Serial micropuncture analysis of glomerular function in two rat models of glomerular sclerosis.

We have recently developed a micropuncture technique to assess repeatedly function of the same nephrons in chronic renal disease and subsequently examine the morphology of their glomeruli by serial thin-section histological analysis. Using this approach, a potential causal linkage between early functional patterns and late structural abnormalities was examined in glomeruli of two established rat models of glomerular sclerosis. The models are (a) puromycin aminonucleoside (PAN) administration in unilaterally nephrectomized Munich-Wistar rats and (b) adriamycin (ADM) treatment in nonnephrectomized Munich-Wistar rats. Single nephron GFR (SNGFR) and glomerular capillary hydraulic pressure (PGC) were measured repeatedly for 8 (PAN rats) or 31 wk (ADM rats). In all animals studied, values for PGC remained at, or slightly below, levels measured before PAN or ADM administration. SNGFR values declined progressively in all glomeruli in PAN rats. Although some glomeruli in ADM rats had an increase in SNGFR above levels observed in nonnephrectomized control rats, these hyperfiltering glomeruli did not have abnormally high PGC nor did they exhibit glomerular sclerosis at the completion of the study. Histological analysis revealed the existence of a significant inverse correlation between the degree of sclerosis and SNGFR assessed at the time of sacrifice in both PAN and ADM groups. Chronic administration of captopril, an angiotensin I converting enzyme inhibitor, in PAN rats substantially attenuated development of glomerular sclerosis without affecting PGC in earlier stages. The observations in these models indicate that glomerular hyperfiltration and hypertension are not required for the development of glomerular sclerosis in renal diseases, and angiotensin I converting enzyme inhibitor can exert its protective effect independently of its effect on glomerular capillary pressure.

Staphylococci-induced human platelet injury mediated by protein A and immunoglobulin G Fc fragment receptor.

Bloodstream infections with staphylococci are accompanied by thromboembolic complications. We have studied the mechanism of the interaction of staphylococci with human blood platelets. Staphylococci that possess protein A, a bacterial receptor for the Fc fragment of immunoglobulin G (IgG), caused aggregation of human platelets in whole plasma accompanied by release of [(3)H]serotonin. These reactions were time and concentration dependent, requiring two or more staphylococci per platelet to give maximal response within 5 min. The interaction between staphylococci and platelets required the presence of cell wall-bound protein A and of IgG with an intact Fc fragment. It did not require an intact complement system. Cell wall-bound protein A (solid phase) was capable of aggregating human platelets in whole plasma. In contrast, free, solubilized protein A (fluid phase) did not cause measurable aggregation, and release of [(3)H]serotonin was reduced. An excess of free, solubilized protein A blocked aggregation of human platelets induced by staphylococci in whole plasma. The role of the Fc fragment of IgG in the staphylococci-human platelet interaction was demonstrated by an experiment in which free, isolated Fc fragment blocked aggregation of platelets in whole plasma induced by staphylococci. Furthermore, binding of (125)I-protein A to human platelets was demonstrated in the presence of complete IgG with intact Fc fragment but not in the presence of the F(ab)(2) fragment. Binding of the protein A-IgG complex to the human platelet Fc receptor was paralleled by the release of [(3)H]serotonin. These results represent a novel example of the interaction of two phylogenetically different Fc receptors, one on prokaryotic staphylococci and the other on human platelets. Their common ligand, IgG, is amplified by one Fc receptor (protein A) to react with another Fc receptor present on human platelets, which results in membrane-mediated aggregation and release reaction occurring in whole plasma. This mechanism can be of significance in the pathomechanism of thromboembolic complications at the site(s) of intravascular staphylococcal infection.

Purification of human erythroid colony-forming units and demonstration of specific binding of erythropoietin.

Morphological and biochemical studies of human colony-forming units-erythroid (CFU-E) have been hindered by their extreme rarity. Since burst-forming units-erythroid (BFU-E) develop into CFU-E, we used normal human blood BFU-E to generate large numbers of highly purified CFU-E in vitro. Using density centrifugation, sheep erythrocyte rosetting, surface immunoglobulin-positive cell depletion, adherence to plastic, and negative panning with monoclonal antibodies, human blood BFU-E were purified from 0.017 to 0.368%, a 22-fold purification with a 43% yield. The panned cells were cultured in methylcellulose with recombinant erythropoietin (rEp) and conditioned medium for 9 d. These cells were then collected and CFU-E were further purified using adherence and density centrifugation. This yielded almost 10(7) erythroid colony forming cells with a purity of 70 +/- 18%. Analysis of these cells by light and electron microscopy showed 94% erythroid cells. The prominent cell was a primitive blast with high nuclear/cytoplasmic ratio, dispersed nuclear chromatin and a distinct large nucleolus. The relation between the number of erythroid colonies and the number of day 9 cells plated in plasma clots was a straight line through the origin with a maximum number of erythroid colonies at 1 U/ml of rEp and no erythroid colonies without rEp. Specific binding with 125I-rEp showed that 60% of the binding was inhibited by excess pure erythropoietin (Ep), but not by albumin, fetal calf serum, and a variety of growth factors or glycoproteins. By days 12-13 of cell culture, when the progenitor cells matured to late erythroblasts, specific binding markedly declined. In this study, human CFU-E have been isolated in sufficient purity to characterize the morphology of these rare cells and in sufficient numbers to measure specific binding of Ep.

"Intact nephrons" as the primary origin of proteinuria in chronic renal disease. Study in the rat model of subtotal nephrectomy.

Single nephron filtration rate of albumin (SNGFRAlb) was measured in remnant nephrons of Munich-Wistar rats 4-6 wk after subtotal nephrectomy (NPX). Serial thin-section histological analysis was then conducted on the same glomeruli by light microscopy. SNGFRAlb ranged from 1 to 15 times normal. However, a direct relationship between abnormalities of structure and function was not seen, e.g. the glomeruli with the fewest structural abnormalities and marked hyperfiltration often had the highest SNGFRAlb. Moreover, the majority of glomeruli had minimal structural abnormalities. Normalization of the markedly elevated glomerular capillary pressure (PGC) in these glomeruli was accomplished by acute intravenous infusion of verapamil, which decreased SNGFRAlb by 9-83% without affecting the single nephron filtration rate of water (SNGFRH2O). 1-2 wk after subtotal NPX, all glomeruli were hyperfiltering and had elevated PGC. The fractional clearance of larger (greater than 36 A) dextrans was selectively increased in these glomeruli that lacked discernible damage by light microscopy. Verapamil normalized PGC, reduced proteinuria to 48 +/- 4% of baseline, and improved glomerular size selectivity without altering SNGFRH2O. Proteinuria after subtotal NPX thus originates largely from glomeruli with minimal structural abnormalities. The defect in size selectivity is largely attributed to the prevailing high PGC, producing large, nonselective channels on the glomerular capillary wall. The observations raise the possibility that in chronic renal diseases, the reduction in proteinuria often seen after therapeutic measures, including antihypertensive medication, may reflect their functional effect on the relatively intact glomeruli rather than their structure-sparing effect on severely damaged glomeruli, which contribute little to the proteinuria.

Malignant lymphomas of follicular center cell origin in man. I. Immunologic studies.

Lymphomas with histologic features indicating a follicular center cell (FCC) origin were analyzed from 26 patients of a group of 45 consecutive non-Hodgkin's lymphoma patinets whose tumors were studied for B- and T-cell characteristics. They were compared with benign, reactive lymphoid tissue from 14 patients. Cell suspensions from biopsy material, blood, or bone marrow were examined for surface Ig and for rosette formation with sheep erythrocytes (E rosettes). Of the 26 patients with FCC lymphomas, 22 had 40% or more Ig-bearing cells; all patients with FCC lymphoma tissues had 25% or less E rosette-forming cells. Cells from most FCC lymphomas of the cleaved type had surfac IgM; those from several FCC lymphomas had both IgM and IgD. Cells from lymphomas of noncleaved cell type had surface IgG or IgA. Light-chain analysis showed that cells from FCC lymphomas bore a predominant light-chain type, which indicated their monoclonal nature. Neoplastic cells from several FCC lymphomas synthesized the surface Ig which they bore. Reactive tissues usually contained fewer Ig-bearing and more E rosette-forming cells than FCC lymphomas; the Ig-bearing cells, with one exception, had a polyclonal distribution. Correlation of histologic and immunologic observations indicates that most lymphomas identified as FCC in origin by light micorscopic criteria mark as B cells with the use of immunologic techniques and that FCC lymphomas are the most common type of non-Hodgkin's lymphoma.

Malignant lymphomas of follicular center cell origin in man. II. Ultrastructural and cytochemical studies.

Tissues from malignant lymphomas with both nodular and diffuse growth patterns, thought by light microscopy to be composed of cells of follicular center cell (FCC) origin, Were examined by electron microscopy; the tumor cells were similar to lymphoid cells found in reactive follicular centers. Tumor cells from neoplasms thought to be composed of cleaved FCC often had more pronounced nuclear folding than did cleaved FCC of reactive follicles, whereas cells in tumors of noncleaved FCC type were indistinguishable from their presumed counterparts in reactive follicles. Large cell noeplasms, previously classified as "histiocytic" lymphomas were composed of cells with ultrastructural characteristics of transformed lymphocytes; they showed neither ultrastructural nor cytochemical features of mononuclear phagocytes. These findings support the concept that a major group of lymphomas arises from lymphocytes of follicular centers.

Cushing's syndrome with small cell carcinoma of the uterine cervix.

A 28 year old white women was found to have a cervical tumor in the 25th week of pregnancy. Pathologic examination revealed a nonkeratinizing small cell carcinoma. After delivery by cesarean section, pelvic lymph node exploration was carried out, and all 15 nodes were free of tumor. Her condition was staged as II-A, and she was treated with local radiation. Metastatic disease became manifest almost a year later and was histologically similar to her primary disease. A Cushingoid appearance was noticed and plasma cortisol levels were elevated. Twenty-four hour urinary 17-hydroxycorticosteroid (17-OHCS) and 17-ketosteroid (17-KS) levels were elevated and failed to suppress with dexamethasone. Plasma adrenocorticotropin (ACTH) level was elevated. Electron microscopic examination of the tumor tissue revealed neurosecretory granules. Immunoperoxidase stains for ACTH were positive. The patient's course was one of progressive decline and eventual death. A literature review revealed two other cases in which carcinoma of the uterine cervix was considered to be the source of ectopic ACTH. Some small cell carcinomas of the cervix may arise from cells of the APUD series. Small cell carcinoma of the uterine cervix may behave differently from the more commonly encountered keratinizing and large cell nonkeratinizing carcinomas of the cervix and may not respond as well to standard therapy. Ectopic hormone production, production of abnormal peptides or of vasoactive amines may be more common in small cell carcinoma of the cervix than is currently recognized, and these products may be clinically useful as tumor markers.

Aortic intimal sarcoma with embolic metastases.

A 46-year-old woman died from massive bowel infarction. At autopsy, a primary sarcoma was found growing along the intimal surface of the aorta at the level of the celiac axis. Tumor emboli were found in distal aortic branches and most abdominal organs. Immunoperoxidase for Factor VIII and electron microscopy (EM) did not support an endothelial origin. EM showed myofibroblastic differentiation. Review of the literature yields an array of diagnostic histologic terms for these tumors, hampering case comparison. The literature does suggest, however, that the clinical presentation of these rare neoplasms correlates nicely with the location and gross morphology of the lesion. We therefore propose a clinicopathologic classification, categorizing the lesions as intimal (obstructive and nonobstructive) and mural. The former are typically pleomorphic sarcomas and are probably of myofibroblastic origin, whereas the latter are usually leiomyosarcomas or fibrosarcomas that probably originate in the media or adventitia.

Comparison of anaplastic large cell Ki-1 lymphomas and microvillous lymphomas in their immunologic and ultrastructural features.

Anaplastic large cell Ki-1 malignant lymphomas (MLs) resemble microvillous lymphoma in having a pleomorphic infiltrate with a prominent sinus growth pattern. Ultrastructural features of anaplastic large cell Ki-1 MLs and their immunologic relationship to the microvillous MLs have not been thoroughly evaluated. We have studied 23 anaplastic large cell Ki-1 MLs immunologically as well as 14 cases ultrastructurally, and compared them with 7 cases of microvillous MLs. Anaplastic large cell Ki-1 MLs were predominantly T-cell in type (13 cases) with three cases marking as B; in seven cases the immunophenotype was not clearly defined. Six microvillous MLs expressed monotypic cytoplasmic or surface immunoglobin and the remaining case had a probable B-cell phenotype (LN-1+, UCHL1-). All microvillous MLs were Ki-1/Ber-H2 (CD30) negative. Epithelial membrane antigen (EMA) marked most anaplastic large cell Ki-1 MLs, except those of B-cell type, whereas all microvillous MLs were EMA negative. By electron microscopy, both lymphomas had features of transformed lymphocytes although anaplastic large cell Ki-1 MLs generally had more nuclear irregularity and variability from cell to cell. Numerous cytoplasmic processes were present in three anaplastic large cell MLs and in all microvillous MLs. The ultrastructural features of the cytoplasmic projections were not sufficiently distinctive to differentiate these two lymphomas. It is apparent that at least two forms of MLs may have a sinus growth pattern and that these MLs cannot be differentiated by morphology alone. Full characterization requires a battery of immunological markers and ultrastructural studies; even then there is overlap of these MLs. The majority of microvillous MLs, are Ki-1-, EMA-, and have a B-cell phenotype, but a small population (21% in this study) of Ki-1+ MLs have numerous cytoplasmic processes. The biological and clinical significance of cytoplasmic projections in these lymphomas are unknown.

Unusual clear cell tumor of the kidney in infancy.

A large renal tumor in a 7-month-old infant was composed entirely of lobules of clear cells with inactive nuclei. Electron microscopy demonstrated features indicative of origin from renal tubules. The infant remains well 15 months after resection.

Splenic marginal zone lymphoma. A distinct B-cell neoplasm.

The splenic marginal zone is a morphologically and perhaps immunologically distinct B-cell compartment. Lymphomas arising from cells of the splenic marginal zone are rare. Here we describe the morphologic, immunologic, and clinical features of 14 cases. Patient age ranged from 35 to 79 years (median, 68 years) with a male-to-female ratio of 1:1.8. The spleen was uniformly enlarged (median, 1,540 g; range, 388-3,845 g) in all patients, the neoplastic infiltrate had a nodular pattern in three cases, nodular and diffuse in seven cases, and diffuse in four cases. The neoplastic cells had small to medium-sized nuclei with round, oval, or slightly indented contours, small eosinophilic nucleoli, and a moderate amount of pale cytoplasm. Extrasplenic involvement was present in 12 patients. Lymph nodes often had a vaguely nodular pattern and preservation of sinuses; bone marrow was infiltrated focally (seven cases) or diffusely (one case). Five patients had hepatic involvement. Ultrastructurally, neoplastic cells differed from other small B cells and resembled normal marginal zone cells by having long, serpentine rough endoplasmic reticulum profiles. All lymphomas marked as B cells and light chain restriction was demonstrated in 12 cases. Bcl-2 protein expression was present in all cases. Most cases (70%) were negative for DBA.44 (CD72). Plasmacytic differentiation was present in three cases. In conclusion, splenic marginal zone lymphoma is a B-cell neoplasm with distinctive clinical, morphologic, immunologic, and ultrastructural characteristics.

Plasmacytic differentiation in parafollicular (monocytoid) B-cell lymphoma. A study of 12 cases.

Parafollicular (or monocytoid) B-cell lymphoma (PBCL) is a recently described low grade lymphoma. The relationship of parafollicular B cells to other B lymphocytes is not known, but the authors observed plasmacytic differentiation in the initial case of PBCL. In this report 12 cases of PBCL were studied by light microscopy and immunophenotypic analysis, and plasmacytic differentiation was found in four cases. This plasmacytic differentiation and the anatomic relationship of the neoplastic cells to reactive follicular centers suggest a functional relationship between these cell types.

Trabecular carcinoma of the skin: a case report.

A case of trabecular carcinoma of the skin in a 62 year old Caucasian male is reported. The lesions occurred on the face, ear, chest, abdominal wall, and the upper extremity over a period of four years. At the light microscopic level the lesions were confused with metastatic carcinoma, lymphoma, and adult neuroblastoma. Ultrastructural study revealed the presence of not only neurosecretory granules but also premelanosomes in the tumor cells.

Mesangial deposition of type I collagen in human glomerulosclerosis.

The presence of type I collagen in both diffuse and nodular diabetic glomerular lesions has been examined using immunohistochemical and electron microscopic techniques. At the ultrastructural level, banded collagen fibrils were observed in the mesangium in all cases of nodular (Kimmelstiel-Wilson) sclerosis and in 60% of the diffuse sclerotic lesions. Antibodies against type I collagen were localized in the fibrotic interstitium and the mesangium in all cases examined. Staining with type I collagen antibodies occurred in glomeruli with intact Bowman's capsules, and was predominantly localized to areas immediately adjacent to mesangial cells. In cases of focal sclerosis of nondiabetic origin, banded collagen fibrils and staining with anti-type I collagen antibody were observed in all cases in which the segmental lesion was presented in the specimen. The pattern of antibody localization in both the diabetic lesions and focal sclerosis differed from that obtained using anti-type IV (basement membrane) collagen antibodies. These results demonstrate that type I collagen is among the extracellular matrix components that comprise the sclerotic glomerular lesions of both diabetic and nondiabetic origin. Furthermore, the spatial localization of this collagen type suggests mesangial cell origin.

Alveolar soft part sarcoma of the uterus.

Two patients with alveolar soft part sarcoma of the uterus are described. One of the sarcomas was a submucosal nodule of the cervix, and the second was a minuscule, incidentally discovered lesion in the corpus. Both lesions contained periodic acid-Schiff-positive, diastase-resistant cytoplasmic granules, and characteristic membrane-bound crystalline inclusion bodies were demonstrated in the cervical lesion.

The extracellular matrix in "sclerosing" follicular center cell lymphomas: an immunohistochemical and ultrastructural study.

"Sclerosis" is frequently seen in follicular center cell (FCC) lymphomas. The mechanism of its deposition, as well as its composition and significance, are unknown. Several clinical studies have suggested that the course of these lymphomas is more indolent than that of lymphomas of the same histologic type without sclerosis. Nine immunologically characterized cleaved FCC lymphomas with sclerosis and 14 reactive lymph nodes with follicular hyperplasia were investigated by special staining methods, electron microscopy, and immunohistochemical studies with antibodies to types I, III, IV, and V collagen, laminin, and fibronectin. The sclerotic tissue in FCC lymphomas stained uniformly with periodic acid-Schiff (PAS) and Masson's stain, with the patterns ranging from delicate filamentous strands to dense doubly refractile bands. Ultrastructurally, the bands of connective tissue were continuous with the adventitia of vessels and composed of varying amounts of banded collagen (types I and III) admixed with filamentous and flocculent material. In all cases the neoplastic lymphocytes were separated from the extra-cellular matrix by fibroblasts and myofibroblasts with long cell processes. Immunohistochemical studies demonstrated intense staining of sclerotic bands with antibodies to fibronectin and type I collagen and, usually, weaker marking with antibodies to types III and V collagen. No significant staining of sclerotic bands was found with antibodies to type IV collagen or with laminin. Weak pericellular staining for type V collagen was present in eight of nine lymphomas and half of the control lymph nodes. These studies suggest that the increased amounts of extracellular matrix in cleaved FCC lymphomas are produced primarily by fibroblasts and myofibroblasts and represent predominantly fibronectin and types I, III, and V collagen. The composition of the sclerotic areas of FCC lymphomas is similar immunohistochemically to that of the capsule and trabeculae of reactive lymph nodes, which are also intimately associated with fibroblasts and myofibroblasts.

The extracellular matrix in large-cell lymphomas: a histopathologic, immunohistochemical, and ultrastructural study.

We studied the histopathologic, immunohistochemical, and ultrastructural features of the extracellular matrix (ECM) in 20 immunologically defined large-cell lymphomas, including immunoblastic sarcoma of B cells (three patients), peripheral T cell lymphoma (five patients), large non-cleaved follicular center cell (FCC) lymphoma (nine patients), and non-marking large-cell lymphoma (three patients). Immunohistochemical studies were performed with antibodies to laminin, fibronectin, and collagen types I, III, IV, and V. The immunologically defined subgroups demonstrated characteristic differences in ECM in light microscopic appearance, composition, and ultrastructural features. Immunoblastic sarcomas of B cells showed delicate intercellular bands that were apparent only at high power but were distinct in exhibiting focal staining for basement membrane elements (laminin and type IV collagen) in addition to type I collagen and fibronectin. Electron microscopically, no basal lamina were apparent, although the collagen fibers were embedded in a dense matrix not seen in the other lymphomas. All peripheral T cell lymphomas exhibited a packeting pattern of intercellular bands and were distinguished by the frequent presence of intense pericellular staining for type V collagen as well as by focal pericellular staining for types I and III collagen. The latter finding corresponded to extensive areas of direct contact between tumor cells and the ECM by electron microscopy. The large non-cleaved FCC lymphomas and the non-marking large-cell lymphomas demonstrated both diffuse and compartmentalizing arrangements of intercellular bands that frequently coexisted and stained predominantly for fibronectin and types I and III collagen. All groups demonstrated myofibroblasts and fibroblasts partially or completely separating the ECM from tumor cells, suggesting that most of the ECM is part of a reaction to these lymphomas. These studies show more variation in light microscopic appearance, composition, and ultrastructural relationships of the intercellular and pericellular ECM than was apparent in earlier studies of cleaved FCC lymphomas.

Hepatosplenic gammadelta T-cell lymphoma: ultrastructural, immunophenotypic, and functional evidence for cytotoxic T lymphocyte differentiation.

Hepatosplenic gammadelta T cell lymphoma (TCL) is a rare, aggressive subset of peripheral TCL that presents with hepatosplenomegaly and cytopenias. Detailed clinicopathological, ultrastructural, and cytogenetic analyses of these lymphomas are limited; functional characteristics of these lymphomas are unknown. We have undertaken a clinicopathological, immunophenotypic, ultrastructural, cytogenetic, and functional analysis of three hepatosplenic gammadelta TCLs. All patients presented with massive hepatosplenomegaly and anemia, thrombocytopenia, or severe neutropenia; terminal blastlike transformation occurred in one patient. Combination chemotherapy had no response in two patients, but induced complete remission in one. gammadelta T cell receptor (TCR) expression and clonal TCRdelta gene rearrangements were documented in each case. Two different subsets of gammadelta TCL were identified based on delta chain variable region usage; two lymphomas were Vdelta1+, whereas the third was negative for both Vdelta1 and Vdelta2. Cytogenetic analysis was performed on two lymphomas; isochromosome 7q and probable trisomy 8 was shown in one of the Vdelta1+ lymphomas, whereas the Vdelta1 negative lymphoma had 14p+ with t(1;14)(q21;p13). NK cell-associated antigens (CD11c, CD16, or CD56) and cytotoxic T lymphocyte (CTL) effector proteins (perforin, granzyme B, TIA-1, and Fas ligand) were expressed by each lymphoma; dense core cytolytic granules were observed by electron microscopy in both lymphomas studied. Functional studies performed in two cases showed TCR-mediated cytolysis of P815 x 2 FcR+ cells induced by anti-CD3 in a redirected cytolysis assay in one of the CD56+, Vdelta1+ lymphomas, whereas IFNgamma secretion was induced by anti-CD3 in the CD56-, Vdelta1 negative lymphoma. These studies show that hepatosplenic gammadelta TCLs have CTL differentiation, retain functional activity in vitro, and are derived from at least two gammadelta T cell subsets.

Melanotic neuroectodermal tumor of infancy. An ophthalmic appearance.

Fullness developed in the left side of a 5-month-old male infant's face in the region of the zygoma. An incisional biopsy specimen showed the mass to be a melanotic neuroectodermal tumor, and radical excision was performed. There has been no recurrence of the tumor one year later. Tumors of this type occur in the face, particularly in the maxilla, and have only rarely been reported around the orbit.

Results of multiparameter studies of T-cell lymphoid neoplasms.

By application of combined structural and functional analyses, most lymphoid neoplasms may be categorized as of T- or B-cell origin. T lymphocyte neoplasms include types of acute and chronic lymphocytic leukemias, certain cutaneous and node-based lymphomas, and lymphomas of thymocytes (convoluted lymphocytic lymphomas). Although much less frequent than B-cell neoplasms, these T-cell neoplasms are important because their recognition has therapeutic and prognostic significance. Relatively specific histopathologic, histochemical, and immunologic criteria have been defined for each neoplasm. These neoplasms are also significant because homogeneous populations of T neoplastic cells have been used successfully in a few cases to study the normal biology of the immune system.