Experimental carcinoma of liver in macaque monkeys exposed to diethylnitrosamine and hepatitis B virus.

The effects of treatments with diethylnitrosamine (DENA) and hepatitis B virus (HBV) on macaque monkeys were investigated by virus serology and by light and electron microscopy. The experimental groups comprised 43 newborn or juvenile cynomolgus and rhesus monkeys of both sexes. HBV neither had a carcinogenic effect nor increased the oncogenic effect of DENA. However, HBV given to juvenile primates before treatment with DENA resulted in subsequent gross and microscopic alterations consistent with mild hepatitis and postnecrotic cirrhosis; multifocal liver carcinoma apparently developed within these cirrhotic nodules. The pathologic findings in the experimental animals were strikingly similar to those observed in liver cancer patients.

Silver staining of nucleolar granules in tumor cells.

With the aid of a simple silver-staining procedure, large numbers and unusual arrays of nucleolar argyrophilic granules were found in Novikoff hepatoma, KB, and HeLa cells. Some of these arrays consisted of linearly arranged discrete granules, and others were in two to three rows each containing three to five granules. Corresponding formations were not found in either the normal or regenerating liver nucleoli which contained an argyrophilic network in which the dark granules were apparently associated with the less dark argyrophilic fibrils of a reticulum. The nucleolar argyrophilic granules were readily identifiable in the separated daughter nuclei of the tumor cells in telophase, suggesting that the increased nucleolar activity of the G1 phase begins in these cells even before cell division has been completed.

Ultrastructural study of pulmonary bleomycin toxicity.

Ultrastructural manifestations of bleomycin A2 toxicity in the human lung were studied in three patients. In addition to the appearance of nucleolar fibrillar centers, an increase in membranous, beaded, and granular nuclear bodies was found in nuclei of type 1, type 2 alveolar epithelial cells, and interstitial fibroblasts in all treated patients. Few such nuclear bodies were found in specimens of untreated patients.

Identification and characterization of a human proliferation-associated nucleolar antigen with a molecular weight of 120,000 expressed in early G1 phase.

Tumor nucleoli were treated with polyclonal antisera to normal human tissue nucleoli to block some determinants common to tumor and normal tissue nucleoli. Immunization of mice with these immune complexes resulted in the development of a monoclonal antibody (FB2) to a novel Mr 120,000 nucleolar proliferation-associated antigen. By indirect immunofluorescence, antibody FB2 produced bright nucleolar staining in a variety of malignant tumors, including cancers of the breast, liver, gastrointestinal tract, genitourinary tract, blood, lymph system, lung, and brain. Although specific nucleolar immunofluorescence was not detectable in most normal tissues, it was detectable in some proliferating nonmalignant tissues including spermatogonia of the testes, ductal regions of hypertrophied prostates, and phytohemagglutinin-stimulated lymphocytes. The Mr 120,000 antigen was not detectable in 48-h serum-deprived HeLa cells but was readily detectable (within 30 min) following serum refeeding. The Mr 120,000 antigen was not detected in retinoic acid-treated HL-60 cells following morphological differentiation but was detectable in 48-h phytohemagglutinin-treated lymphocytes. These studies suggest that the Mr 120,000 antigen is a proliferation-associated antigen which plays a role in the early G1 phase of the cell cycle.

A nucleolar antigen found in a broad range of human malignant tumor specimens.

With rabbit antibodies to nuclear 0.01 M Tris-HCl, pH 8, extract or "nucleolar preparations" of human HeLa S3 cells and fluorescein-labeled goat anti-rabbit antibodies, bright nucleolar immunofluorescence was observed in 61 or 63 human adenocarcinomas, squamous cell carcinomas, sarcomas, hematological neoplasms, and other malignant tumors. With these antibodies, nucleolar immunofluorescence was not found in 23 normal tissue specimens, 10 benign adenomas and hyperplastic tissues, and 8 specimens of inflammatory diseases. In the nontumorous tissues examined, positive nucelolar fluorescence was found in a few sections of a gastric ulcer and chronic ulcerative colitis which have been known propensities for malignant change; these areas may have been undergoing focal malignant changes.

Nuclear and nucleolar ultrastructure of Sézary cells.

Sézary cells were studied in the peripheral blood and characteristic skin lesions of the Sézary syndrome and mycosis fungoides by transmission electron microscopy to obtain more information on their nuclear and nucleolar ultrastructure. Sézary cells contain nucleoli with nucleolonemas or ring-shaped nucleoli similar to those of lymphoblasts and mature lymphocytes. "Maturation asynchrony" of the nucleolus and cytoplasm was evident in some cells that contain large numbers of ribosomes and ring-shaped nucleoli and in other cells that contain nucleoi with nucleolonemas and few ribosomes. The maturation asynchrony of the nucleolus and the cytoplasm, the presence of mitochondrion-like inclusion bodies in the nucleus, and fusion of mitochondria with the nucleus in Sézary cells are ultrastructural abnormalities of this neoplastic lymphocytic variant. The presence of the intranuclear "mitochondrion-like" inclusion body and nuclear rodlets in Sézary cells were exceptional findings.

Identification and partial characterization of a nucleolar antigen with a molecular weight of 145,000 found in a broad range of human cancers.

Previous studies in our laboratory have indicated the presence of nucleolar antigens in tumors which were not detected in normal tissues. Some of the polyclonal antisera produced in these studies were shown to identify a Mr, 145,000 nucleolar antigen on immunoblots of tumor nucleoli but not in normal human liver nucleoli. A monoclonal antibody to a Mr 145,000 nucleolar protein (p145) was produced by immunization of mice with a nucleolar extract of HeLa cells which is enriched with this antigen. The monoclonal antibody showed bright nucleolar immunofluorescence localization in a broad range of human tumors including cancers of the gastrointestinal tract, genitourinary tract, lung, liver, muscle, cartilage, and blood. The p145 nucleolar antigen was not detected in most normal human tissues or in benign tumors, with only weak nucleolar staining observed in spermatogonia of the testes and in ductal regions of some hypertrophied prostates. Nucleolar antigen p145 was extracted from HeLa cell nucleoli by homogenization in a 0.01 M Tris buffer containing 0.2% deoxycholate. On sucrose density gradient centrifugation, the antigen remained sedimented with the nucleolar ribonucleoprotein fraction. Nucleolar antigen p145 was released from ribonucleoproteins following treatment with 4 M guanidinium hydrochloride or RNase. Peptide mapping of nucleolar antigen p145 showed that it was distinct from other known nucleolar antigens. Although it remains to be determined if the p145 antigen plays a role in cell transformation, maintenance of the malignant phenotype, or in cell division, it may have value as a tumor marker or as a therapeutic target.

Detection of a serum DNA-binding protein associated with cancer.

An accompanying report describes the purification and partial characterization of a unique DNA-binding protein (Mr 64,000; pI 5.9) that is present in human sera. This report gives the results of assays of sera from patients for the bleomycin inhibitor protein (BIP) using the Pseudomonas bacteriophage covalently closed circular DNA fluorescence technique standardized for DNA breakage induced by bleomycin. The results of the BIP assays were expressed by values of specific activity of inhibition. One arbitrary unit of inhibitory activity was defined as equivalent to the amount of serum protein required to cause 50% inhibition of DNA degradation using standard conditions of the DNA breakage assay. The mean values of specific activity of inhibition (SAI) for groups of healthy individuals (n = 26), patients with nonmalignant diseases (n = 33), and patients with malignant diseases (n = 83) were 12.60 +/- 4.69 (S.E.), 12.53 +/- 3.17, and 2.40 +/- 0.84 units/mg, respectively. Mean SAI values for patients with cancers of various types were: solid tumors (n = 46), 2.44 +/- 0.86; leukemias (n = 24), 2.59 +/- 0.96; and lymphomas (n = 18), 2.07 +/- 0.64. The decrease in BIP activity was not correlated with sex, age, or prior chemotherapy. Mean SAI values of male (n = 29) and female (n = 59) patients with cancer were 2.61 +/- 0.87 and 2.30 +/- 0.83 units/mg, respectively. Mean SAI values for different age groups were: 0 to 40 years (n = 21), 2.05 +/- 0.68 units/mg; 41 to 70 years (n = 56), 2.59 +/- 0.68 units/mg; and greater than 70 years (n = 11), 2.12 +/- 0.67 units/mg. Cancer patients with and without prior chemotherapy had mean SAI values of 2.97 +/- 0.85 (n = 23) and 2.20 +/- 0.86 units/mg (n = 65), respectively. Linear regression analysis comparing SAI values and serum protein levels showed no correlation (r = 0.21). These results suggest the decrease of the BIP is associated with malignant disease. Additional controlled studies are required before the significance of this association can be adequately assessed.

Viral expressions in Reed-Sternberg cells.

In the murine system natural hybridoma formation was observed first in 1968-9. In the #620 to 818 system a mouse leukemia virus-(MLV-) producer diploid lymphoma cell fused with an immune plasma cell. The tetraploid fusion product cells grew in suspension cultures and as ascites tumors in mice and continued the production of MLV particles and MLV-neutralizing antibodies. Analogy between the #620 to 818 system and the origin of RS cells is proposed. Indirect evidence suggests retroviral infection of the mononuclear HD cell which presumably is an interdigitating reticulum (IR) cell. Reactive B and T cells interact in an abnormal manner and fuse with the retrovirally infected IR cell. The fusion product cells display hyperdiploidy and a disarray of markers as IR markers are lost due to dedifferentiation (and regained upon differentiation induction) and B and/or T cell markers are gained. Conventional theories for the origin of RS cells fail to explain the great heterogeneity of their markers. Derivation of RS cells from IR cells and B and/or T lymphocytes as natural hybridomas offers plausible explanation for all the features of RS cells.

Myeloma-like lesions of the kidney. Occurrence in a case of acinic cell adenocarcinoma of the pancreas.

A 54-year-old man with acinic cell adenocarcinoma of the pancreas died of oliguric renal failure associated with myeloma-like renal lesions. Electron microscopical study of the tumor cells disclosed rich rough-surfaced endoplasmic reticulum and membrane-bound secretory granules, which indicated active protein synthesis and suggested that abnormal proteins produced by the tumor cells were the underlying cause of the renal lesions. Rapid deterioration of renal function ensued after intravenous pyelography, as is usual in the syndrome of myeloma-like lesions of the kidneys. This case presents further evidence for the occurrence of "myeloma kidney" in association with tumors other than plasma cell myeloma.

Membranous nephropathy with epithelial crescents in a patient with pulmonary sarcoidosis.

Renal failure in patients with pulmonary or extrapulmonary sarcoidosis has been attributed to interstitial disease. Reports of cases of primary glomerular abnormality with renal failure in patients with sarcoidosis are rare. We describe a patient with pulmonary sarcoidosis and renal failure due to membranous nephropathy with epithelial crescents. A review of primary glomerular involvement in patients with sarcoidosis and the association of immune complexes in the pathogenesis of the two diseases is discussed.

Thrombotic thrombocytopenic purpura complicating Legionnaires' disease.

A case of Legionella pneumophila infection complicated by thrombotic thrombocytopenic purpura (TTP) was confirmed at autopsy by the demonstration of the organism in lung tissue, and by the finding of widespread intravascular and subendothelial thrombi associated with microinfarctions in all major organs examined. In addition to the typical hematologic abnormalities of TTP, the patient was found to have a low serum C3 level and elevated levels of immune complexes as measured by the liquid phase C1q binding assay. We suggest that the L pneumophilia infection caused endothelial damage and/or platelet aggregation, perhaps as a consequence of complement activation, thus initiating the sequence of events leading to extensive microvascular thromboses.

Effect of plasma exchange on circulating immune complexes and antibody formation in patients treated with cyclophosphamide and prednisone.

A patient with systemic lupus erythematosus (SLE) and a patient with an immune complex disease resembling Goodpasture's syndrome were treated with cyclophosphamide, prednisone and repeated plasma exchanges. Circulating immune complexes decreased, and symptoms of central nervous system disease remitted for up to 15 to 20 days after plasma exchange in the patient with SLE. In vitro lymphocyte blastogenic responses to antigens were also transiently increased on two occasions following treatment. In the second patient, decreases in circulating immune complexes and clinical improvement were ascribed chiefly to immunosuppressive drug treatment. Serum antibody to keyhole limpet hemocyanin was relatively unaffected by plasma exchange in both patients. These results suggest that plasma exchange may help to deplete circulating immune complexes or alter the equilibrium between soluble antigen and antibody which causes complexes to form and circulate. It may be less effective in reducing circulating antibody levels in patients who continue to produce new antibody.

The usefulness of electron microscopy in the diagnosis of human tumors.

Ultrastructural features of neoplastic cells can provide clues for correct diagnosis when light microscopy fails. Secretory granules are characteristic in the following tumors: mucin granules in poorly differentiated adenocarcinomas, zymogen granules in acinic cell carcinomas, lysosomal granules in prostatic carcinomas, melanin granules in malignant melanoma, carcinoid, islet cell tumors, pheochromocytoma, and neuroblastoma granules in the corresponding neoplasms. Among cytoplasmic organelles, rough surfaced endoplasmic reticulum characterizes adrenocortical, ovarian, and hepatocellular carcinomas and plasmacytomas. Tonofibrils are characteristic of squamous cell carcinomas. Glycogen deposits distinguish Ewing's sarcoma from lymphoreticular neoplasms. Intercellular relationships and membrane specialization are important features in the differential diagnosis of various undifferentiated tumors. The frequent resolution of difficult diagnostic problems by electron microscopy outweighs the disadvantages of this technique, such as the expense and time required.

Acquired immune deficiency syndrome: postmortem findings.

The autopsies of 13 male homosexuals with acquired immune deficiency syndrome (AIDS) were reviewed. All patients had laboratory evidence of cellular immune dysfunction. The most common diagnoses made were disseminated cytomegalovirus infection in 12 patients and Kaposi's sarcoma in 10. All patients infected with cytomegalovirus had pulmonary compromise. The adrenal glands and gastrointestinal tract also were involved often by cytomegalovirus. Cytomegalovirus infection of organs uncommonly affected such as heart, meninges, cerebrum, and peripheral nerves was documented in two patients. Skin most frequently was involved by Kaposi's sarcoma, followed by gastrointestinal tract and lymph nodes. Two patients had visceral and/or nodal Kaposi's sarcoma with no skin compromise. Other important diagnoses were Pneumocystis carinii pneumonia, cryptosporidiosis, fungal infections, toxoplasmosis, and brain lymphoma. The cause of death was due to one or more infections in most patients. Kaposi's sarcoma did not contribute substantially to the cause of death, except in one patient with massive multifocal and multiorgan involvement.

Zinc binding by glycosaminoglycans.

Seven different standard GAGs were shown to bind various amounts of 65Zn by gel filtration on Sephadex G-25 at pH 4.0 and pH 7.0. "Molar" zinc-binding ratios, calculated on the basis of molar bound 65Zn per hexuronic acid and sulfate of each GAG, were significantly different for C4S, C6S, DS, HA, HS, at the two pHs; however, there was no difference for HP and KS. Moreover, while the binding ratios of C4S, C6S, and DS were higher, those of HA and HS were lower at pH 4.0 than at pH 7.0. The order of increasing zinc-binding ratios was (i) HS, KS, HA, C6S, C4S, DS, HP at pH 4.0 and (ii) C6S, KS, C4S, DS, HS, HP, HA at pH 7.0.

Prostatic adenoma of ductal origin.

A case of prostatic adenoma believed to originate from the prostatic duct is described. There were morphologic similarities to basal cell adenomas of salivary glands, and it was concluded that the tumor is a benign counterpart of "salivary gland" carcinomas, rarely observed in the prostate.

Bidimensional electrophoresis in the diagnosis of mucopolysaccharidosis.

Using the modified bidimensional electrophoresis method, characteristic patterns of urinary glycosaminoglycans were obtained for nine different syndromes of mucopolysaccharidoses and normal. Each pattern, portrayed on a 55 mm square cellulose acetate membrane, was visualized by staining the electrophoretically separated GAGs with alcian blue. The method, tested with 38 cases, was precise and sensitive; the first direction of electrophoresis was run for 25 min and the second direction for 60 min. Because the method is qualitative, the usual quantitative measurements and calculations are circumvented. Moreover, any irregular pattern can readily be verified by repeating the bidimensional electrophoresis. Thus, the clarity of the pattern itself offers assurance of its diagnostic reliabilty; consequently, false negative and false positive results should be minimized.

IgA nephropathy in a patient with ankylosing spondylitis and a solitary kidney.

We describe a patient with ankylosing spondylitis and psoriasis who was found to have IgA nephropathy in a solitary kidney. Renal biopsy demonstrated mesangial proliferation and interstitial nephritis with mesangial deposition of IgA. Although the renal disease and the rheumatic disease could have been present together by chance association, evidence is presented to suggest a possible common pathogenesis.

The effect of HIV infection on phagocytosis and killing of Staphylococcus aureus by human pulmonary alveolar macrophages.

Pulmonary alveolar macrophages (PAM) play a central role in host defense against pulmonary infection. The authors studied the number, viability, and ultrastructure of PAM recovered by bronchoalveolar lavage from normal and HIV-infected subjects, and their ability to phagocytose and kill Staphylococcus aureus. PAM from HIV-infected subjects who did not have pneumonia were present in greater numbers and phagocytosed significantly more opsonized Staphylococcus aureus (32.5% and 27.3% for nonsmokers and smokers, respectively) than did PAM from healthy controls (19.5% and 18.2%). In 15 patients with AIDS and pneumonia (due to Pneumocystis carinii in 13/15), viability of PAM and their phagocytic capacity were significantly reduced; in smokers with AIDS and pneumonia, the PAM yield was also dramatically decreased. Killing of S. aureus was similar by PAM from all patient groups. HIV infection was associated with the electron microscopic finding in PAM of extensively ruffled PAM cell-surfaces and ingestion of lymphocytes. Thus, HIV infection stimulates the phagocytic capacity and produces morphologic changes consistent with the possibility that PAM are activated by this retroviral infection. In patients with AIDS who develop pneumonia, especially in smokers, the number, viability and phagocytic capacity of PAM are significantly decreased; our study could not determine whether this diminished activity reflects evolution of the HIV infection or a secondary effect of the pneumonia.