNA antibodies were detected in all classes (IgG, IgM) of antibodies studied and the anti-DNA antibodies were found to be unrestricted with respect to IgG isotypes and within a given (IgG2a) isotype. The data indicate that the spontaneous immune response to DNA in NZB/W mice reflects activation of several autoreactive clones and that, overall, anti-DNA activity in the plasma or renal eluate is a poor predictor of extent of renal disease.

In a recent report we described a syndrome, identical to bowel-bypass syndrome, that occurred in four patients who had not had bypass surgery. Herein, circulating immune complexes (CICs) and neutrophil migration are evaluated in three of those four patients to test the hypothesis that the cutaneous lesions might have resulted from interaction between immune complex-mediated vessel damage and increased neutrophil migration. In vitro assays indicated that CICs were present in one of two patients and "histamine trap" test evidence for CICs was present in both patients tested. Although serum from the three patients appeared to increase neutrophil movement, statistically significant increases were not observed when data were pooled in this small study group. Preliminary results suggest that immune complex-mediated vessel damage, followed by extensive accumulation of neutrophils, may cause the pustular vasculitis in the bowel-associated dermatosis-arthritis syndrome.

To verify whether an increase in glomerular permeability precedes the deposition of immune complexes in the capillary wall, the following were studied in mice with lupus nephritis: 1) urinary proteins; 2) glomerular transfer of IgG, albumin, and anionic ferritin (AF) (isoelectric point, 4.2-4.6); and 3) anionic groups of the capillary wall as detected by binding of cationized ferritin (CF) (isoelectric point, 7.5-8.6). The glomeruli were investigated by immunofluorescence, immunoelectron, and transmission electron-microscopic studies. Urinary proteins were quantitated and characterized by electrophoresis. When mice were 5 months of age, (the time at which pathologic proteinuria was first detected), immune deposits were few and were confined to the mesangium. Although AF molecules were largely retained at the level of the lamina rara interna, focal leakage of albumin and IgG was detected across capillary walls that were not found to contain immune deposits. Focal and irregular loss of anionic groups, reflected by decreased binding of CF molecules, occurred in the laminae rarae of the basement membrane. Foot processes of glomeruli incubated with CF showed no loss of polyanion. We conclude that the increase in glomerular permeability that precedes deposition of immune complexes is due, in part, to focal loss of anionic groups of the basement membrane.

Pustular vasculitis is a new disease concept that links cutaneous, and possibly systemic, aspects of Behçet's, bowel bypass, bowel-associated dermatosis-arthritis, and disseminated gonorrhea syndromes. The pathomechanism of pustular vasculitic lesion generation may relate to circulating immune complex (CIC)-mediated vessel damage and serum enhancement of neutrophil migration. Thalidomide, an oral pharmaceutical available on strict protocol, has therapeutic effects based on proposed modulation of CIC- and neutrophil-mediated cytotoxicity. Thalidomide therapy was started for four patients with significant morbidity from Behçet's syndrome and for one patient with bowel-associated dermatosis-arthritis syndrome. Clinical benefit was dramatic in all patients who completed sequential four-week "on" and "off" thalidomide therapeutic cycles. In three of four patients, in vivo testing for CIC after histamine injection immunopathology converted from positive (immunoreactant deposition in dermal vasculature [four hours after histamine] and CIC-mediated vasculitis [24 hours after histamine]) to negative during therapy. No effects were noted on neutrophil migration or on the LFA-1/Mac-1/p150,95 family of glycoproteins associated with neutrophil adherence as assessed qualitatively by tritium labelling of neutrophil cell surfaces. In this small patient group, thalidomide was a clinically effective, safe (with rigid monitoring) therapy whose mechanism of action may relate more to inhibitory effects on CIC-induced vasculitis than to effects on neutrophil-mediated cytotoxicity.

We determined the avidity of antiDNA antibodies in plasma of NZB/W mice when nephritis became apparent, when such mice developed chronic glomerulonephritis, and in mice whose nephritis was arrested, or attenuated, by immunosuppression. Antibody avidity was estimated by the rate of dissociation of 125IDNA-antiDNA complexes when excess unlabelled DNA was added to the plasma. In all groups of mice studied, the relative contributions of high and low avidity antiDNA antibodies were comparable and, overall, high avidity antibodies predominated. The results indicate that the avidity of circulating antiDNA antibodies did not reflect the extent and type of glomerular involvement, and was not predictive of clinical course.

A circulating immune complex-mediated pathogenesis for lesions of secondary syphilis has been postulated. Textbook descriptions of a lymphoplasmacytic histopathologic picture have contradicted a role for circulating immune complexes in lesion pathogenesis. Four patients with early cutaneous lesions of secondary syphilis were studied. All four patients had serum Raji cell and/or Clq binding assay evidence for circulating immune complexes. Three patients showed a neutrophilic vascular reaction on histologic study of early lesions. The patients studied had immunofluorescence microscopic evidence of immunoreactant deposition in dermal blood vessels (4 hours) and/or a neutrophilic vascular reaction (24 hours) after intradermal histamine injection. Dieterle staining of lesional tissue from all patients showed the presence of treponemal organisms in dermal blood vessels. This new preliminary evidence adds some support to a circulating immune complex-mediated pathogenesis of cutaneous lesions in human secondary syphilis.

The charge-based barrier of the glomerular capillary filter was investigated in normal mice and in mice with immune complex lupus nephritis. Cationized ferritin (CF), pI 7.7 To 8.5, was used as a molecular probe of fixed anionic sites. Mice with various degrees of proteinuria and severity of glomerulonephritis were systemically injected with CF and their glomeruli studied ultrastructurally employing morphometric methods. A decreased and erratic localization of CF was observed in the lamina rara externa, in the intervening basement membrane between immune deposits, and in basement membrane projections, areas previously shown to be abnormally permeably to a large anionic protein. In small numbers, CF molecules were found in residual epithelial slits and in the urinary space. In normal mice, CF regularly labeled the laminae rarae of the glomerular basement membrane and the slit pore area but not leak into the urinary space. Such differences in CF localization in capillary loops of proteinuric and normal mice were confirmed by morphometric estimate of particle counts. Focal areas of increased permeability to anionic protein are deficient of and/or exhibit a disorderly redistribution of fixed anionic sites.

The nephrotoxic and cytoproliferative side effects observed in a patient with Streptozotocin-treated, multiple hormone-secreting, pancreatic islet cell carcinoma are described. Streptozotocin induced prolonged partial remission of the patient's multiendocrine syndrome but resulted in progressive azotemia, which was controlled by temporary hemodialysis. A renal biopsy, the first to be reported in detail in such a condition, demonstrated a tubulo-interstitial nephritis and a glomerular alteration consisting of cellular nodules. At autopsy there were numerous bilateral renal cortical spindle cell "tumors" and cellular aggregates in glomeruli. These findings suggest that the tumorigenic effects of Streptozotocin demonstrated in animals may also occur in man.

A 26-year-old woman presented with the classic manifestations of malignant atrophic papulosis, a rare disease of unknown cause. We report the results of our immunologic studies, which may help to explain why treatment with systemic immunosuppressant therapy has not been effective.

A body of evidence suggests that in membranoproliferative glomerulonephritis (MPGN), complement is activated by the alternate pathway. Therefore, deposition of early components of complement should not be expected in glomeruli. The renal tissues of 16 patients--13 with classic MPGN and 3 with dense deposit disease, a variant of MPGN--were studied by light and electron microscopy and by means of elution and immunofluorescence for the localization of complement (C1q, C4, and C3), immunoglobulins (1gG, IgM, and 1gA), and other serum proteins. Variable amounts of C3, C4 and/or C1q, and IgM were detected in the glomeruli of all patients, whereas IgG and IgA were present, respectively, in 15 of 16 and 6 of 16 patients. Deposits were localized in mesangium and in peripheral capillary loops in a typical lobular distribution. The specificity of each antiserum was verified by immunodiffusion, immunoelectrophoresis, and blocking experiments utilizing unlabeled antibody. Glomerular-bound IgG was eluted with acid citrate buffer, suggesting that IgG might be complexed with antigen(s) in glomerular deposits. By light microscopy, lesions ranged from focal proliferation and lobulation to more severe involvement with typical splitting of glomerular basement membranes, sclerosis, and less frequently, crescent formation. Ultrastructurally, all patients with classic MPGN exhibited mesangial and subendothelial deposits, and in 5 of these patients, subepithelial deposits were demonstrated. With the exception of ultrastructural lesions, patients with the dense deposit variant lacked distinguishable features when compared with those with classic MPGN. The significance of these findings is discussed in relation to a) activation of complement and the possible role of an immune complex mechanism and b) the variability of the morphologic expression.

Walker ascites tumor cells and an extract derived from such cells (tumor angiogenesis factor, TAF) were injected into the subcutaneous tissue of rats by using a dorsal air sac technique. At intervals thereafter, thymidine-(3)H was injected into the air sac and the tissues were examined by autoradiography and electron microscopy. Autoradiographs of 1micro thick Epon sections showed thymidine-(3)H labeling in endothelial cells of small vessels 1-3 mm from the site of implantation, as early as 6-8 hr after exposure to live tumor cells At this time interval endothelial cells appeared histologically normal. DNA synthesis by endothelium subsequently increased and within 48 hr new blood vessel formation was detected. The presence of thymidine-(3)H-labeled endothelial nuclei, endothelial mitoses, and regenerating-type endothelium was confirmed by electron microscopy. TAF also induced neovascularization and endothelial cell DNA synthesis after 48 hr. A similar response was not evoked in saline controls. Formic acid, which elicited a more intense inflammatory response, was associated with less endothelial labeling and neovascularization at the times studied. Pericytes and other connective tissue cells were also stimulated by live tumor cells and TAF. The mechanism of new blood vessel formation induced by tumors is still unknown but our findings argue against cytoplasmic contact or nonspecific inflammation as prerequisites for tumor angiogenesis.

The early local vasoproliferative response induced by live tumor cells and an extract derived from such cells was studied in rat subcutaneous tissue by means of electron microscopy and ultrastructural autoradiography after local injections of tritium-labeled thymidine. DNA synthesis was localized in endothelial cells, pericytes, and perivascular cells 6 to 8 hours after exposure to 10(6) live Walker ascites tumor cells. At this time, DNA-synthesizing endothelial cells in parent vessels exhibited a continuous basement membrane and could not be readily differentiated, ultrastructurally, from control endothelium. At 48 to 50 hours, the number of labeled cells increased and there was ultrastructural evidence of regenerating endothelium: marked increase in ribosomes and endoplasmic reticulum, scarce or absent pinocytotic vesicles, attenuated or discontinuous basement membrane and marked irregularities in cytoplasmic surfaces. Labeled endothelial cells were present in parent vessels, as well as along newly formed sprouts. Autoradiographic and ultrastructural findings after tumor extract or live tumor cells at 48 hours were similar. Evidence was also presented that cells which were recognizable as pericytes, by ultrastructural criteria and by their localization within the basement membrane, were capable of DNA synthesis and mitosis.